http://2014.igem.org/wiki/index.php?title=Special:Contributions/Mf1512&feed=atom&limit=50&target=Mf1512&year=&month=2014.igem.org - User contributions [en]2024-03-28T21:39:25ZFrom 2014.igem.orgMediaWiki 1.16.5http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2015-02-02T16:05:15Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
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<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
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<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
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<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus igem</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and a new strain G. xylinus igem, creating a genetic toolbox of consisting of five new plasmid backbones and around 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
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<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em>.</li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain. </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem.</li> <br />
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<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em>.</li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a platform for genetic engineering of new cellulose-based biomaterials.</li><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
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<h2>Introduction</h2><br />
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<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
In the middle of the summer, we isolated a new strain of Gluconacetobacter (named G. xylinus igem, which we previously called G. xylinus KI for Kombucha isolated ) from Kombucha tea and characterized its properties fully. We found it to be more amenable for genetic engineering due to higher transformation efficiencies than <em>G. xylinus</em> ATCC 53582 strain (see below), so we continued work on this strain, including sequencing its genome due to potential economic importance (it is a key component in the popular Kombucha tea). <br />
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<h2>Aims</h2><br />
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<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and igem strains and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
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<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
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<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000 for "simple" to sequence genomes, but can far exceed that for repeat-rich, GC-rich or otherwise problematic genomes. <br />
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In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing, and were able to sequence two genomes for less than £1000. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of <em> G.xylinus </em> resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and igem in a single run, paired-end 250bp reads using standard flow cell) on an Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure 1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. <br />
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As a result, we are proud to present here the first genomes sequenced in iGEM. The genome of <em>G. xylinus igem </em> strain is approximately 3.4Mbp in size, with a GC content of 63.52% (see Figure 2). Autoannotation using Prokka (Seemann 2014) identified in total 3052 putative coding sequences, 59 tRNA sequences and 24 rRNA and other RNA sequences. The genome <em> G. xylinus</em> ATCC53582 is approximately 2.8Mbp in size, with a GC content of 59.82% (see Figure 3). Autoannotation using Prokka (Seemann 2014) identified in total 2491 putative coding sequences, 46 tRNA sequences and 17 rRNA and other RNA sequences. We are currently performing on the comparitive genomics of these genomes and related genomes, and will properly publish our results in a peer-reviewed journal. The full genome sequences, as well as detailed information about <em>G. xylinus</em> igem strain and genome can be found on the Registry as part <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1321306">BBa_K1321306</a><br />
and <em>G. xylinus</em> ATCC53582 as part <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1321305. "> BBa_K1321305 </a><br />
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<img class="image-left" src="https://static.igem.org/mediawiki/2014/f/f5/IC14_G.xylinus_ATCC_53582_genome_overview.jpg"><br />
<figcaption> Figure 2. Overview of the genome of G.xylinus ATCC 53582. The genome is approximately 2.8Mbp in size, with a GC content of around 60% (internal circle) and approximately 2500 coding sequences (external circle; blue bands denote single CDSs). Note that this overview should be taken as a guide, as further analysis may reveal a more accurate organization of the contigs. </figcaption><br />
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<img class="image-left" src="https://static.igem.org/mediawiki/2014/9/9b/IC14_G.xylinus_igem_genome_view.png"><br />
<figcaption> Figure 3. Overview of the genome of G.xylinus igem. The genome is approximately 3.4Mbp in size, with a GC content of around 63% (internal circle) and approximately 3000 coding sequences (external circle; blue bands denote single CDSs. Note that this overview should be taken as a guide, as further analysis may reveal a more accurate organization of the contigs. </figcaption><br />
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<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and igem strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and igem strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus igem strains to increase cellulose productivity and decrease its cost. In total, by cloning widely used parts into our <em>G. xylinus</em> shuttle vectors we created a toolkit consisting of:<br />
<ul><br />
<li> 5 new shuttle vectors </li> <br />
<li> 15 Anderson promoters </li><br />
<li> Fluorescent proteins RFP and sfGFP </li><br />
<li> 5 chromoproteins </li><br />
<li> 11 CBD fusion proteins </li> <br />
<li> Vitreoscilla hemoglobin gene </li> <br />
<li> TetR and Arabinose inducible promoters </li><br />
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<p> Because the toolkit is in the non-standard pSEVA331-Bb backbone, it can not be housed in the Parts Registry, however in order to make it accessible for the synthetic biology community, we have made it freely available on request.<br />
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<h3>Characterization of G.xylinus ATCC 53582 and igem strains</h3><br />
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<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
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<p> Cellulose productivity of G.xylinus ATCC 53582 and igem on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the igem strain is approximately one fifth of that of ATCC 53582 strain (see Figure 4 for comparison of ATCC53582 and igem strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for igem cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus). <br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 4. Cellulose production of G.xylinus ATCC 53582 and igem (KI) strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
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<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure 5.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, igem strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 5 for comparison of ATCC and igem strains and details of he experiment). This could be due to the niche igem strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
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<figcaption> Figure 5. Cellulose productivity of G.xylinum ATCC 53582 and igem (KI) strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
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<p> Natural antibiotics resistance of ATCC 53582 and igem strains </p><br />
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<figcaption> Figure 6. Natural antibiotics resistance of G.xylinus ATCC 53582 and igem (KI) strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and igem seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
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<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 6 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and igem strains). However, when using high cell densities for plating (after transformations), antibiotic-resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus igem is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the igem strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
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<h3>Plasmid backbones</h3><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 7. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus igem after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
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<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and igem strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus igem and ATCC 53583 (See Figure 7). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
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<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
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<h3>Anderson promoters</h3><br />
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<p> We inserted 15 Anderson promoter-RFP coding devices into pSEVA331-Bb backbone, transformed them into G.xylinus igem strain in order to determine their relative strengths in Gluconacetobacter (see Figure 8 for some of them: <br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/1/1a/IC14_Composite_image_correct.jpg"><br />
<figcaption>Figure 8. Different Anderson promoter-RFP constructs expressed in G.xylinus. Left- wild-type negative control. Right- G.xylinus igem transformed with pSEVA331Bb-Anderson promoter constructs. Plates grown at 30C for 7 days before imageing under blue light. </figcaption><br />
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<h3>Vitreoscilla hemoglobin</h3><br />
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<p><i>G. xylinus</i> is obligatively aerobic because of which cellulose production is reduced below the medium surface in static culture[1] due to reduced oxygen concentrations, reducing the overall volume of the pellicle that is biologically active. Transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i> (another obligate aerobe that expresses this protein in oxygen-poor environments) has been shown to result in increased the metabolic activity and cellulose production of G.xylinus [2].<br />
Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure 9. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem wild type cells and cells transformed with pSEVA331-BBa_K1321200 plasmid were cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days, after which OD600 was measured. Samples were diluted 1:1 in HS-cellulase medium before measurement. Negative controls (HS-cellulase without inoculations) showed no growth. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
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<p>We cloned the Vhb gene behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS (part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a>) in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure 9) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
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<p>We also made a hypothesis of the metabolic pathway involving the interaction of vHb, which could potentially explain its effect on enhancing bacterial cellulose yield. As described in the diagram above, vHb plays a role as oxygen storage: it will actively capture oxygen molecules when when the oxygen concentration is high in the cell whereas release oxygen molecules when it is low. As such, the presence of vHb will smooth out the oxygen level within the cell and therefore provides it with a relatively constant oxygen supply. Considering the fact that oxygen also bind to protein complex cG PDE (cyclic GMP phosphodiesterase), where the binding will impose an upregulating effect on bacterial cellulose production, a constant oxygen level maintained by vHb will inevitably contribute to enhance bacterial cellulose yield.</p><br />
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<figcaption>Figure 10. Effects of vHb interaction on cellular oxygen level.</figcaption><br />
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<h2>References</h2><br />
<ul><br />
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<li>[1] https://www.researchgate.net/ publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2015-02-02T15:50:04Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
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<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
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<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus igem</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and a new strain G. xylinus igem, creating a genetic toolbox of consisting of five new plasmid backbones and around 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
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<h2>Key Achievements </h2><br />
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<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em>.</li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain. </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem.</li> <br />
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<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em>.</li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a platform for genetic engineering of new cellulose-based biomaterials.</li><br />
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<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
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<h2>Introduction</h2><br />
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<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
In the middle of the summer, we isolated a new strain of Gluconacetobacter (named G. xylinus igem, which we previously called G. xylinus KI for Kombucha isolated ) from Kombucha tea and characterized its properties fully. We found it to be more amenable for genetic engineering due to higher transformation efficiencies than <em>G. xylinus</em> ATCC 53582 strain (see below), so we continued work on this strain, including sequencing its genome due to potential economic importance (it is a key component in the popular Kombucha tea). <br />
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<h2>Aims</h2><br />
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<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and igem strains and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
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<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
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<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000 for "simple" to sequence genomes, but can far exceed that for repeat-rich, GC-rich or otherwise problematic genomes. <br />
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In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing, and were able to sequence two genomes for less than £1000. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of <em> G.xylinus </em> resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and igem in a single run, paired-end 250bp reads using standard flow cell) on an Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure 1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. <br />
As a result, we are proud to present here the first genomes sequenced in iGEM. The genome of <em>G. xylinus<em> igem strain is approximately 3.4Mbp in size, with a GC content of 63.52% (see Figure 2). Autoannotation using Prokka (Seemann 2014) identified in total 3052 putative coding sequences, 59 tRNA sequences and 24 rRNA and other RNA sequences. The genome of <em> G. xylinus <em> ATCC53582 is approximately 2.8Mbp in size, with a GC content of 59.82% (see Figure 2). Autoannotation using Prokka (Seemann 2014) identified in total 2491 putative coding sequences, 46 tRNA sequences and 17 rRNA and other RNA sequences. We are currently performing on the comparitive genomics of these genomes and related genomes, and will properly publish our results in a peer-reviewed journal. The full genome sequences, as well as detailed information about <em>G. xylinus<em> igem strain and genome can be found on the Registry as part BBa_K1321305 http://parts.igem.org/wiki/index.php?title=Part:BBa_K1321306 and <em>G. xylinus<em> ATCC53582 as part BBa_K1321305 http://parts.igem.org/wiki/index.php?title=Part:BBa_K1321305. <br />
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<img class="image-left" src="https://static.igem.org/mediawiki/2014/f/f5/IC14_G.xylinus_ATCC_53582_genome_overview.jpg"><br />
<figcaption> Figure 2. Overview of the genome of G.xylinus ATCC 53582. The genome is approximately 2.8Mbp in size, with a GC content of around 60% (internal circle) and approximately 2500 coding sequences (external circle; blue bands denote single CDSs). Note that this overview should be taken as a guide, as further analysis may reveal a more accurate organization of the contigs. </figcaption><br />
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<img class="image-right" src="https://static.igem.org/mediawiki/2014/9/9b/IC14_G.xylinus_igem_genome_view.png"><br />
<figcaption> Figure 3. Overview of the genome of G.xylinus igem. The genome is approximately 3.4Mbp in size, with a GC content of around 63% (internal circle) and approximately 3000 coding sequences (external circle; blue bands denote single CDSs. Note that this overview should be taken as a guide, as further analysis may reveal a more accurate organization of the contigs. </figcaption><br />
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<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and igem strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and igem strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus igem strains to increase cellulose productivity and decrease its cost. In total, by cloning widely used parts into our <em>G. xylinus</em> shuttle vectors we created a toolkit consisting of:<br />
<ul><br />
<li> 5 new shuttle vectors </li> <br />
<li> 15 Anderson promoters </li><br />
<li> Fluorescent proteins RFP and sfGFP </li><br />
<li> 5 chromoproteins </li><br />
<li> 11 CBD fusion proteins </li> <br />
<li> Vitreoscilla hemoglobin gene </li> <br />
<li> TetR and Arabinose inducible promoters </li><br />
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<p> Because the toolkit is in the non-standard pSEVA331-Bb backbone, it can not be housed in the Parts Registry, however in order to make it accessible for the synthetic biology community, we have made it freely available on request.<br />
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<h3>Characterization of G.xylinus ATCC 53582 and igem strains</h3><br />
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<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
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<p> Cellulose productivity of G.xylinus ATCC 53582 and igem on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the igem strain is approximately one fifth of that of ATCC 53582 strain (see Figure 4 for comparison of ATCC53582 and igem strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for igem cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus). <br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 4. Cellulose production of G.xylinus ATCC 53582 and igem (KI) strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
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<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure 5.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, igem strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 5 for comparison of ATCC and igem strains and details of he experiment). This could be due to the niche igem strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
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<figcaption> Figure 5. Cellulose productivity of G.xylinum ATCC 53582 and igem (KI) strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
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<p> Natural antibiotics resistance of ATCC 53582 and igem strains </p><br />
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<figcaption> Figure 6. Natural antibiotics resistance of G.xylinus ATCC 53582 and igem (KI) strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and igem seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
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<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 6 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and igem strains). However, when using high cell densities for plating (after transformations), antibiotic-resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus igem is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the igem strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
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<h3>Plasmid backbones</h3><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 7. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus igem after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
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<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and igem strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus igem and ATCC 53583 (See Figure 7). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
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<h3>Anderson promoters</h3><br />
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<p> We inserted 15 Anderson promoter-RFP coding devices into pSEVA331-Bb backbone, transformed them into G.xylinus igem strain in order to determine their relative strengths in Gluconacetobacter (see Figure 8 for some of them: <br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/1/1a/IC14_Composite_image_correct.jpg"><br />
<figcaption>Figure 8. Different Anderson promoter-RFP constructs expressed in G.xylinus. Left- wild-type negative control. Right- G.xylinus igem transformed with pSEVA331Bb-Anderson promoter constructs. Plates grown at 30C for 7 days before imageing under blue light. </figcaption><br />
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<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p><i>G. xylinus</i> is obligatively aerobic because of which cellulose production is reduced below the medium surface in static culture[1] due to reduced oxygen concentrations, reducing the overall volume of the pellicle that is biologically active. Transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i> (another obligate aerobe that expresses this protein in oxygen-poor environments) has been shown to result in increased the metabolic activity and cellulose production of G.xylinus [2].<br />
Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure 9. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem wild type cells and cells transformed with pSEVA331-BBa_K1321200 plasmid were cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days, after which OD600 was measured. Samples were diluted 1:1 in HS-cellulase medium before measurement. Negative controls (HS-cellulase without inoculations) showed no growth. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
</figure><br />
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<p>We cloned the Vhb gene behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS (part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a>) in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure 9) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
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<p>We also made a hypothesis of the metabolic pathway involving the interaction of vHb, which could potentially explain its effect on enhancing bacterial cellulose yield. As described in the diagram above, vHb plays a role as oxygen storage: it will actively capture oxygen molecules when when the oxygen concentration is high in the cell whereas release oxygen molecules when it is low. As such, the presence of vHb will smooth out the oxygen level within the cell and therefore provides it with a relatively constant oxygen supply. Considering the fact that oxygen also bind to protein complex cG PDE (cyclic GMP phosphodiesterase), where the binding will impose an upregulating effect on bacterial cellulose production, a constant oxygen level maintained by vHb will inevitably contribute to enhance bacterial cellulose yield.</p><br />
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<figcaption>Figure 10. Effects of vHb interaction on cellular oxygen level.</figcaption><br />
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<h2>References</h2><br />
<ul><br />
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<li>[1] https://www.researchgate.net/ publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2015-02-02T15:46:37Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
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<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
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<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
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<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus igem</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and a new strain G. xylinus igem, creating a genetic toolbox of consisting of five new plasmid backbones and around 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
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<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em>.</li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain. </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem.</li> <br />
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<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em>.</li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a platform for genetic engineering of new cellulose-based biomaterials.</li><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
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<h2>Introduction</h2><br />
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<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
In the middle of the summer, we isolated a new strain of Gluconacetobacter (named G. xylinus igem, which we previously called G. xylinus KI for Kombucha isolated ) from Kombucha tea and characterized its properties fully. We found it to be more amenable for genetic engineering due to higher transformation efficiencies than <em>G. xylinus</em> ATCC 53582 strain (see below), so we continued work on this strain, including sequencing its genome due to potential economic importance (it is a key component in the popular Kombucha tea). <br />
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<section id="aim"><br />
<h2>Aims</h2><br />
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<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and igem strains and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
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<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
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<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000 for "simple" to sequence genomes, but can far exceed that for repeat-rich, GC-rich or otherwise problematic genomes. <br />
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In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing, and were able to sequence two genomes for less than £1000. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of <em> G.xylinus </em> resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and igem in a single run, paired-end 250bp reads using standard flow cell) on an Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure 1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. <br />
As a result, we are proud to present here the first genomes sequenced in iGEM. The genome of <em>G. xylinus<em> igem strain is approximately 3.4Mbp in size, with a GC content of 63.52% (see Figure 2). Autoannotation using Prokka (Seemann 2014) identified in total 3052 putative coding sequences, 59 tRNA sequences and 24 rRNA and other RNA sequences. The genome of <em> G. xylinus <em> ATCC53582 is approximately 2.8Mbp in size, with a GC content of 59.82% (see Figure 2). Autoannotation using Prokka (Seemann 2014) identified in total 2491 putative coding sequences, 46 tRNA sequences and 17 rRNA and other RNA sequences. We are currently performing on the comparitive genomics of these genomes and related genomes, and will properly publish our results in a peer-reviewed journal. The full genome sequences, as well as detailed information about <em>G. xylinus<em> igem strain and genome can be found on the Registry as part BBa_K1321305 http://parts.igem.org/wiki/index.php?title=Part:BBa_K1321306 and <em>G. xylinus<em> ATCC53582 as part BBa_K1321305 http://parts.igem.org/wiki/index.php?title=Part:BBa_K1321305. <br />
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<img class="image-left" src="https://static.igem.org/mediawiki/2014/f/f5/IC14_G.xylinus_ATCC_53582_genome_overview.jpg"><br />
<figcaption> Figure 2. Overview of the genome of G.xylinus ATCC 53582. The genome is approximately 2.8Mbp in size, with a GC content of around 60% (internal circle) and approximately 2500 coding sequences (external circle; blue bands denote single CDSs). Note that this overview should be taken as a guide, as further analysis may reveal a more accurate organization of the contigs. </figcaption><br />
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<img class="image-right" src="https://static.igem.org/mediawiki/2014/9/9b/IC14_G.xylinus_igem_genome_view.png"><br />
<figcaption> Figure 3. Overview of the genome of G.xylinus igem. The genome is approximately 3.4Mbp in size, with a GC content of around 63% (internal circle) and approximately 3000 coding sequences (external circle; blue bands denote single CDSs. Note that this overview should be taken as a guide, as further analysis may reveal a more accurate organization of the contigs. </figcaption><br />
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<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and igem strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and igem strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus igem strains to increase cellulose productivity and decrease its cost. In total, by cloning widely used parts into our <em>G. xylinus</em> shuttle vectors we created a toolkit consisting of:<br />
<ul><br />
<li> 5 new shuttle vectors </li> <br />
<li> 15 Anderson promoters </li><br />
<li> Fluorescent proteins RFP and sfGFP </li><br />
<li> 5 chromoproteins </li><br />
<li> 11 CBD fusion proteins </li> <br />
<li> Vitreoscilla hemoglobin gene </li> <br />
<li> TetR and Arabinose inducible promoters </li><br />
</ul><br />
</p><br />
<p> Because the toolkit is in the non-standard pSEVA331-Bb backbone, it can not be housed in the Parts Registry, however in order to make it accessible for the synthetic biology community, we have made it freely available on request.<br />
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<h3>Characterization of G.xylinus ATCC 53582 and igem strains</h3><br />
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<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
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<p> Cellulose productivity of G.xylinus ATCC 53582 and igem on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the igem strain is approximately one fifth of that of ATCC 53582 strain (see Figure 2 for comparison of ATCC53582 and igem strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for igem cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus). <br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 2. Cellulose production of G.xylinus ATCC 53582 and igem (KI) strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
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<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure 3.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, igem strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 3 for comparison of ATCC and igem strains and details of he experiment). This could be due to the niche igem strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
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<figcaption> Figure 3. Cellulose productivity of G.xylinum ATCC 53582 and igem (KI) strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
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<p> Natural antibiotics resistance of ATCC 53582 and igem strains </p><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 4. Natural antibiotics resistance of G.xylinus ATCC 53582 and igem (KI) strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and igem seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
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<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 4 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and igem strains). However, when using high cell densities for plating (after transformations), antibiotic-resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus igem is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the igem strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
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<h3>Plasmid backbones</h3><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 5. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus igem after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
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<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and igem strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus igem and ATCC 53583 (See Figure 5). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
</ul><br />
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<h3>Anderson promoters</h3><br />
<div><br />
<p> We inserted 15 Anderson promoter-RFP coding devices into pSEVA331-Bb backbone, transformed them into G.xylinus igem strain in order to determine their relative strengths in Gluconacetobacter (see Figure 6 for some of them: <br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/1/1a/IC14_Composite_image_correct.jpg"><br />
<figcaption>Figure 6. Different Anderson promoter-RFP constructs expressed in G.xylinus. Left- wild-type negative control. Right- G.xylinus igem transformed with pSEVA331Bb-Anderson promoter constructs. Plates grown at 30C for 7 days before imageing under blue light. </figcaption><br />
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<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p><i>G. xylinus</i> is obligatively aerobic because of which cellulose production is reduced below the medium surface in static culture[1] due to reduced oxygen concentrations, reducing the overall volume of the pellicle that is biologically active. Transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i> (another obligate aerobe that expresses this protein in oxygen-poor environments) has been shown to result in increased the metabolic activity and cellulose production of G.xylinus [2].<br />
Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure 7. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem wild type cells and cells transformed with pSEVA331-BBa_K1321200 plasmid were cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days, after which OD600 was measured. Samples were diluted 1:1 in HS-cellulase medium before measurement. Negative controls (HS-cellulase without inoculations) showed no growth. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
</figure><br />
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<p>We cloned the Vhb gene behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS (part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a>) in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure 7) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
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<p>We also made a hypothesis of the metabolic pathway involving the interaction of vHb, which could potentially explain its effect on enhancing bacterial cellulose yield. As described in the diagram above, vHb plays a role as oxygen storage: it will actively capture oxygen molecules when when the oxygen concentration is high in the cell whereas release oxygen molecules when it is low. As such, the presence of vHb will smooth out the oxygen level within the cell and therefore provides it with a relatively constant oxygen supply. Considering the fact that oxygen also bind to protein complex cG PDE (cyclic GMP phosphodiesterase), where the binding will impose an upregulating effect on bacterial cellulose production, a constant oxygen level maintained by vHb will inevitably contribute to enhance bacterial cellulose yield.</p><br />
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<figcaption>Figure 8. Effects of vHb interaction on cellular oxygen level.</figcaption><br />
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<h2>References</h2><br />
<ul><br />
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<li>[1] https://www.researchgate.net/ publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2015-02-02T15:43:38Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
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<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
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<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
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<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus igem</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and a new strain G. xylinus igem, creating a genetic toolbox of consisting of five new plasmid backbones and around 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
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<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em>.</li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain. </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem.</li> <br />
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<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em>.</li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a platform for genetic engineering of new cellulose-based biomaterials.</li><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
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<h2>Introduction</h2><br />
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<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
In the middle of the summer, we isolated a new strain of Gluconacetobacter (named G. xylinus igem, which we previously called G. xylinus KI for Kombucha isolated ) from Kombucha tea and characterized its properties fully. We found it to be more amenable for genetic engineering due to higher transformation efficiencies than <em>G. xylinus</em> ATCC 53582 strain (see below), so we continued work on this strain, including sequencing its genome due to potential economic importance (it is a key component in the popular Kombucha tea). <br />
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<section id="aim"><br />
<h2>Aims</h2><br />
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<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and igem strains and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
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<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
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<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000 for "simple" to sequence genomes, but can far exceed that for repeat-rich, GC-rich or otherwise problematic genomes. <br />
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In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing, and were able to sequence two genomes for less than £1000. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of <em> G.xylinus </em> resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and igem in a single run, paired-end 250bp reads using standard flow cell) on an Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure 1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. <br />
As a result, we are proud to present here the first genomes sequenced in iGEM. The genome of <em>G. xylinus<em> igem strain is approximately 3.4Mbp in size, with a GC content of 63.52% (see Figure 2). Autoannotation using Prokka (Seemann 2014) identified in total 3052 putative coding sequences, 59 tRNA sequences and 24 rRNA and other RNA sequences. The genome of <em> G. xylinus <em> ATCC53582 is approximately 2.8Mbp in size, with a GC content of 59.82% (see Figure 2). Autoannotation using Prokka (Seemann 2014) identified in total 2491 putative coding sequences, 46 tRNA sequences and 17 rRNA and other RNA sequences. We are currently performing on the comparitive genomics of these genomes and related genomes, and will properly publish our results in a peer-reviewed journal. The full genome sequences, as well as detailed information about <em>G. xylinus<em> igem strain and genome can be found on the Registry as part BBa_K1321305 http://parts.igem.org/wiki/index.php?title=Part:BBa_K1321306 and <em>G. xylinus<em> ATCC53582 as part BBa_K1321305 http://parts.igem.org/wiki/index.php?title=Part:BBa_K1321305. <br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f5/IC14_G.xylinus_ATCC_53582_genome_overview.jpg"><br />
<figcaption> Figure 2. Overview of the genome of G.xylinus ATCC 53582. The genome is approximately 2.8Mbp in size, with a GC content of around 60% (internal circle) and approximately 2500 coding sequences (external circle; blue bands denote single CDSs). Note that this overview should be taken as a guide, as further analysis may reveal a more accurate organization of the contigs. </figcaption><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/9/9b/IC14_G.xylinus_igem_genome_view.png"><br />
<figcaption> Figure 3. Overview of the genome of G.xylinus igem. The genome is approximately 3.4Mbp in size, with a GC content of around 63% (internal circle) and approximately 3000 coding sequences (external circle; blue bands denote single CDSs. Note that this overview should be taken as a guide, as further analysis may reveal a more accurate organization of the contigs. </figcaption><br />
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<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and igem strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and igem strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus igem strains to increase cellulose productivity and decrease its cost. In total, by cloning widely used parts into our <em>G. xylinus</em> shuttle vectors we created a toolkit consisting of:<br />
<ul><br />
<li> 5 new shuttle vectors </li> <br />
<li> 15 Anderson promoters </li><br />
<li> Fluorescent proteins RFP and sfGFP </li><br />
<li> 5 chromoproteins </li><br />
<li> 11 CBD fusion proteins </li> <br />
<li> Vitreoscilla hemoglobin gene </li> <br />
<li> TetR and Arabinose inducible promoters </li><br />
</ul><br />
</p><br />
<p> Because the toolkit is in the non-standard pSEVA331-Bb backbone, it can not be housed in the Parts Registry, however in order to make it accessible for the synthetic biology community, we have made it freely available on request.<br />
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<h3>Characterization of G.xylinus ATCC 53582 and igem strains</h3><br />
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<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
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<p> Cellulose productivity of G.xylinus ATCC 53582 and igem on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the igem strain is approximately one fifth of that of ATCC 53582 strain (see Figure 2 for comparison of ATCC53582 and igem strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for igem cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus). <br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 2. Cellulose production of G.xylinus ATCC 53582 and igem (KI) strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
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<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure 3.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, igem strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 3 for comparison of ATCC and igem strains and details of he experiment). This could be due to the niche igem strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 3. Cellulose productivity of G.xylinum ATCC 53582 and igem (KI) strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
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</p> <br />
<p> Natural antibiotics resistance of ATCC 53582 and igem strains </p><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 4. Natural antibiotics resistance of G.xylinus ATCC 53582 and igem (KI) strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and igem seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
</figure><br />
<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 4 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and igem strains). However, when using high cell densities for plating (after transformations), antibiotic-resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus igem is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the igem strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
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<h3>Plasmid backbones</h3><br />
<div><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 5. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus igem after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
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<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and igem strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus igem and ATCC 53583 (See Figure 5). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
</ul><br />
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<h3>Anderson promoters</h3><br />
<div><br />
<p> We inserted 15 Anderson promoter-RFP coding devices into pSEVA331-Bb backbone, transformed them into G.xylinus igem strain in order to determine their relative strengths in Gluconacetobacter (see Figure 6 for some of them: <br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/1/1a/IC14_Composite_image_correct.jpg"><br />
<figcaption>Figure 6. Different Anderson promoter-RFP constructs expressed in G.xylinus. Left- wild-type negative control. Right- G.xylinus igem transformed with pSEVA331Bb-Anderson promoter constructs. Plates grown at 30C for 7 days before imageing under blue light. </figcaption><br />
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<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p><i>G. xylinus</i> is obligatively aerobic because of which cellulose production is reduced below the medium surface in static culture[1] due to reduced oxygen concentrations, reducing the overall volume of the pellicle that is biologically active. Transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i> (another obligate aerobe that expresses this protein in oxygen-poor environments) has been shown to result in increased the metabolic activity and cellulose production of G.xylinus [2].<br />
Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure 7. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem wild type cells and cells transformed with pSEVA331-BBa_K1321200 plasmid were cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days, after which OD600 was measured. Samples were diluted 1:1 in HS-cellulase medium before measurement. Negative controls (HS-cellulase without inoculations) showed no growth. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
</figure><br />
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<p>We cloned the Vhb gene behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS (part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a>) in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure 7) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
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<p>We also made a hypothesis of the metabolic pathway involving the interaction of vHb, which could potentially explain its effect on enhancing bacterial cellulose yield. As described in the diagram above, vHb plays a role as oxygen storage: it will actively capture oxygen molecules when when the oxygen concentration is high in the cell whereas release oxygen molecules when it is low. As such, the presence of vHb will smooth out the oxygen level within the cell and therefore provides it with a relatively constant oxygen supply. Considering the fact that oxygen also bind to protein complex cG PDE (cyclic GMP phosphodiesterase), where the binding will impose an upregulating effect on bacterial cellulose production, a constant oxygen level maintained by vHb will inevitably contribute to enhance bacterial cellulose yield.</p><br />
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<img class="image-full" src="https://lh5.googleusercontent.com/OkLBTdjMaO6PjVewhOFchlpEd3x4REDL53KYLp_sSxkD35RU4U8Gh21PJKUSfTA1GZ7ShEayn0Y=w751-h608"><br />
<figcaption>Figure 8. Effects of vHb interaction on cellular oxygen level.</figcaption><br />
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<h2>References</h2><br />
<ul><br />
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<li>[1] https://www.researchgate.net/ publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/File:IC14_G.xylinus_igem_genome_view.pngFile:IC14 G.xylinus igem genome view.png2015-02-02T15:42:16Z<p>Mf1512: </p>
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<div></div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2015-02-02T15:40:10Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
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<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
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<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
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<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus igem</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and a new strain G. xylinus igem, creating a genetic toolbox of consisting of five new plasmid backbones and around 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
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<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em>.</li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain. </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem.</li> <br />
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<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em>.</li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a platform for genetic engineering of new cellulose-based biomaterials.</li><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
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<h2>Introduction</h2><br />
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<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
In the middle of the summer, we isolated a new strain of Gluconacetobacter (named G. xylinus igem, which we previously called G. xylinus KI for Kombucha isolated ) from Kombucha tea and characterized its properties fully. We found it to be more amenable for genetic engineering due to higher transformation efficiencies than <em>G. xylinus</em> ATCC 53582 strain (see below), so we continued work on this strain, including sequencing its genome due to potential economic importance (it is a key component in the popular Kombucha tea). <br />
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<section id="aim"><br />
<h2>Aims</h2><br />
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<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and igem strains and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
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<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
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<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000 for "simple" to sequence genomes, but can far exceed that for repeat-rich, GC-rich or otherwise problematic genomes. <br />
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In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing, and were able to sequence two genomes for less than £1000. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of <em> G.xylinus </em> resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and igem in a single run, paired-end 250bp reads using standard flow cell) on an Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure 1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. <br />
As a result, we are proud to present here the first genomes sequenced in iGEM. The genome of <em>G. xylinus<em> igem strain is approximately 3.4Mbp in size, with a GC content of 63.52% (see Figure 2). Autoannotation using Prokka (Seemann 2014) identified in total 3052 putative coding sequences, 59 tRNA sequences and 24 rRNA and other RNA sequences. The genome of <em> G. xylinus <em> ATCC53582 is approximately 2.8Mbp in size, with a GC content of 59.82% (see Figure 2). Autoannotation using Prokka (Seemann 2014) identified in total 2491 putative coding sequences, 46 tRNA sequences and 17 rRNA and other RNA sequences. We are currently performing on the comparitive genomics of these genomes and related genomes, and will properly publish our results in a peer-reviewed journal. The full genome sequences, as well as detailed information about <em>G. xylinus<em> igem strain and genome can be found on the Registry as part BBa_K1321305 http://parts.igem.org/wiki/index.php?title=Part:BBa_K1321306 and <em>G. xylinus<em> ATCC53582 as part BBa_K1321305 http://parts.igem.org/wiki/index.php?title=Part:BBa_K1321305. <br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f5/IC14_G.xylinus_ATCC_53582_genome_overview.jpg"><br />
<figcaption> Figure2. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
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<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and igem strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and igem strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus igem strains to increase cellulose productivity and decrease its cost. In total, by cloning widely used parts into our <em>G. xylinus</em> shuttle vectors we created a toolkit consisting of:<br />
<ul><br />
<li> 5 new shuttle vectors </li> <br />
<li> 15 Anderson promoters </li><br />
<li> Fluorescent proteins RFP and sfGFP </li><br />
<li> 5 chromoproteins </li><br />
<li> 11 CBD fusion proteins </li> <br />
<li> Vitreoscilla hemoglobin gene </li> <br />
<li> TetR and Arabinose inducible promoters </li><br />
</ul><br />
</p><br />
<p> Because the toolkit is in the non-standard pSEVA331-Bb backbone, it can not be housed in the Parts Registry, however in order to make it accessible for the synthetic biology community, we have made it freely available on request.<br />
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<div class="accordion"><br />
<h3>Characterization of G.xylinus ATCC 53582 and igem strains</h3><br />
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<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
</p><br />
<p> Cellulose productivity of G.xylinus ATCC 53582 and igem on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the igem strain is approximately one fifth of that of ATCC 53582 strain (see Figure 2 for comparison of ATCC53582 and igem strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for igem cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus). <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 2. Cellulose production of G.xylinus ATCC 53582 and igem (KI) strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
</figure><br />
</p> <br />
<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure 3.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, igem strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 3 for comparison of ATCC and igem strains and details of he experiment). This could be due to the niche igem strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 3. Cellulose productivity of G.xylinum ATCC 53582 and igem (KI) strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
</figure><br />
</p> <br />
<p> Natural antibiotics resistance of ATCC 53582 and igem strains </p><br />
<p></p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 4. Natural antibiotics resistance of G.xylinus ATCC 53582 and igem (KI) strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and igem seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
</figure><br />
<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 4 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and igem strains). However, when using high cell densities for plating (after transformations), antibiotic-resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus igem is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the igem strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
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<h3>Plasmid backbones</h3><br />
<div><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 5. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus igem after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
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<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and igem strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus igem and ATCC 53583 (See Figure 5). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
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<h3>Anderson promoters</h3><br />
<div><br />
<p> We inserted 15 Anderson promoter-RFP coding devices into pSEVA331-Bb backbone, transformed them into G.xylinus igem strain in order to determine their relative strengths in Gluconacetobacter (see Figure 6 for some of them: <br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/1/1a/IC14_Composite_image_correct.jpg"><br />
<figcaption>Figure 6. Different Anderson promoter-RFP constructs expressed in G.xylinus. Left- wild-type negative control. Right- G.xylinus igem transformed with pSEVA331Bb-Anderson promoter constructs. Plates grown at 30C for 7 days before imageing under blue light. </figcaption><br />
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<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p><i>G. xylinus</i> is obligatively aerobic because of which cellulose production is reduced below the medium surface in static culture[1] due to reduced oxygen concentrations, reducing the overall volume of the pellicle that is biologically active. Transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i> (another obligate aerobe that expresses this protein in oxygen-poor environments) has been shown to result in increased the metabolic activity and cellulose production of G.xylinus [2].<br />
Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure 7. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem wild type cells and cells transformed with pSEVA331-BBa_K1321200 plasmid were cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days, after which OD600 was measured. Samples were diluted 1:1 in HS-cellulase medium before measurement. Negative controls (HS-cellulase without inoculations) showed no growth. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
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<p>We cloned the Vhb gene behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS (part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a>) in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure 7) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
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<p>We also made a hypothesis of the metabolic pathway involving the interaction of vHb, which could potentially explain its effect on enhancing bacterial cellulose yield. As described in the diagram above, vHb plays a role as oxygen storage: it will actively capture oxygen molecules when when the oxygen concentration is high in the cell whereas release oxygen molecules when it is low. As such, the presence of vHb will smooth out the oxygen level within the cell and therefore provides it with a relatively constant oxygen supply. Considering the fact that oxygen also bind to protein complex cG PDE (cyclic GMP phosphodiesterase), where the binding will impose an upregulating effect on bacterial cellulose production, a constant oxygen level maintained by vHb will inevitably contribute to enhance bacterial cellulose yield.</p><br />
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<img class="image-full" src="https://lh5.googleusercontent.com/OkLBTdjMaO6PjVewhOFchlpEd3x4REDL53KYLp_sSxkD35RU4U8Gh21PJKUSfTA1GZ7ShEayn0Y=w751-h608"><br />
<figcaption>Figure 8. Effects of vHb interaction on cellular oxygen level.</figcaption><br />
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<h2>References</h2><br />
<ul><br />
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<li>[1] https://www.researchgate.net/ publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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<h1><em>G. xylinus</em></h1><br />
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<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
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<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
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<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
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<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus igem</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and a new strain G. xylinus igem, creating a genetic toolbox of consisting of five new plasmid backbones and around 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
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<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em>.</li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain. </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem.</li> <br />
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<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em>.</li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a platform for genetic engineering of new cellulose-based biomaterials.</li><br />
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<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
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<h2>Introduction</h2><br />
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<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
In the middle of the summer, we isolated a new strain of Gluconacetobacter (named G. xylinus igem, which we previously called G. xylinus KI for Kombucha isolated ) from Kombucha tea and characterized its properties fully. We found it to be more amenable for genetic engineering due to higher transformation efficiencies than <em>G. xylinus</em> ATCC 53582 strain (see below), so we continued work on this strain, including sequencing its genome due to potential economic importance (it is a key component in the popular Kombucha tea). <br />
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<section id="aim"><br />
<h2>Aims</h2><br />
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<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and igem strains and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
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<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
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<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000 for "simple" to sequence genomes, but can exceed far that for repeat-rich, GC-rich or otherwise problematic genomes. <br />
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In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing, and were able to sequence two genomes for less than £1000. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of <em> G.xylinus </em> resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and igem in a single run, paired-end 250bp reads using standard flow cell) on an Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure 1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. <br />
As a result, we are proud to present here the first genomes sequenced in iGEM. The genome of <em>G. xylinus<em> igem strain is approximately 3.4Mbp in size, with a GC content of 63.52% (see Figure 2). Autoannotation using Prokka (Seemann 2014) identified in total 3052 putative coding sequences, 59 tRNA sequences and 24 rRNA and other RNA sequences. The genome of <em> G. xylinus <em> ATCC53582 is approximately 2.8Mbp in size, with a GC content of 59.82% (see Figure 2). Autoannotation using Prokka (Seemann 2014) identified in total 2491 putative coding sequences, 46 tRNA sequences and 17 rRNA and other RNA sequences. We are currently performing on the comparitive genomics of these genomes and related genomes, and will properly publish our results in a peer-reviewed journal. The full genome sequences, as well as detailed information about <em>G. xylinus<em> igem strain and genome can be found on the Registry as part BBa_K1321305 http://parts.igem.org/wiki/index.php?title=Part:BBa_K1321306 and <em>G. xylinus<em> ATCC53582 as part BBa_K1321305 http://parts.igem.org/wiki/index.php?title=Part:BBa_K1321305. <br />
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<figcaption> Figure2. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
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<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and igem strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and igem strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus igem strains to increase cellulose productivity and decrease its cost. In total, by cloning widely used parts into our <em>G. xylinus</em> shuttle vectors we created a toolkit consisting of:<br />
<ul><br />
<li> 5 new shuttle vectors </li> <br />
<li> 15 Anderson promoters </li><br />
<li> Fluorescent proteins RFP and sfGFP </li><br />
<li> 5 chromoproteins </li><br />
<li> 11 CBD fusion proteins </li> <br />
<li> Vitreoscilla hemoglobin gene </li> <br />
<li> TetR and Arabinose inducible promoters </li><br />
</ul><br />
</p><br />
<p> Because the toolkit is in the non-standard pSEVA331-Bb backbone, it can not be housed in the Parts Registry, however in order to make it accessible for the synthetic biology community, we have made it freely available on request.<br />
</p><br />
<div class="accordion"><br />
<h3>Characterization of G.xylinus ATCC 53582 and igem strains</h3><br />
<div><br />
<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
</p><br />
<p> Cellulose productivity of G.xylinus ATCC 53582 and igem on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the igem strain is approximately one fifth of that of ATCC 53582 strain (see Figure 2 for comparison of ATCC53582 and igem strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for igem cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus). <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 2. Cellulose production of G.xylinus ATCC 53582 and igem (KI) strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
</figure><br />
</p> <br />
<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure 3.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, igem strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 3 for comparison of ATCC and igem strains and details of he experiment). This could be due to the niche igem strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 3. Cellulose productivity of G.xylinum ATCC 53582 and igem (KI) strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
</figure><br />
</p> <br />
<p> Natural antibiotics resistance of ATCC 53582 and igem strains </p><br />
<p></p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 4. Natural antibiotics resistance of G.xylinus ATCC 53582 and igem (KI) strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and igem seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
</figure><br />
<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 4 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and igem strains). However, when using high cell densities for plating (after transformations), antibiotic-resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus igem is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the igem strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
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<h3>Plasmid backbones</h3><br />
<div><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 5. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus igem after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
</figure><br />
<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and igem strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus igem and ATCC 53583 (See Figure 5). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
</ul><br />
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<h3>Anderson promoters</h3><br />
<div><br />
<p> We inserted 15 Anderson promoter-RFP coding devices into pSEVA331-Bb backbone, transformed them into G.xylinus igem strain in order to determine their relative strengths in Gluconacetobacter (see Figure 6 for some of them: <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/1/1a/IC14_Composite_image_correct.jpg"><br />
<figcaption>Figure 6. Different Anderson promoter-RFP constructs expressed in G.xylinus. Left- wild-type negative control. Right- G.xylinus igem transformed with pSEVA331Bb-Anderson promoter constructs. Plates grown at 30C for 7 days before imageing under blue light. </figcaption><br />
</figure><br />
</p><br />
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<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p><i>G. xylinus</i> is obligatively aerobic because of which cellulose production is reduced below the medium surface in static culture[1] due to reduced oxygen concentrations, reducing the overall volume of the pellicle that is biologically active. Transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i> (another obligate aerobe that expresses this protein in oxygen-poor environments) has been shown to result in increased the metabolic activity and cellulose production of G.xylinus [2].<br />
Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
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<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure 7. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem wild type cells and cells transformed with pSEVA331-BBa_K1321200 plasmid were cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days, after which OD600 was measured. Samples were diluted 1:1 in HS-cellulase medium before measurement. Negative controls (HS-cellulase without inoculations) showed no growth. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
</figure><br />
<br />
<p>We cloned the Vhb gene behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS (part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a>) in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure 7) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
<br />
<p>We also made a hypothesis of the metabolic pathway involving the interaction of vHb, which could potentially explain its effect on enhancing bacterial cellulose yield. As described in the diagram above, vHb plays a role as oxygen storage: it will actively capture oxygen molecules when when the oxygen concentration is high in the cell whereas release oxygen molecules when it is low. As such, the presence of vHb will smooth out the oxygen level within the cell and therefore provides it with a relatively constant oxygen supply. Considering the fact that oxygen also bind to protein complex cG PDE (cyclic GMP phosphodiesterase), where the binding will impose an upregulating effect on bacterial cellulose production, a constant oxygen level maintained by vHb will inevitably contribute to enhance bacterial cellulose yield.</p><br />
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<img class="image-full" src="https://lh5.googleusercontent.com/OkLBTdjMaO6PjVewhOFchlpEd3x4REDL53KYLp_sSxkD35RU4U8Gh21PJKUSfTA1GZ7ShEayn0Y=w751-h608"><br />
<figcaption>Figure 8. Effects of vHb interaction on cellular oxygen level.</figcaption><br />
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<h2>References</h2><br />
<ul><br />
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<li>[1] https://www.researchgate.net/ publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/Mass_Production_and_ProcessingTeam:Imperial/Mass Production and Processing2014-10-18T03:59:31Z<p>Mf1512: </p>
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<h1>Mass Production and Processing</h1><br />
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<li><a data-scroll href="#introduction">Introduction</a><br />
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<h2>Introduction</h2><br />
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<figcaption>Figure 1: Blue dyed bacterial cellulose</figcaption><br />
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<p> Bacterial cellulose (BC) exhibits a <a href="https://2014.igem.org/Team:Imperial/Project_Background">multitude of different properties</a> depending on the processing, growth conditions, functionalisation and strain used (Bismarck 2013) for production of the material. Acquiring large quantities of cellulose produced would allow testing of a broad variety of cellulose processing methods and functionalisation steps. </p><br />
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<p> By mass producing cellulose this enables a better understanding of what material properties can be realistically produced during the short duration of iGEM. More importantly, it improves the likelihood of finding suitable processing candidates for the project’s aim of making a customisable ultrafiltration membrane, at the same time as allowing room for creativity and exploration of the remarkable properties of cellulose. </p><br />
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<h3>Minimum requirements</h3><br />
<ol><br />
<li>Treatment of BC requires killing the cells, particularly if the cells are genetically engineered, which is the aim for putting the customisable in ultrafiltration membranes.</li><br />
<li>Based on brainstorming with Central Saint Martins student Zuzana, removing the colour of BC is required as it looks displeasing to the eye otherwise, and seems counter-intuituve to filter clean water with cellulose coloured like turbid water. </li><br />
<li>Removal of the smell of BC has also been raised as a requirement, particularly by producers who work in close contact with the processing facilities.</li><br />
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<h3>Mass Production Methods</h3><br />
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<figure class="content-image image-right image-small"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/0/04/IC14-post-prod-fig-2-pellicle-comparison.png > <br />
<figcaption>Figure 2. left: A granular pellicle, right: even pellicle</figcaption> <br />
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<p>Setting up the mass production of cellulose was done according to the <a href="https://2014.igem.org/Team:Imperial/Protocols#gluconacetobacter">Kombucha media protocol</a> , which involved setting up 61 trays with media and G. xylinus and yeast co-culture as shown in figure 3. The trays were left to grow up over 7 days, after which diminishing pellicle growth was detected. Upon harvesting, the pellicles were sorted according to granular pellicles (see figure 2 left) and even pellicles (figure 2 right). All pellicles were kept in distilled water in large plastic buckets or containers. </p><br />
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<p>Below shows the general workflow we employed to mass produce our cellulose and illustrates the process of manufacturing biomaterials with significantly different properties despite originating from the same BC source.<br />
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<img class="content-image image-full" src="https://static.igem.org/mediawiki/2014/b/bf/IC14-mass_production2.png"> <br />
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<h2>Results</h2><br />
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<p>We have made bacterial cellulose of a quality high enough to be used for water filtration in an ultrafiltration membrane setting. From different trial and error, we tried making leather cellulose that can be used a potential fabric garment. We produced bacterial cellulose on a scale larger than any previous iGEM team has produced a biomaterial in the Manufacturing track before. This has provided us with opportunities to try treating cellulose with different low tech solutions that have been identified as invaluable to our collaborating Chemical Engineer Dr. Koonyang Lee. In his own words, we have produced more bacterial cellulose than he did during both his PhD and postdoc on BC, and come up with some simple solutions for practical issues. The ideas of ours that Dr. Lee has decided to carry on include using silicon coated baking paper in the protocol for measuring yield of BC to avoid BC sticking to the surface, and effectively using a non-bleach fabric stain remover to clean the BC thereby reducing the harmful impact of research in this area. </p><br />
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<h2>Appendices </h2><br />
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<li>0.1M NaOH for 60 min at 120C: Produces the most white cellulose but the process has been shown to produce some yellow/brownish cellulose if the film that covers the bottom of the pellicle was not removed before treatment.<br />
</li><br />
<li>1M NaHCO3 for 60 min at 120C: Produces quite poor results, 3 samples have been tested and even after 4 hours of treatment at 120C the samples were less white than similar samples treated in distilled water for the same duration.<br />
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<li> Heat treatment in distilled water at 120C: 3 samples still contained some residual brownish tint after 4 hours of incubation, but the samples produced were considerably whiter than those treated with baking soda solution<br />
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<li> Air drying without treatment first: Produces brownish cellulose with high moisture content left, material is flexible<br />
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<li>120C Oven drying without press, without treatment for 180 min: Produces brittle paper like cellulose, it is fragile, brownish and prone to tears.<br />
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<li>Oven drying with press (1l Duran bottle on top of two tiles) after NaOH treatment for 120 min: Produces flexible more plastic cellulose capable of being shaped into a cone that filters water through. The cellulose was capable of immersion into water, which produced wet cellulose that could be reshaped and redried. <br />
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<li>NaOH for 20 min at 120 C, followed by blendering: produces cellulose that seems like it is much less ductile. Disadvantage: the functionalisation will be blended just like the cellulose, so the proteins may be broken down mechanically. <br />
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<li>NaOH for 20 min at 120 C, followed by blendering: produces cellulose that seems like it is much less ductile. Disadvantage: the functionalisation will be blended just like the cellulose, so the proteins may be broken down mechanically. <br />
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<li>Distilled water treatment over 48 hours: Produced more white cellulose than what was harvested. The distilled water turned yellow giving evidence that the surface of cellulose actually did dissolve some of the medium’s colour<br />
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<li>60 C incubation in tightly wrapped autoclave tape: New tape was applied every 3-8 hours during a 36 hour period. The pressure allowed water to escape and create a compact material of high hardness. Quite a promising result for hard cellulose.<br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2014-10-18T03:59:31Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
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<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
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<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus igem</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and a new strain G. xylinus igem, creating a genetic toolbox of consisting of five new plasmid backbones and around 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
</div><br />
<div class="pure-u-1-2"><br />
<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em>.</li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain. </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem.</li> <br />
<br />
<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em>.</li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a platform for genetic engineering of new cellulose-based biomaterials.</li><br />
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</ul><br />
</div><br />
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</div><br />
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</section><br />
<section id="introduction"><br />
<br/><br />
<figure class="content-image image-full"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
</figure><br />
<h2>Introduction</h2><br />
<br />
<br />
<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
In the middle of the summer, we isolated a new strain of Gluconacetobacter (named G. xylinus igem, which we previously called G. xylinus KI for Kombucha isolated ) from Kombucha tea and characterized its properties fully. We found it to be more amenable for genetic engineering due to higher transformation efficiencies than <em>G. xylinus</em> ATCC 53582 strain (see below), so we continued work on this strain, including sequencing its genome due to potential economic importance (it is a key component in the popular Kombucha tea). <br />
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</p><br />
<br />
</section><br />
<section id="aim"><br />
<h2>Aims</h2><br />
<br />
<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and igem strains and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
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</section><br />
<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
</figure><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £2500.<br />
<br />
In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing, and were able to sequence two genomes for less than £1000. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of <em> G.xylinus </em> resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and igem in a single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. We are currently in the process of completing the bioinformatics analyses of the genomes, and will publish the full genome sequences soon.<br />
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</p><br />
</section><br />
<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and igem strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and igem strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus igem strains to increase cellulose productivity and decrease its cost. In total, by cloning widely used parts into our <em>G. xylinus</em> shuttle vectors we created a toolkit consisting of:<br />
<ul><br />
<li> 5 new shuttle vectors </li> <br />
<li> 15 Anderson promoters </li><br />
<li> Fluorescent proteins RFP and sfGFP </li><br />
<li> 5 chromoproteins </li><br />
<li> 11 CBD fusion proteins </li> <br />
<li> Vitreoscilla hemoglobin gene </li> <br />
<li> TetR and Arabinose inducible promoters </li><br />
</ul><br />
</p><br />
<p> Because the toolkit is in the non-standard pSEVA331-Bb backbone, it can not be housed in the Parts Registry, however in order to make it accessible for the synthetic biology community, we have made it freely available on request.<br />
</p><br />
<div class="accordion"><br />
<h3>Characterization of G.xylinus ATCC 53582 and igem strains</h3><br />
<div><br />
<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
</p><br />
<p> Cellulose productivity of G.xylinus ATCC 53582 and igem on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the igem strain is approximately one fifth of that of ATCC 53582 strain (see Figure 2 for comparison of ATCC53582 and igem strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for igem cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus). <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 2. Cellulose production of G.xylinus ATCC 53582 and igem (KI) strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
</figure><br />
</p> <br />
<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure 3.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, igem strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 3 for comparison of ATCC and igem strains and details of he experiment). This could be due to the niche igem strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 3. Cellulose productivity of G.xylinum ATCC 53582 and igem (KI) strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
</figure><br />
</p> <br />
<p> Natural antibiotics resistance of ATCC 53582 and igem strains </p><br />
<p></p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 4. Natural antibiotics resistance of G.xylinus ATCC 53582 and igem (KI) strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and igem seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
</figure><br />
<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 4 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and igem strains). However, when using high cell densities for plating (after transformations), antibiotic-resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus igem is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the igem strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
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</div><br />
<h3>Plasmid backbones</h3><br />
<div><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 5. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus igem after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
</figure><br />
<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and igem strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus igem and ATCC 53583 (See Figure 5). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
</ul><br />
</div><br />
<h3>Anderson promoters</h3><br />
<div><br />
<p> We inserted 15 Anderson promoter-RFP coding devices into pSEVA331-Bb backbone, transformed them into G.xylinus igem strain in order to determine their relative strengths in Gluconacetobacter (see Figure 6 for some of them: <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/1/1a/IC14_Composite_image_correct.jpg"><br />
<figcaption>Figure 6. Different Anderson promoter-RFP constructs expressed in G.xylinus. Left- wild-type negative control. Right- G.xylinus igem transformed with pSEVA331Bb-Anderson promoter constructs. Plates grown at 30C for 7 days before imageing under blue light. </figcaption><br />
</figure><br />
</p><br />
</div><br />
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<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p><i>G. xylinus</i> is obligatively aerobic because of which cellulose production is reduced below the medium surface in static culture[1] due to reduced oxygen concentrations, reducing the overall volume of the pellicle that is biologically active. Transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i> (another obligate aerobe that expresses this protein in oxygen-poor environments) has been shown to result in increased the metabolic activity and cellulose production of G.xylinus [2].<br />
Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
<br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure 7. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem wild type cells and cells transformed with pSEVA331-BBa_K1321200 plasmid were cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days, after which OD600 was measured. Samples were diluted 1:1 in HS-cellulase medium before measurement. Negative controls (HS-cellulase without inoculations) showed no growth. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
</figure><br />
<br />
<p>We cloned the Vhb gene behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS (part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a>) in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure 7) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
<br />
<p>We also made a hypothesis of the metabolic pathway involving the interaction of vHb, which could potentially explain its effect on enhancing bacterial cellulose yield. As described in the diagram above, vHb plays a role as oxygen storage: it will actively capture oxygen molecules when when the oxygen concentration is high in the cell whereas release oxygen molecules when it is low. As such, the presence of vHb will smooth out the oxygen level within the cell and therefore provides it with a relatively constant oxygen supply. Considering the fact that oxygen also bind to protein complex cG PDE (cyclic GMP phosphodiesterase), where the binding will impose an upregulating effect on bacterial cellulose production, a constant oxygen level maintained by vHb will inevitably contribute to enhance bacterial cellulose yield.</p><br />
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<figure class="content-image image-left"><br />
<img class="image-full" src="https://lh5.googleusercontent.com/OkLBTdjMaO6PjVewhOFchlpEd3x4REDL53KYLp_sSxkD35RU4U8Gh21PJKUSfTA1GZ7ShEayn0Y=w751-h608"><br />
<figcaption>Figure 8. Effects of vHb interaction on cellular oxygen level.</figcaption><br />
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<section id="references"><br />
<h2>References</h2><br />
<ul><br />
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<li>[1] https://www.researchgate.net/ publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/Mass_Production_and_ProcessingTeam:Imperial/Mass Production and Processing2014-10-18T03:57:44Z<p>Mf1512: </p>
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<h1>Mass Production and Processing</h1><br />
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<li><a data-scroll href="#introduction">Introduction</a><br />
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<h2>Introduction</h2><br />
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<img class="image-full" src=https://static.igem.org/mediawiki/2014/7/7b/IC14-Post-prod-fig-1-blue.png ><br />
<figcaption>Figure 1: Blue dyed bacterial cellulose</figcaption><br />
</figure><br />
<br />
<p> Bacterial cellulose (BC) exhibits a <a href="https://2014.igem.org/Team:Imperial/Project_Background">multitude of different properties</a> depending on the processing, growth conditions, functionalisation and strain used (Bismarck 2013) for production of the material. Acquiring large quantities of cellulose produced would allow testing of a broad variety of cellulose processing methods and functionalisation steps. </p><br />
<br />
<p> By mass producing cellulose this enables a better understanding of what material properties can be realistically produced during the short duration of iGEM. More importantly, it improves the likelihood of finding suitable processing candidates for the project’s aim of making a customisable ultrafiltration membrane, at the same time as allowing room for creativity and exploration of the remarkable properties of cellulose. </p><br />
<br />
<h3>Minimum requirements</h3><br />
<ol><br />
<li>Treatment of BC requires killing the cells, particularly if the cells are genetically engineered, which is the aim for putting the customisable in ultrafiltration membranes.</li><br />
<li>Based on brainstorming with Central Saint Martins student Zuzana, removing the colour of BC is required as it looks displeasing to the eye otherwise, and seems counter-intuituve to filter clean water with cellulose coloured like turbid water. </li><br />
<li>Removal of the smell of BC has also been raised as a requirement, particularly by producers who work in close contact with the processing facilities.</li><br />
</ol><br />
<br />
<h3>Mass Production Methods</h3><br />
<br />
<figure class="content-image image-right image-small"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/0/04/IC14-post-prod-fig-2-pellicle-comparison.png > <br />
<figcaption>Figure 2. left: A granular pellicle, right: even pellicle</figcaption> <br />
</figure> <br />
<br />
<p>Setting up the mass production of cellulose was done according to the <a href="https://2014.igem.org/Team:Imperial/Protocols#gluconacetobacter">Kombucha media protocol</a> , which involved setting up 61 trays with media and G. xylinus and yeast co-culture as shown in figure 3. The trays were left to grow up over 7 days, after which diminishing pellicle growth was detected. Upon harvesting, the pellicles were sorted according to granular pellicles (see figure 2 left) and even pellicles (figure 2 right). All pellicles were kept in distilled water in large plastic buckets or containers. </p><br />
<br />
<p>Below shows the general workflow we employed to mass produce our cellulose and illustrates the process of manufacturing biomaterials with significantly different properties despite originating from the same BC source.<br />
</p><br />
<br />
<img class="content-image image-full" src="https://static.igem.org/mediawiki/2014/b/bf/IC14-mass_production2.png"> <br />
<figure class="content-image image-full"><br />
<br />
</section><br />
</section><br />
</div><br />
<div class="pure-u-1-1"><br />
<section id="discussion"><br />
<h2>Results</h2><br />
<br />
<p>We have made water filter grade bacterial cellulose. From different trial and error, we tried making leather cellulose that can be used a potential fabric garment. We produced bacterial cellulose on a scale larger than any previous iGEM team has produced a biomaterial in the Manufacturing track. This has provided us with opportunities to try treating cellulose with different low tech solutions that have been identified as invaluable to our collaborating Chemical Engineer Dr. Koonyang Lee. In his own words, we have produced more bacterial cellulose than he did during both his PhD and postdoc on BC, and come up with some simple solutions for practical issues. The ideas of ours that Dr. Lee has decided to carry on include using silicon coated baking paper in the protocol for measuring yield of BC to avoid BC sticking to the surface, and effectively using a non-bleach fabric stain remover to clean the BC thereby reducing the harmful impact of research in this area. </p><br />
</section><br />
<br />
<br />
</section><br />
</div><br />
<div class="pure-u-1-1"><br />
<h2>Appendices </h2><br />
<ul><br />
<li>0.1M NaOH for 60 min at 120C: Produces the most white cellulose but the process has been shown to produce some yellow/brownish cellulose if the film that covers the bottom of the pellicle was not removed before treatment.<br />
</li><br />
<li>1M NaHCO3 for 60 min at 120C: Produces quite poor results, 3 samples have been tested and even after 4 hours of treatment at 120C the samples were less white than similar samples treated in distilled water for the same duration.<br />
</li><br />
<li> Heat treatment in distilled water at 120C: 3 samples still contained some residual brownish tint after 4 hours of incubation, but the samples produced were considerably whiter than those treated with baking soda solution<br />
<br />
</li><br />
<li> Air drying without treatment first: Produces brownish cellulose with high moisture content left, material is flexible<br />
<br />
</li><br />
<li>120C Oven drying without press, without treatment for 180 min: Produces brittle paper like cellulose, it is fragile, brownish and prone to tears.<br />
<br />
</li><br />
<li>Oven drying with press (1l Duran bottle on top of two tiles) after NaOH treatment for 120 min: Produces flexible more plastic cellulose capable of being shaped into a cone that filters water through. The cellulose was capable of immersion into water, which produced wet cellulose that could be reshaped and redried. <br />
<br />
</li><br />
<br />
<li>NaOH for 20 min at 120 C, followed by blendering: produces cellulose that seems like it is much less ductile. Disadvantage: the functionalisation will be blended just like the cellulose, so the proteins may be broken down mechanically. <br />
<br />
<br />
</li><br />
<li>NaOH for 20 min at 120 C, followed by blendering: produces cellulose that seems like it is much less ductile. Disadvantage: the functionalisation will be blended just like the cellulose, so the proteins may be broken down mechanically. <br />
<br />
<br />
</li><br />
<li>Distilled water treatment over 48 hours: Produced more white cellulose than what was harvested. The distilled water turned yellow giving evidence that the surface of cellulose actually did dissolve some of the medium’s colour<br />
</li><br />
<br />
<li>60 C incubation in tightly wrapped autoclave tape: New tape was applied every 3-8 hours during a 36 hour period. The pressure allowed water to escape and create a compact material of high hardness. Quite a promising result for hard cellulose.<br />
</li><br />
<br />
<br />
</ul><br />
<br />
<br />
</div><br />
</body><br />
</html><br />
{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/Mass_Production_and_ProcessingTeam:Imperial/Mass Production and Processing2014-10-18T03:57:08Z<p>Mf1512: </p>
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<h1>Mass Production and Processing</h1><br />
<div id="navWrap"><br />
<div id="subNav"><br />
<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
</li><br />
<br />
<li><a data-scroll href="#references">References</a><br />
</li><br />
</ul><br />
<br class="clearLeft" /><br />
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</div><br />
<br />
<section id="introduction"><br />
<h2>Introduction</h2><br />
<br/> <br />
<br />
<figure class="content-image image-right"><br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/7/7b/IC14-Post-prod-fig-1-blue.png ><br />
<figcaption>Figure 1: Blue dyed bacterial cellulose</figcaption><br />
</figure><br />
<br />
<p> Bacterial cellulose (BC) exhibits a <a href="https://2014.igem.org/Team:Imperial/Project_Background">multitude of different properties</a> depending on the processing, growth conditions, functionalisation and strain used (Bismarck 2013) for production of the material. Acquiring large quantities of cellulose produced would allow testing of a broad variety of cellulose processing methods and functionalisation steps. </p><br />
<br />
<p> By mass producing cellulose this enables a better understanding of what material properties can be realistically produced during the short duration of iGEM. More importantly, it improves the likelihood of finding suitable processing candidates for the project’s aim of making a customisable ultrafiltration membrane, at the same time as allowing room for creativity and exploration of the remarkable properties of cellulose. </p><br />
<br />
<h3>Minimum requirements</h3><br />
<ol><br />
<li>Treatment of BC requires killing the cells, particularly if the cells are genetically engineered, which is the aim for putting the customisable in ultrafiltration membranes.</li><br />
<li>Based on brainstorming with Central Saint Martins student Zuzana, removing the colour of BC is required as it looks displeasing to the eye otherwise, and seems counter-intuituve to filter clean water with cellulose coloured like turbid water. </li><br />
<li>Removal of the smell of BC has also been raised as a requirement, particularly by producers who work in close contact with the processing facilities.</li><br />
</ol><br />
<br />
<h3>Mass Production Methods</h3><br />
<br />
<figure class="content-image image-right image-small"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/0/04/IC14-post-prod-fig-2-pellicle-comparison.png > <br />
<figcaption>Figure 2. left: A granular pellicle, right: even pellicle</figcaption> <br />
</figure> <br />
<br />
<p>Setting up the mass production of cellulose was done according to the <a href="https://2014.igem.org/Team:Imperial/Protocols#gluconacetobacter">Kombucha media protocol</a> , which involved setting up 61 trays with media and G. xylinus and yeast co-culture as shown in figure 3. The trays were left to grow up over 7 days, after which diminishing pellicle growth was detected. Upon harvesting, the pellicles were sorted according to granular pellicles (see figure 2 left) and even pellicles (figure 2 right). All pellicles were kept in distilled water in large plastic buckets or containers. </p><br />
<br />
<p>Below shows the general workflow we employed to mass produce our cellulose and illustrates the process of manufacturing biomaterials with significantly different properties despite originating from the same BC source.<br />
</p><br />
<br />
<img class="content-image image-full" src="https://static.igem.org/mediawiki/2014/b/bf/IC14-mass_production2.png"> <br />
<figure class="content-image image-full"><br />
<br />
</section><br />
</section><br />
</div><br />
<div class="pure-u-1-1"><br />
<section id="discussion"><br />
<h2>Results</h2><br />
<br />
<p>We have made water filter grade bacterial cellulose. From different trial and error, we tried making leather cellulose that can be used a potential fabric garment. We produced bacterial cellulose on a scale larger than any previous iGEM team has produced a biomaterial in the Manufacturing track. This has provided us with opportunities to try treating cellulose with different low tech solutions that have been identified as invaluable to our collaborating Chemical Engineer Dr. Koonyang Lee. In his own words, we have produced more bacterial cellulose than he did during both his PhD and postdoc on BC, and come up with some simple solutions for practical issues. The ideas of ours that Dr. Lee has decided to carry on include using silicon coated baking paper in the protocol for measuring yield of BC to avoid BC sticking to the surface, and effectively using a non-bleach fabric stain remover to clean the BC thereby reducing the harmful impact of research in this area. </p><br />
</section><br />
<br />
<br />
</section><br />
</div><br />
<div class="pure-u-1-1"><br />
<h2>Key Achievements </h2><br />
<ul><br />
<li>0.1M NaOH for 60 min at 120C: Produces the most white cellulose but the process has been shown to produce some yellow/brownish cellulose if the film that covers the bottom of the pellicle was not removed before treatment.<br />
</li><br />
<li>1M NaHCO3 for 60 min at 120C: Produces quite poor results, 3 samples have been tested and even after 4 hours of treatment at 120C the samples were less white than similar samples treated in distilled water for the same duration.<br />
</li><br />
<li> Heat treatment in distilled water at 120C: 3 samples still contained some residual brownish tint after 4 hours of incubation, but the samples produced were considerably whiter than those treated with baking soda solution<br />
<br />
</li><br />
<li> Air drying without treatment first: Produces brownish cellulose with high moisture content left, material is flexible<br />
<br />
</li><br />
<li>120C Oven drying without press, without treatment for 180 min: Produces brittle paper like cellulose, it is fragile, brownish and prone to tears.<br />
<br />
</li><br />
<li>Oven drying with press (1l Duran bottle on top of two tiles) after NaOH treatment for 120 min: Produces flexible more plastic cellulose capable of being shaped into a cone that filters water through. The cellulose was capable of immersion into water, which produced wet cellulose that could be reshaped and redried. <br />
<br />
</li><br />
<br />
<li>NaOH for 20 min at 120 C, followed by blendering: produces cellulose that seems like it is much less ductile. Disadvantage: the functionalisation will be blended just like the cellulose, so the proteins may be broken down mechanically. <br />
<br />
<br />
</li><br />
<li>NaOH for 20 min at 120 C, followed by blendering: produces cellulose that seems like it is much less ductile. Disadvantage: the functionalisation will be blended just like the cellulose, so the proteins may be broken down mechanically. <br />
<br />
<br />
</li><br />
<li>Distilled water treatment over 48 hours: Produced more white cellulose than what was harvested. The distilled water turned yellow giving evidence that the surface of cellulose actually did dissolve some of the medium’s colour<br />
</li><br />
<br />
<li>60 C incubation in tightly wrapped autoclave tape: New tape was applied every 3-8 hours during a 36 hour period. The pressure allowed water to escape and create a compact material of high hardness. Quite a promising result for hard cellulose.<br />
</li><br />
<br />
<br />
</ul><br />
<br />
<br />
</div><br />
</body><br />
</html><br />
{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2014-10-18T03:54:51Z<p>Mf1512: </p>
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<div class="pure-u-1-1 main"><br />
<h1><em>G. xylinus</em></h1><br />
<div id="navWrap"><br />
<div id="subNav"><br />
<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
</li><br />
<li><a data-scroll href="#aim">Aims</a><br />
</li><br />
<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
</li><br />
<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
</li><br />
<li><a data-scroll href="#references">References</a><br />
</li><br />
</ul><br />
<br class="clearLeft" /><br />
</div><br />
</div><br />
<section id="overview"><br />
<div class="pure-g"><br />
<div class="pure-u-1-2"><br />
<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus igem</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and a new strain G. xylinus igem, creating a genetic toolbox of consisting of five new plasmid backbones and around 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
</div><br />
<div class="pure-u-1-2"><br />
<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em>.</li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain. </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem.</li> <br />
<br />
<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em>.</li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a platform for genetic engineering of new cellulose-based biomaterials.</li><br />
<br />
<br />
<br />
<br />
<br />
</ul><br />
</div><br />
<br />
<br />
</div><br />
<br />
</section><br />
<section id="introduction"><br />
<br/><br />
<figure class="content-image image-full"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
</figure><br />
<h2>Introduction</h2><br />
<br />
<br />
<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
In the middle of the summer, we isolated a new strain of Gluconacetobacter (named G. xylinus igem, previously named G. xylinus KI for Kombucha isolate ) from Kombucha tea and characterized its properties fully. We found it to be more amenable for genetic engineering than <em>G. xylinus</em> ATCC 53582 strain (see below), so we continued work on this strain, including sequencing its genome due to potential economic importance (it is a key component in the popular Kombucha tea). <br />
<br />
</p><br />
<br />
</section><br />
<section id="aim"><br />
<h2>Aims</h2><br />
<br />
<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and igem strains and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
<br />
<br />
</section><br />
<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
</figure><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.<br />
<br />
In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and igem in a single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. We are currently in the process of completing the bioinformatics analyses of the genomes, and will publish the full genome sequences soon.<br />
<br />
</p><br />
</section><br />
<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and igem strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and igem strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus igem strains to increase cellulose productivity and decrease its cost. In total, by cloning widely used parts into our <em>G. xylinus</em> shuttle vectors we created a toolkit consisting of:<br />
<ul><br />
<li> 5 new shuttle vectors </li> <br />
<li> 15 Anderson promoters </li><br />
<li> Fluorescent proteins RFP and sfGFP </li><br />
<li> 5 chromoproteins </li><br />
<li> 11 CBD fusion proteins </li> <br />
<li> Vitreoscilla hemoglobin gene </li> <br />
<li> TetR and Arabinose inducible promoters </li><br />
</ul><br />
</p><br />
<p> Because the toolkit is in the non-standard pSEVA331-Bb backbone, it can not be housed in the Parts Registry, however in order to make it accessible for the synthetic biology community, we have made it freely available on request.<br />
</p><br />
<div class="accordion"><br />
<h3>Characterization of G.xylinus ATCC 53582 and igem strains</h3><br />
<div><br />
<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
</p><br />
<p> Cellulose productivity of G.xylinus ATCC 53582 and igem on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the igem strain is approximately one fifth of that of ATCC 53582 strain (see Figure 2 for comparison of ATCC53582 and igem strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for igem cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus). <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 2. Cellulose production of G.xylinus ATCC 53582 and igem (KI) strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
</figure><br />
</p> <br />
<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure 3.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, igem strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 3 for comparison of ATCC and igem strains and details of he experiment). This could be due to the niche igem strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 3. Cellulose productivity of G.xylinum ATCC 53582 and igem (KI) strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
</figure><br />
</p> <br />
<p> Natural antibiotics resistance of ATCC 53582 and igem strains </p><br />
<p></p><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 4. Natural antibiotics resistance of G.xylinus ATCC 53582 and igem (KI) strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and igem seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
</figure><br />
<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 4 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and igem strains). However, when using high cell densities for plating (after transformations), antibiotic-resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus igem is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the igem strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
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</div><br />
<h3>Plasmid backbones</h3><br />
<div><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 5. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus igem after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
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<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and igem strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus igem and ATCC 53583 (See Figure 5). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
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<h3>Anderson promoters</h3><br />
<div><br />
<p> We inserted 15 Anderson promoter-RFP coding devices into pSEVA331-Bb backbone, transformed them into G.xylinus igem strain in order to determine their relative strengths in Gluconacetobacter (see Figure 6 for some of them: <br />
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<figcaption>Figure 6. Different Anderson promoter-RFP constructs expressed in G.xylinus. Left- wild-type negative control. Right- G.xylinus igem transformed with pSEVA331Bb-Anderson promoter constructs. Plates grown at 30C for 7 days before imageing under blue light. </figcaption><br />
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<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p><i>G. xylinus</i> is obligatively aerobic because of which cellulose production is reduced below the medium surface in static culture[1] due to reduced oxygen concentrations, reducing the overall volume of the pellicle that is biologically active. Transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i> (another obligate aerobe that expresses this protein in oxygen-poor environments) has been shown to result in increased the metabolic activity and cellulose production of G.xylinus [2].<br />
Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure 7. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem wild type cells and cells transformed with pSEVA331-BBa_K1321200 plasmid were cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days, after which OD600 was measured. Samples were diluted 1:1 in HS-cellulase medium before measurement. Negative controls (HS-cellulase without inoculations) showed no growth. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
</figure><br />
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<p>We cloned the Vhb gene behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS (part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a>) in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure 7) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
<br />
<p>We also made a hypothesis of the metabolic pathway involving the interaction of vHb, which could potentially explain its effect on enhancing bacterial cellulose yield. As described in the diagram above, vHb plays a role as oxygen storage: it will actively capture oxygen molecules when when the oxygen concentration is high in the cell whereas release oxygen molecules when it is low. As such, the presence of vHb will smooth out the oxygen level within the cell and therefore provides it with a relatively constant oxygen supply. Considering the fact that oxygen also bind to protein complex cG PDE (cyclic GMP phosphodiesterase), where the binding will impose an upregulating effect on bacterial cellulose production, a constant oxygen level maintained by vHb will inevitably contribute to enhance bacterial cellulose yield.</p><br />
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<img class="image-full" src="https://lh5.googleusercontent.com/OkLBTdjMaO6PjVewhOFchlpEd3x4REDL53KYLp_sSxkD35RU4U8Gh21PJKUSfTA1GZ7ShEayn0Y=w751-h608"><br />
<figcaption>Figure 8. Effects of vHb interaction on cellular oxygen level.</figcaption><br />
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<h2>References</h2><br />
<ul><br />
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<li>[1] https://www.researchgate.net/ publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/Mass_Production_and_ProcessingTeam:Imperial/Mass Production and Processing2014-10-18T03:51:19Z<p>Mf1512: </p>
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<h1>Mass Production and Processing</h1><br />
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<li><a data-scroll href="#introduction">Introduction</a><br />
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<h2>Introduction</h2><br />
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<p> Bacterial cellulose (BC) exhibits a <a href="https://2014.igem.org/Team:Imperial/Project_Background">multitude of different properties</a> depending on the processing, growth conditions, functionalisation and strain used (Bismarck 2013) for production of the material. Acquiring large quantities of cellulose produced would allow testing of a broad variety of cellulose processing methods and functionalisation steps. </p><br />
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<p> By mass producing cellulose this enables a better understanding of what material properties can be realistically produced during the short duration of iGEM. More importantly, it improves the likelihood of finding suitable processing candidates for the project’s aim of making a customisable ultrafiltration membrane, at the same time as allowing room for creativity and exploration of the remarkable properties of cellulose. </p><br />
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<h3>Minimum requirements</h3><br />
<ol><br />
<li>Treatment of BC requires killing the cells, particularly if the cells are genetically engineered, which is the aim for putting the customisable in ultrafiltration membranes.</li><br />
<li>Based on brainstorming with Central Saint Martins student Zuzana, removing the colour of BC is required as it looks displeasing to the eye otherwise, and seems counter-intuituve to filter clean water with cellulose coloured like turbid water. </li><br />
<li>Removal of the smell of BC has also been raised as a requirement, particularly by producers who work in close contact with the processing facilities.</li><br />
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<h3>Mass Production Methods</h3><br />
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<img class="image-full" src=https://static.igem.org/mediawiki/2014/0/04/IC14-post-prod-fig-2-pellicle-comparison.png > <br />
<figcaption>Figure 2. left: A granular pellicle, right: even pellicle</figcaption> <br />
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<p>Setting up the mass production of cellulose was done according to the <a href="https://2014.igem.org/Team:Imperial/Protocols#gluconacetobacter">Kombucha media protocol</a> , which involved setting up 61 trays with media and G. xylinus and yeast co-culture as shown in figure 3. The trays were left to grow up over 7 days, after which diminishing pellicle growth was detected. Upon harvesting, the pellicles were sorted according to granular pellicles (see figure 2 left) and even pellicles (figure 2 right). All pellicles were kept in distilled water in large plastic buckets or containers. </p><br />
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<p>Below shows the general workflow we employed to mass produce our cellulose and illustrates the process of manufacturing biomaterials with significantly different properties despite originating from the same BC source.<br />
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<h2>Results</h2><br />
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<p>We have made water filter grade bacterial cellulose. From different trial and error, we tried making leather cellulose that can be used a potential fabric garment. We produced bacterial cellulose on a scale larger than any previous iGEM team has produced a biomaterial in the Manufacturing track. This has provided us with opportunities to try treating cellulose with different low tech solutions that have been identified as invaluable to our collaborating Chemical Engineer Dr. Koonyang Lee. In his own words, we have produced more bacterial cellulose than he did during both his PhD and postdoc on BC, and come up with some simple solutions for practical issues. The ideas of ours that Dr. Lee has decided to carry on include using silicon coated baking paper in the protocol for measuring yield of BC to avoid BC sticking to the surface, and effectively using a non-bleach fabric stain remover to clean the BC thereby reducing the harmful impact of research in this area. </p><br />
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<h2>Key Achievements </h2><br />
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<li>Introduced a new aspect to the manufacturing track in terms of quantitatively characterising the mechanical properties of our product, bacterial cellulose<br />
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<li>Quantified the tensile stress-strain properties of our bacterial cellulose.<br />
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<li>Discovered that our bacterial cellulose can sustain an order of magnitude higher water pressures than those typically used for ultrafiltration membranes.<br />
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<li>Established that non-blended compared to blended bacterial cellulose was significantly more ductile, and would have longer life time as a water filter, which informed the choice of cellulose for the final product.<br />
</li><br />
<li>Tested and analysed 20 samples of bacterial cellulose test pieces.<br />
</li><br />
<li>Created a size and quality optimised gif for visualisation of the experiment and written a protocol for future iGEM teams to do the same.<br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2014-10-18T03:49:39Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
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<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
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<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus igem</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and igem, creating a genetic toolbox of consisting of five new plasmid backbones and around 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
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<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em></li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem</li> <br />
<br />
<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em></li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a powerful platform for the synthesis of new cellulose-based biomaterials and water filters</li><br />
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<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
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<h2>Introduction</h2><br />
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<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
In the middle of the summer, we isolated a new strain of Gluconacetobacter (named G. xylinus igem, previously named G. xylinus KI for Kombucha isolate ) from Kombucha tea and characterized its properties fully. We found it to be more amenable for genetic engineering than <em>G. xylinus</em> ATCC 53582 strain (see below), so we continued work on this strain, including sequencing its genome due to potential economic importance (it is a key component in the popular Kombucha tea). <br />
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<h2>Aims</h2><br />
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<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and igem strains and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
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<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
</figure><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.<br />
<br />
In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and igem in a single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. We are currently in the process of completing the bioinformatics analyses of the genomes, and will publish the full genome sequences soon.<br />
<br />
</p><br />
</section><br />
<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and igem strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and igem strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus igem strains to increase cellulose productivity and decrease its cost. In total, by cloning widely used parts into our <em>G. xylinus</em> shuttle vectors we created a toolkit consisting of:<br />
<ul><br />
<li> 5 new shuttle vectors </li> <br />
<li> 15 Anderson promoters </li><br />
<li> Fluorescent proteins RFP and sfGFP </li><br />
<li> 5 chromoproteins </li><br />
<li> 11 CBD fusion proteins </li> <br />
<li> Vitreoscilla hemoglobin gene </li> <br />
<li> TetR and Arabinose inducible promoters </li><br />
</ul><br />
</p><br />
<p> Because the toolkit is in the non-standard pSEVA331-Bb backbone, it can not be housed in the Parts Registry, however in order to make it accessible for the synthetic biology community, we have made it freely available on request.<br />
</p><br />
<div class="accordion"><br />
<h3>Characterization of G.xylinus ATCC 53582 and igem strains</h3><br />
<div><br />
<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
</p><br />
<p> Cellulose productivity of G.xylinus ATCC 53582 and igem on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the igem strain is approximately one fifth of that of ATCC 53582 strain (see Figure 2 for comparison of ATCC53582 and igem strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for igem cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus). <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 2. Cellulose production of G.xylinus ATCC 53582 and igem (KI) strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
</figure><br />
</p> <br />
<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure 3.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, igem strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 3 for comparison of ATCC and igem strains and details of he experiment). This could be due to the niche igem strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 3. Cellulose productivity of G.xylinum ATCC 53582 and igem (KI) strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
</figure><br />
</p> <br />
<p> Natural antibiotics resistance of ATCC 53582 and igem strains </p><br />
<p></p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 4. Natural antibiotics resistance of G.xylinus ATCC 53582 and igem (KI) strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and igem seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
</figure><br />
<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 4 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and igem strains). However, when using high cell densities for plating (after transformations), antibiotic-resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus igem is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the igem strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
<br />
<br />
</div><br />
<h3>Plasmid backbones</h3><br />
<div><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 5. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus igem after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
</figure><br />
<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and igem strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus igem and ATCC 53583 (See Figure 5). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
</ul><br />
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<h3>Anderson promoters</h3><br />
<div><br />
<p> We inserted 15 Anderson promoter-RFP coding devices into pSEVA331-Bb backbone, transformed them into G.xylinus igem strain in order to determine their relative strengths in Gluconacetobacter (see Figure 6 for some of them: <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/1/1a/IC14_Composite_image_correct.jpg"><br />
<figcaption>Figure 6. Different Anderson promoter-RFP constructs expressed in G.xylinus. Left- wild-type negative control. Right- G.xylinus igem transformed with pSEVA331Bb-Anderson promoter constructs. Plates grown at 30C for 7 days before imageing under blue light. </figcaption><br />
</figure><br />
</p><br />
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<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p><i>G. xylinus</i> is obligatively aerobic because of which cellulose production is reduced below the medium surface in static culture[1] due to reduced oxygen concentrations, reducing the overall volume of the pellicle that is biologically active. Transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i> (another obligate aerobe that expresses this protein in oxygen-poor environments) has been shown to result in increased the metabolic activity and cellulose production of G.xylinus [2].<br />
Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
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<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure 7. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem wild type cells and cells transformed with pSEVA331-BBa_K1321200 plasmid were cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days, after which OD600 was measured. Samples were diluted 1:1 in HS-cellulase medium before measurement. Negative controls (HS-cellulase without inoculations) showed no growth. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
</figure><br />
<br />
<p>We cloned the Vhb gene behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS (part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a>) in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure 7) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
<br />
<p>We also made a hypothesis of the metabolic pathway involving the interaction of vHb, which could potentially explain its effect on enhancing bacterial cellulose yield. As described in the diagram above, vHb plays a role as oxygen storage: it will actively capture oxygen molecules when when the oxygen concentration is high in the cell whereas release oxygen molecules when it is low. As such, the presence of vHb will smooth out the oxygen level within the cell and therefore provides it with a relatively constant oxygen supply. Considering the fact that oxygen also bind to protein complex cG PDE (cyclic GMP phosphodiesterase), where the binding will impose an upregulating effect on bacterial cellulose production, a constant oxygen level maintained by vHb will inevitably contribute to enhance bacterial cellulose yield.</p><br />
<br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://lh5.googleusercontent.com/OkLBTdjMaO6PjVewhOFchlpEd3x4REDL53KYLp_sSxkD35RU4U8Gh21PJKUSfTA1GZ7ShEayn0Y=w751-h608"><br />
<figcaption>Figure Z. Effects of vHb interaction on cellular oxygen level.</figcaption><br />
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<h2>References</h2><br />
<ul><br />
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<li>[1] https://www.researchgate.net/ publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/Mass_Production_and_ProcessingTeam:Imperial/Mass Production and Processing2014-10-18T03:48:14Z<p>Mf1512: </p>
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<p> Bacterial cellulose (BC) exhibits a <a href="https://2014.igem.org/Team:Imperial/Project_Background">multitude of different properties</a> depending on the processing, growth conditions, functionalisation and strain used (Bismarck 2013) for production of the material. Acquiring large quantities of cellulose produced would allow testing of a broad variety of cellulose processing methods and functionalisation steps. </p><br />
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<p> By mass producing cellulose this enables a better understanding of what material properties can be realistically produced during the short duration of iGEM. More importantly, it improves the likelihood of finding suitable processing candidates for the project’s aim of making a customisable ultrafiltration membrane, at the same time as allowing room for creativity and exploration of the remarkable properties of cellulose. </p><br />
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<h3>Minimum requirements</h3><br />
<ol><br />
<li>Treatment of BC requires killing the cells, particularly if the cells are genetically engineered, which is the aim for putting the customisable in ultrafiltration membranes.</li><br />
<li>Based on brainstorming with Central Saint Martins student Zuzana, removing the colour of BC is required as it looks displeasing to the eye otherwise, and seems counter-intuituve to filter clean water with cellulose coloured like turbid water. </li><br />
<li>Removal of the smell of BC has also been raised as a requirement, particularly by producers who work in close contact with the processing facilities.</li><br />
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<h3>Mass Production Methods</h3><br />
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<img class="image-full" src=https://static.igem.org/mediawiki/2014/0/04/IC14-post-prod-fig-2-pellicle-comparison.png > <br />
<figcaption>Figure 2. left: A granular pellicle, right: even pellicle</figcaption> <br />
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<p>Setting up the mass production of cellulose was done according to the <a href="https://2014.igem.org/Team:Imperial/Protocols#gluconacetobacter">Kombucha media protocol</a> , which involved setting up 61 trays with media and G. xylinus and yeast co-culture as shown in figure 3. The trays were left to grow up over 7 days, after which diminishing pellicle growth was detected. Upon harvesting, the pellicles were sorted according to granular pellicles (see figure 2 left) and even pellicles (figure 2 right). All pellicles were kept in distilled water in large plastic buckets or containers. </p><br />
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<p>Below shows the general workflow we employed to mass produce our cellulose and illustrates the process of manufacturing biomaterials with significantly different properties despite originating from the same BC source.<br />
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<p>We have made water filter grade bacterial cellulose. From different trial and error, we tried making leather cellulose that can be used a potential fabric garment. We produced bacterial cellulose on a scale larger than any previous iGEM team has produced a biomaterial in the Manufacturing track. This has provided us with opportunities to try treating cellulose with different low tech solutions that have been identified as invaluable to our collaborating Chemical Engineer Dr. Koonyang Lee. In his own words, we have produced more bacterial cellulose than he did during both his PhD and postdoc on BC, and come up with some simple solutions for practical issues. The ideas of ours that Dr. Lee has decided to carry on include using silicon coated baking paper in the protocol for measuring yield of BC to avoid BC sticking to the surface, and effectively using a non-bleach fabric stain remover to clean the BC thereby reducing the harmful impact of research in this area. </p><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2014-10-18T03:48:04Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
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<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
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<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
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<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus igem</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and igem, creating a genetic toolbox of consisting of five new plasmid backbones and around 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
</div><br />
<div class="pure-u-1-2"><br />
<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em></li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem</li> <br />
<br />
<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em></li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a powerful platform for the synthesis of new cellulose-based biomaterials and water filters</li><br />
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</ul><br />
</div><br />
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</div><br />
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</section><br />
<section id="introduction"><br />
<br/><br />
<figure class="content-image image-full"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
</figure><br />
<h2>Introduction</h2><br />
<br />
<br />
<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
In the middle of the summer, we isolated a new strain of Gluconacetobacter (named G. xylinus igem, previously named G. xylinus KI for Kombucha isolate ) from Kombucha tea and characterized its properties fully. We found it to be more amenable for genetic engineering than <em>G. xylinus</em> ATCC 53582 strain (see below), so we continued work on this strain, including sequencing its genome due to potential economic importance (it is a key component in the popular Kombucha tea). <br />
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</p><br />
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</section><br />
<section id="aim"><br />
<h2>Aims</h2><br />
<br />
<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and igem strains and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
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</section><br />
<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
</figure><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.<br />
<br />
In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and igem in a single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. We are currently in the process of completing the bioinformatics analyses of the genomes, and will publish the full genome sequences soon.<br />
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</p><br />
</section><br />
<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and igem strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and igem strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus igem strains to increase cellulose productivity and decrease its cost. In total, by cloning widely used parts into our <em>G. xylinus</em> shuttle vectors we created a toolkit consisting of:<br />
<ul><br />
<li> 5 new shuttle vectors </li> <br />
<li> 15 Anderson promoters </li><br />
<li> Fluorescent proteins RFP and sfGFP </li><br />
<li> 5 chromoproteins </li><br />
<li> 11 CBD fusion proteins </li> <br />
<li> Vitreoscilla hemoglobin gene </li> <br />
<li> TetR and Arabinose inducible promoters </li><br />
</ul><br />
</p><br />
<p> Because the toolkit is in the non-standard pSEVA331-Bb backbone, it can not be housed in the Parts Registry, however in order to make it accessible for the synthetic biology community, we have made it freely available on request.<br />
</p><br />
<div class="accordion"><br />
<h3>Characterization of G.xylinus ATCC 53582 and igem strains</h3><br />
<div><br />
<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
</p><br />
<p> Cellulose productivity of G.xylinus ATCC 53582 and igem on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the igem strain is approximately one fifth of that of ATCC 53582 strain (see Figure 2 for comparison of ATCC53582 and igem strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for igem cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus). <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 2. Cellulose production of G.xylinus ATCC 53582 and igem (KI) strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
</figure><br />
</p> <br />
<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure 3.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, igem strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 3 for comparison of ATCC and igem strains and details of he experiment). This could be due to the niche igem strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 3. Cellulose productivity of G.xylinum ATCC 53582 and igem (KI) strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
</figure><br />
</p> <br />
<p> Natural antibiotics resistance of ATCC 53582 and igem strains </p><br />
<p></p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 4. Natural antibiotics resistance of G.xylinus ATCC 53582 and igem (KI) strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and igem seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
</figure><br />
<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 4 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and igem strains). However, when using high cell densities for plating (after transformations), antibiotic-resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus igem is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the igem strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
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</div><br />
<h3>Plasmid backbones</h3><br />
<div><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 5. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus igem after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
</figure><br />
<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and igem strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus igem and ATCC 53583 (See Figure 5). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
</ul><br />
</div><br />
<h3>Anderson promoters</h3><br />
<div><br />
<p> We inserted 15 Anderson promoter-RFP coding devices into pSEVA331-Bb backbone, transformed them into G.xylinus igem strain in order to determine their relative strengths in Gluconacetobacter (see Figure 6 for some of them: <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/1/1a/IC14_Composite_image_correct.jpg"><br />
<figcaption>Figure 6. Different Anderson promoter-RFP constructs expressed in G.xylinus. Left- wild-type negative control. Right- G.xylinus igem transformed with pSEVA331Bb-Anderson promoter constructs. Plates grown at 30C for 7 days before imageing under blue light. </figcaption><br />
</figure><br />
</p><br />
</div><br />
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<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p><i>G. xylinus</i> is obligatively aerobic because of which cellulose production is reduced below the medium surface in static culture[1] due to reduced oxygen concentrations, reducing the overall volume of the pellicle that is biologically active. Transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i> (another obligate aerobe that expresses this protein in oxygen-poor environments) has been shown to result in increased the metabolic activity and cellulose production of G.xylinus [2].<br />
Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
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<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure 7. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem wild type cells and cells transformed with pSEVA331-BBa_K1321200 plasmid were cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days, after which OD600 was measured. Samples were diluted 1:1 in HS-cellulase medium before measurement. Negative controls (HS-cellulase without inoculations) showed no growth. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
</figure><br />
<br />
<p>We cloned the Vhb gene behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS (part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a>) in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure 7) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
<br />
<p>We also made a hypothesis of the metabolic pathway involving the interaction of vHb, which could potentially explain its effect on enhancing bacterial cellulose yield. As described in the diagram above, vHb plays a role as oxygen storage: it will actively capture oxygen molecules when when the oxygen concentration is high in the cell whereas release oxygen molecules when it is low. As such, the presence of vHb will smooth out the oxygen level within the cell and therefore provides it with a relatively constant oxygen supply. Considering the fact that oxygen also bind to protein complex cG PDE (cyclic GMP phosphodiesterase), where the binding will impose an upregulating effect on bacterial cellulose production, a constant oxygen level maintained by vHb will inevitably contribute to enhance bacterial cellulose yield.</p><br />
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<figure class="content-image image-left"><br />
<img class="image-full" src="https://lh5.googleusercontent.com/OkLBTdjMaO6PjVewhOFchlpEd3x4REDL53KYLp_sSxkD35RU4U8Gh21PJKUSfTA1GZ7ShEayn0Y=w751-h608"><br />
<figcaption>Figure Z. Effects of vHb interaction on cellular oxygen level.</figcaption><br />
</figure> <br />
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id="references"><br />
<h2>References</h2><br />
<ul><br />
<br />
<li>[1] https://www.researchgate.net/ publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2014-10-18T03:46:37Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
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<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
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<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
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<li><a data-scroll href="#results">Results</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
<div class="pure-g"><br />
<div class="pure-u-1-2"><br />
<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus igem</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and igem, creating a genetic toolbox of consisting of five new plasmid backbones and around 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
</div><br />
<div class="pure-u-1-2"><br />
<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em></li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem</li> <br />
<br />
<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em></li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a powerful platform for the synthesis of new cellulose-based biomaterials and water filters</li><br />
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</ul><br />
</div><br />
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</div><br />
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</section><br />
<section id="introduction"><br />
<br/><br />
<figure class="content-image image-full"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
</figure><br />
<h2>Introduction</h2><br />
<br />
<br />
<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
In the middle of the summer, we isolated a new strain of Gluconacetobacter (named G. xylinus igem, previously named G. xylinus KI for Kombucha isolate ) from Kombucha tea and characterized its properties fully. We found it to be more amenable for genetic engineering than <em>G. xylinus</em> ATCC 53582 strain (see below), so we continued work on this strain, including sequencing its genome due to potential economic importance (it is a key component in the popular Kombucha tea). <br />
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</p><br />
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</section><br />
<section id="aim"><br />
<h2>Aims</h2><br />
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<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and igem strains and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
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</section><br />
<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
</figure><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.<br />
<br />
In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and igem in a single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. We are currently in the process of completing the bioinformatics analyses of the genomes, and will publish the full genome sequences soon.<br />
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</p><br />
</section><br />
<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and igem strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and igem strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus igem strains to increase cellulose productivity and decrease its cost. In total, by cloning widely used parts into our <em>G. xylinus</em> shuttle vectors we created a toolkit consisting of:<br />
<ul><br />
<li> 5 new shuttle vectors </li> <br />
<li> 15 Anderson promoters </li><br />
<li> Fluorescent proteins RFP and sfGFP </li><br />
<li> 5 chromoproteins </li><br />
<li> 11 CBD fusion proteins </li> <br />
<li> Vitreoscilla hemoglobin gene </li> <br />
<li> TetR and Arabinose inducible promoters </li><br />
</ul><br />
</p><br />
<p> Because the toolkit is in the non-standard pSEVA331-Bb backbone, it can not be housed in the Parts Registry, however in order to make it accessible for the synthetic biology community, we have made it freely available on request.<br />
</p><br />
<div class="accordion"><br />
<h3>Characterization of G.xylinus ATCC 53582 and igem strains</h3><br />
<div><br />
<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
</p><br />
<p> Cellulose productivity of G.xylinus ATCC 53582 and igem on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the igem strain is approximately one fifth of that of ATCC 53582 strain (see Figure 2 for comparison of ATCC53582 and igem strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for igem cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus). <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 2. Cellulose production of G.xylinus ATCC 53582 and igem (KI) strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
</figure><br />
</p> <br />
<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure 3.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, igem strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 3 for comparison of ATCC and igem strains and details of he experiment). This could be due to the niche igem strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 3. Cellulose productivity of G.xylinum ATCC 53582 and igem (KI) strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
</figure><br />
</p> <br />
<p> Natural antibiotics resistance of ATCC 53582 and igem strains </p><br />
<p></p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 4. Natural antibiotics resistance of G.xylinus ATCC 53582 and igem (KI) strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and igem seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
</figure><br />
<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 4 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and igem strains). However, when using high cell densities for plating (after transformations), antibiotic-resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus igem is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the igem strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
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</div><br />
<h3>Plasmid backbones</h3><br />
<div><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 5. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus igem after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
</figure><br />
<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and igem strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus igem and ATCC 53583 (See Figure 5). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
</ul><br />
</div><br />
<h3>Anderson promoters</h3><br />
<div><br />
<p> We inserted 15 Anderson promoter-RFP coding devices into pSEVA331-Bb backbone, transformed them into G.xylinus igem strain in order to determine their relative strengths in Gluconacetobacter (see Figure 6 for some of them: <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/1/1a/IC14_Composite_image_correct.jpg"><br />
<figcaption>Figure 6. Different Anderson promoter-RFP constructs expressed in G.xylinus. Left- wild-type negative control. Right- G.xylinus igem transformed with pSEVA331Bb-Anderson promoter constructs. Plates grown at 30C for 7 days before imageing under blue light. </figcaption><br />
</figure><br />
</p><br />
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<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p><i>G. xylinus</i> is obligatively aerobic because of which cellulose production is reduced below the medium surface in static culture[1] due to reduced oxygen concentrations, reducing the overall volume of the pellicle that is biologically active. Transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i> (another obligate aerobe that expresses this protein in oxygen-poor environments) has been shown to result in increased the metabolic activity and cellulose production of G.xylinus [2].<br />
Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
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<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure 7. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem wild type cells and cells transformed with pSEVA331-BBa_K1321200 plasmid were cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days, after which OD600 was measured. Samples were diluted 1:1 in HS-cellulase medium before measurement. Negative controls (HS-cellulase without inoculations) showed no growth. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
</figure><br />
<br />
<p>We cloned the Vhb gene behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS (part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a>) in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure 7) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
<br />
<p>We also made a hypothesis of the metabolic pathway involving the interaction of vHb, which could potentially explain its effect on enhancing bacterial cellulose yield. As described in the diagram above, vHb plays a role as oxygen storage: it will actively capture oxygen molecules when when the oxygen concentration is high in the cell whereas release oxygen molecules when it is low. As such, the presence of vHb will smooth out the oxygen level within the cell and therefore provides it with a relatively constant oxygen supply. Considering the fact that oxygen also bind to protein complex cG PDE (cyclic GMP phosphodiesterase), where the binding will impose an upregulating effect on bacterial cellulose production, a constant oxygen level maintained by vHb will inevitably contribute to enhance bacterial cellulose yield.</p><br />
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<figure class="content-image image-left"><br />
<img class="image-full" src="https://lh5.googleusercontent.com/OkLBTdjMaO6PjVewhOFchlpEd3x4REDL53KYLp_sSxkD35RU4U8Gh21PJKUSfTA1GZ7ShEayn0Y=w751-h608"><br />
<figcaption>Figure Z. Effects of vHb interaction on cellular oxygen level.</figcaption><br />
</figure> <br />
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</div><br />
<section id="results"><br />
<h2>Results</h2><br />
<p>We have isolated a new strain of Acetobacter from kombucha, and determined its growth rate, cellulose productivity, and natural antibiotic resistance. We have also developed a set of methods for genetic engineering of this strain and have determined five new plasmids capable of replication in this strain. Thus we are the first to genetically engineer it.</p><br />
<p>We have discovered four new plasmids capable of replication in Acetobacter species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K - both in G.xylinus ATCC 53582 strain, and in the strain we isolated from Kombucha. We have biobricked these plasmids, with the exception of pBAV1K, which had been originally engineered to be biobrick compatible (however, pBAV1K can not be uploaded to parts registry due to binding materials-transfer agreements).<br />
</p><br />
</section><br />
<br />
<section id="references"><br />
<h2>References</h2><br />
<ul><br />
<br />
<li>[1] https://www.researchgate.net/ publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2014-10-18T03:45:52Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
</li><br />
<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
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<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
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<li><a data-scroll href="#engineering">Engineering</a><br />
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<li><a data-scroll href="#results">Results</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
<div class="pure-g"><br />
<div class="pure-u-1-2"><br />
<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus igem</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and igem, creating a genetic toolbox of consisting of five new plasmid backbones and around 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
</div><br />
<div class="pure-u-1-2"><br />
<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em></li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem</li> <br />
<br />
<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em></li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a powerful platform for the synthesis of new cellulose-based biomaterials and water filters</li><br />
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</section><br />
<section id="introduction"><br />
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<figure class="content-image image-full"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
</figure><br />
<h2>Introduction</h2><br />
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<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
In the middle of the summer, we isolated a new strain of Gluconacetobacter (named G. xylinus igem, previously named G. xylinus KI for Kombucha isolate ) from Kombucha tea and characterized its properties fully. We found it to be more amenable for genetic engineering than <em>G. xylinus</em> ATCC 53582 strain (see below), so we continued work on this strain, including sequencing its genome due to potential economic importance (it is a key component in the popular Kombucha tea). <br />
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</p><br />
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</section><br />
<section id="aim"><br />
<h2>Aims</h2><br />
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<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and igem strains and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
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</section><br />
<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
</figure><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.<br />
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In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and igem in a single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. We are currently in the process of completing the bioinformatics analyses of the genomes, and will publish the full genome sequences soon.<br />
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</p><br />
</section><br />
<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and igem strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and igem strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus igem strains to increase cellulose productivity and decrease its cost. In total, by cloning widely used parts into our <em>G. xylinus</em> shuttle vectors we created a toolkit consisting of:<br />
<ul><br />
<li> 5 new shuttle vectors </li> <br />
<li> 15 Anderson promoters </li><br />
<li> Fluorescent proteins RFP and sfGFP </li><br />
<li> 5 chromoproteins </li><br />
<li> 11 CBD fusion proteins </li> <br />
<li> Vitreoscilla hemoglobin gene </li> <br />
<li> TetR and Arabinose inducible promoters </li><br />
</ul><br />
</p><br />
<p> Because the toolkit is in the non-standard pSEVA331-Bb backbone, it can not be housed in the Parts Registry, however in order to make it accessible for the synthetic biology community, we have made it freely available on request.<br />
</p><br />
<div class="accordion"><br />
<h3>Characterization of G.xylinus ATCC 53582 and igem strains</h3><br />
<div><br />
<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
</p><br />
<p> Cellulose productivity of G.xylinus ATCC 53582 and igem on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the igem strain is approximately one fifth of that of ATCC 53582 strain (see Figure 2 for comparison of ATCC53582 and igem strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for igem cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus). <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 2. Cellulose production of G.xylinus ATCC 53582 and igem (KI) strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
</figure><br />
</p> <br />
<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure 3.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, igem strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 3 for comparison of ATCC and igem strains and details of he experiment). This could be due to the niche igem strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 3. Cellulose productivity of G.xylinum ATCC 53582 and igem (KI) strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
</figure><br />
</p> <br />
<p> Natural antibiotics resistance of ATCC 53582 and igem strains </p><br />
<p></p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 4. Natural antibiotics resistance of G.xylinus ATCC 53582 and igem (KI) strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and igem seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
</figure><br />
<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 4 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and igem strains). However, when using high cell densities for plating (after transformations), antibiotic-resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus igem is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the igem strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
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</div><br />
<h3>Plasmid backbones</h3><br />
<div><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 5. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus igem after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
</figure><br />
<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and igem strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus igem and ATCC 53583 (See Figure 5). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
</ul><br />
</div><br />
<h3>Anderson promoters</h3><br />
<div><br />
<p> We inserted 15 Anderson promoter-RFP coding devices into pSEVA331-Bb backbone, transformed them into G.xylinus igem strain in order to determine their relative strengths in Gluconacetobacter (see Figure 6 for some of them: <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/1/1a/IC14_Composite_image_correct.jpg"><br />
<figcaption>Figure 6. Different Anderson promoter-RFP constructs expressed in G.xylinus. Left- wild-type negative control. Right- G.xylinus igem transformed with pSEVA331Bb-Anderson promoter constructs. Plates grown at 30C for 7 days before imageing under blue light. </figcaption><br />
</figure><br />
</p><br />
</div><br />
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<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p><i>G. xylinus</i> is obligatively aerobic because of which cellulose production is reduced below the medium surface in static culture[1] due to reduced oxygen concentrations, reducing the overall volume of the pellicle that is biologically active. Transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i> (another obligate aerobe that expresses this protein in oxygen-poor environments) has been shown to result in increased the metabolic activity and cellulose production of G.xylinus [2].<br />
Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
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<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure 7. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem wild type cells and cells transformed with pSEVA331-BBa_K1321200 plasmid were cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days, after which OD600 was measured. Samples were diluted 1:1 in HS-cellulase medium before measurement. Negative controls (HS-cellulase without inoculations) showed no growth. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
</figure><br />
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<p>We cloned the Vhb gene behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS (part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a>) in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure 7) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
<br />
<p>We also made a hypothesis of the metabolic pathway involving the interaction of vHb, which could potentially explain its effect on enhancing bacterial cellulose yield. As described in the diagram above, vHb plays a role as oxygen storage: it will actively capture oxygen molecules when when the oxygen concentration is high in the cell whereas release oxygen molecules when it is low. As such, the presence of vHb will smooth out the oxygen level within the cell and therefore provides it with a relatively constant oxygen supply. Considering the fact that oxygen also bind to protein complex cG PDE (cyclic GMP phosphodiesterase), where the binding will impose an upregulating effect on bacterial cellulose production, a constant oxygen level maintained by vHb will inevitably contribute to enhance bacterial cellulose yield.</p><br />
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<figure class="content-image image-left"><br />
<img class="image-full" src="https://lh5.googleusercontent.com/OkLBTdjMaO6PjVewhOFchlpEd3x4REDL53KYLp_sSxkD35RU4U8Gh21PJKUSfTA1GZ7ShEayn0Y=w751-h608"><br />
<figcaption>Figure Z. Effects of vHb interaction on cellular oxygen level.</figcaption><br />
</figure> <br />
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</div><br />
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</div><br />
<section id="results"><br />
<h2>Results</h2><br />
<p>We have isolated a new strain of Acetobacter from kombucha, and determined its growth rate, cellulose productivity, and natural antibiotic resistance. We have also developed a set of methods for genetic engineering of this strain and have determined five new plasmids capable of replication in this strain. Thus we are the first to genetically engineer it.</p><br />
<p>We have discovered four new plasmids capable of replication in Acetobacter species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K - both in G.xylinus ATCC 53582 strain, and in the strain we isolated from Kombucha. We have biobricked these plasmids, with the exception of pBAV1K, which had been originally engineered to be biobrick compatible (however, pBAV1K can not be uploaded to parts registry due to binding materials-transfer agreements).<br />
</p><br />
</section><br />
<br />
<section id="references"><br />
<h2>References</h2><br />
<ul><br />
<br />
<li>[1] https://www.researchgate.net/ publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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<ul><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2014-10-18T03:43:43Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
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<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
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<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
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<li><a data-scroll href="#engineering">Engineering</a><br />
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<li><a data-scroll href="#results">Results</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
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<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus igem</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and igem, creating a genetic toolbox of consisting of five new plasmid backbones and around 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
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<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em></li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem</li> <br />
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<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em></li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a powerful platform for the synthesis of new cellulose-based biomaterials and water filters</li><br />
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<section id="introduction"><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
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<h2>Introduction</h2><br />
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<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
In the middle of the summer, we isolated a new strain of Gluconacetobacter (named G. xylinus igem, previously named G. xylinus KI for Kombucha isolate ) from Kombucha tea and characterized its properties fully. We found it to be more amenable for genetic engineering than <em>G. xylinus</em> ATCC 53582 strain (see below), so we continued work on this strain, including sequencing its genome due to potential economic importance (it is a key component in the popular Kombucha tea). <br />
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<section id="aim"><br />
<h2>Aims</h2><br />
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<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and Kombucha-isolated strain and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
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<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
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<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.<br />
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In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and igem in a single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. We are currently in the process of completing the bioinformatics analyses of the genomes, and will publish the full genome sequences soon.<br />
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<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and igem strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and igem strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus igem strains to increase cellulose productivity and decrease its cost. In total, by cloning widely used parts into our <em>G. xylinus</em> shuttle vectors we created a toolkit consisting of:<br />
<ul><br />
<li> 5 new shuttle vectors </li> <br />
<li> 15 Anderson promoters </li><br />
<li> Fluorescent proteins RFP and sfGFP </li><br />
<li> 5 chromoproteins </li><br />
<li> 11 CBD fusion proteins </li> <br />
<li> Vitreoscilla hemoglobin gene </li> <br />
<li> TetR and Arabinose inducible promoters </li><br />
</ul><br />
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<p> Because the toolkit is in the non-standard pSEVA331-Bb backbone, it can not be housed in the Parts Registry, however in order to make it accessible for the synthetic biology community, we have made it freely available on request.<br />
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<h3>Characterization of G.xylinus ATCC 53582 and igem strains</h3><br />
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<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
</p><br />
<p> Cellulose productivity of G.xylinus ATCC 53582 and igem on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the igem strain is approximately one fifth of that of ATCC 53582 strain (see Figure 2 for comparison of ATCC53582 and igem strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for igem cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus). <br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 2. Cellulose production of G.xylinus ATCC 53582 and igem (KI) strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
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<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure 3.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, igem strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 3 for comparison of ATCC and igem strains and details of he experiment). This could be due to the niche igem strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 3. Cellulose productivity of G.xylinum ATCC 53582 and igem (KI) strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
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<p> Natural antibiotics resistance of ATCC 53582 and igem strains </p><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 4. Natural antibiotics resistance of G.xylinus ATCC 53582 and igem (KI) strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and igem seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
</figure><br />
<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 4 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and igem strains). However, when using high cell densities for plating (after transformations), antibiotic-resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus igem is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the igem strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
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<h3>Plasmid backbones</h3><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 5. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus igem after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
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<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and igem strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus igem and ATCC 53583 (See Figure 5). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
</ul><br />
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<h3>Anderson promoters</h3><br />
<div><br />
<p> We inserted 15 Anderson promoter-RFP coding devices into pSEVA331-Bb backbone, transformed them into G.xylinus igem strain in order to determine their relative strengths in Gluconacetobacter (see Figure 6 for some of them: <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/1/1a/IC14_Composite_image_correct.jpg"><br />
<figcaption>Figure 6. Different Anderson promoter-RFP constructs expressed in G.xylinus. Left- wild-type negative control. Right- G.xylinus igem transformed with pSEVA331Bb-Anderson promoter constructs. Plates grown at 30C for 7 days before imageing under blue light. </figcaption><br />
</figure><br />
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<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p><i>G. xylinus</i> is obligatively aerobic because of which cellulose production is reduced below the medium surface in static culture[1] due to reduced oxygen concentrations, reducing the overall volume of the pellicle that is biologically active. Transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i> (another obligate aerobe that expresses this protein in oxygen-poor environments) has been shown to result in increased the metabolic activity and cellulose production of G.xylinus [2].<br />
Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
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<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure 7. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem wild type cells and cells transformed with pSEVA331-BBa_K1321200 plasmid were cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days, after which OD600 was measured. Samples were diluted 1:1 in HS-cellulase medium before measurement. Negative controls (HS-cellulase without inoculations) showed no growth. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
</figure><br />
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<p>We cloned the Vhb gene behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS (part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a>) in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure 7) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
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<p>We also made a hypothesis of the metabolic pathway involving the interaction of vHb, which could potentially explain its effect on enhancing bacterial cellulose yield. As described in the diagram above, vHb plays a role as oxygen storage: it will actively capture oxygen molecules when when the oxygen concentration is high in the cell whereas release oxygen molecules when it is low. As such, the presence of vHb will smooth out the oxygen level within the cell and therefore provides it with a relatively constant oxygen supply. Considering the fact that oxygen also bind to protein complex cG PDE (cyclic GMP phosphodiesterase), where the binding will impose an upregulating effect on bacterial cellulose production, a constant oxygen level maintained by vHb will inevitably contribute to enhance bacterial cellulose yield.</p><br />
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<figure class="content-image image-left"><br />
<img class="image-full" src="https://lh5.googleusercontent.com/OkLBTdjMaO6PjVewhOFchlpEd3x4REDL53KYLp_sSxkD35RU4U8Gh21PJKUSfTA1GZ7ShEayn0Y=w751-h608"><br />
<figcaption>Figure Z. Effects of vHb interaction on cellular oxygen level.</figcaption><br />
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<section id="results"><br />
<h2>Results</h2><br />
<p>We have isolated a new strain of Acetobacter from kombucha, and determined its growth rate, cellulose productivity, and natural antibiotic resistance. We have also developed a set of methods for genetic engineering of this strain and have determined five new plasmids capable of replication in this strain. Thus we are the first to genetically engineer it.</p><br />
<p>We have discovered four new plasmids capable of replication in Acetobacter species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K - both in G.xylinus ATCC 53582 strain, and in the strain we isolated from Kombucha. We have biobricked these plasmids, with the exception of pBAV1K, which had been originally engineered to be biobrick compatible (however, pBAV1K can not be uploaded to parts registry due to binding materials-transfer agreements).<br />
</p><br />
</section><br />
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<section id="references"><br />
<h2>References</h2><br />
<ul><br />
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<li>[1] https://www.researchgate.net/ publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2014-10-18T03:42:18Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
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<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
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<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
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<li><a data-scroll href="#engineering">Engineering</a><br />
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<li><a data-scroll href="#results">Results</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
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<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus igem</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and igem, creating a genetic toolbox of consisting of five new plasmid backbones and around 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
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<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em></li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem</li> <br />
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<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em></li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a powerful platform for the synthesis of new cellulose-based biomaterials and water filters</li><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
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<h2>Introduction</h2><br />
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<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
In the middle of the summer, we isolated a new strain of Gluconacetobacter (named G. xylinus igem, previously named G. xylinus KI for Kombucha isolate ) from Kombucha tea and characterized its properties fully. We found it to be more amenable for genetic engineering than <em>G. xylinus</em> ATCC 53582 strain (see below), so we continued work on this strain, including sequencing its genome due to potential economic importance (it is a key component in the popular Kombucha tea). <br />
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<section id="aim"><br />
<h2>Aims</h2><br />
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<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and Kombucha-isolated strain and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
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<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
</figure><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.<br />
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In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and igem in a single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. We are currently in the process of completing the bioinformatics analyses of the genomes, and will publish the full genome sequences soon.<br />
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<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and igem strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and igem strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus igem strains to increase cellulose productivity and decrease its cost. In total, by cloning widely used parts into our <em>G. xylinus</em> shuttle vectors we created a toolkit consisting of:<br />
<ul><br />
<li> 5 new shuttle vectors </li> <br />
<li> 15 Anderson promoters </li><br />
<li> Fluorescent proteins RFP and sfGFP </li><br />
<li> 5 chromoproteins </li><br />
<li> 11 CBD fusion proteins </li> <br />
<li> Vitreoscilla hemoglobin gene </li> <br />
<li> TetR and Arabinose inducible promoters </li><br />
</ul><br />
</p><br />
<p> Because the toolkit is in the non-standard pSEVA331-Bb backbone, it can not be housed in the Parts Registry, however in order to make it accessible for the synthetic biology community, we have made it freely available on request.<br />
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<h3>Characterization of G.xylinus ATCC 53582 and igem strains</h3><br />
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<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
</p><br />
<p> Cellulose productivity of G.xylinus ATCC 53582 and igem on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the igem strain is approximately one fifth of that of ATCC 53582 strain (see Figure 2 for comparison of ATCC53582 and igem strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for igem cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus). <br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 2. Cellulose production of G.xylinus ATCC 53582 and igem strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
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<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure 3.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, igem strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 3 for comparison of ATCC and igem strains and details of he experiment). This could be due to the niche igem strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 3. Cellulose productivity of G.xylinum ATCC 53582 and igem (KI) strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
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<p> Natural antibiotics resistance of ATCC 53582 and igem strains </p><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 4. Natural antibiotics resistance of G.xylinus ATCC 53582 and igem (KI) strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and igem seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
</figure><br />
<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 4 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and igem strains). However, when using high cell densities for plating (after transformations), antibiotic-resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus igem is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the igem strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
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<h3>Plasmid backbones</h3><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 5. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus igem after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
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<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and igem strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus igem and ATCC 53583 (See Figure 5). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
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<h3>Anderson promoters</h3><br />
<div><br />
<p> We inserted 15 Anderson promoter-RFP coding devices into pSEVA331-Bb backbone, transformed them into G.xylinus igem strain in order to determine their relative strengths in Gluconacetobacter (see Figure 6 for some of them: <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/1/1a/IC14_Composite_image_correct.jpg"><br />
<figcaption>Figure 6. Different Anderson promoter-RFP constructs expressed in G.xylinus. Left- wild-type negative control. Right- G.xylinus igem transformed with pSEVA331Bb-Anderson promoter constructs. Plates grown at 30C for 7 days before imageing under blue light. </figcaption><br />
</figure><br />
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<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p><i>G. xylinus</i> is obligatively aerobic because of which cellulose production is reduced below the medium surface in static culture[1] due to reduced oxygen concentrations, reducing the overall volume of the pellicle that is biologically active. Transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i> (another obligate aerobe that expresses this protein in oxygen-poor environments) has been shown to result in increased the metabolic activity and cellulose production of G.xylinus [2].<br />
Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
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<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure 7. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem wild type cells and cells transformed with pSEVA331-BBa_K1321200 plasmid were cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days, after which OD600 was measured. Samples were diluted 1:1 in HS-cellulase medium before measurement. Negative controls (HS-cellulase without inoculations) showed no growth. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
</figure><br />
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<p>We cloned the Vhb gene behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS (part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a>) in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure 7) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
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<p>We also made a hypothesis of the metabolic pathway involving the interaction of vHb, which could potentially explain its effect on enhancing bacterial cellulose yield. As described in the diagram above, vHb plays a role as oxygen storage: it will actively capture oxygen molecules when when the oxygen concentration is high in the cell whereas release oxygen molecules when it is low. As such, the presence of vHb will smooth out the oxygen level within the cell and therefore provides it with a relatively constant oxygen supply. Considering the fact that oxygen also bind to protein complex cG PDE (cyclic GMP phosphodiesterase), where the binding will impose an upregulating effect on bacterial cellulose production, a constant oxygen level maintained by vHb will inevitably contribute to enhance bacterial cellulose yield.</p><br />
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<figure class="content-image image-left"><br />
<img class="image-full" src="https://lh5.googleusercontent.com/OkLBTdjMaO6PjVewhOFchlpEd3x4REDL53KYLp_sSxkD35RU4U8Gh21PJKUSfTA1GZ7ShEayn0Y=w751-h608"><br />
<figcaption>Figure Z. Effects of vHb interaction on cellular oxygen level.</figcaption><br />
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<section id="results"><br />
<h2>Results</h2><br />
<p>We have isolated a new strain of Acetobacter from kombucha, and determined its growth rate, cellulose productivity, and natural antibiotic resistance. We have also developed a set of methods for genetic engineering of this strain and have determined five new plasmids capable of replication in this strain. Thus we are the first to genetically engineer it.</p><br />
<p>We have discovered four new plasmids capable of replication in Acetobacter species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K - both in G.xylinus ATCC 53582 strain, and in the strain we isolated from Kombucha. We have biobricked these plasmids, with the exception of pBAV1K, which had been originally engineered to be biobrick compatible (however, pBAV1K can not be uploaded to parts registry due to binding materials-transfer agreements).<br />
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</section><br />
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<h2>References</h2><br />
<ul><br />
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<li>[1] https://www.researchgate.net/ publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2014-10-18T03:38:18Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
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<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
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<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
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<li><a data-scroll href="#engineering">Engineering</a><br />
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<li><a data-scroll href="#results">Results</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
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<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus KI</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and KI, creating a genetic toolbox of consisting of five new plasmid backbones and around 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
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<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em></li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem</li> <br />
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<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em></li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a powerful platform for the synthesis of new cellulose-based biomaterials and water filters</li><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
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<h2>Introduction</h2><br />
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<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
In the middle of the summer, we isolated a new strain of Gluconacetobacter (named G. xylinus igem, previously named G. xylinus KI for Kombucha isolate ) from Kombucha tea and characterized its properties fully. We found it to be more amenable for genetic engineering than <em>G. xylinus</em> ATCC 53582 strain (see below), so we continued work on this strain, including sequencing its genome due to potential economic importance (it is a key component in the popular Kombucha tea). <br />
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<section id="aim"><br />
<h2>Aims</h2><br />
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<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and Kombucha-isolated strain and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
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<h2>Genome sequencing</h2><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
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<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.<br />
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In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. We are currently in the process of completing the bioinformatics analyses of the genomes, and will publish the full genome sequences soon.<br />
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<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and KI strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and KI strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus KI strains to increase cellulose productivity and decrease its cost. In total, by cloning widely used parts into our <em>G. xylinus</em> shuttle vectors we created a toolkit consisting of:<br />
<ul><br />
<li> 5 new shuttle vectors </li> <br />
<li> 15 Anderson promoters </li><br />
<li> Fluorescent proteins RFP and sfGFP </li><br />
<li> 5 chromoproteins </li><br />
<li> 11 CBD fusion proteins </li> <br />
<li> Vitreoscilla hemoglobin gene </li> <br />
<li> TetR and Arabinose inducible promoters </li><br />
</ul><br />
</p><br />
<p> Because the toolkit is in the non-standard pSEVA331-Bb backbone, it can not be housed in the Parts Registry, however in order to make it accessible for the synthetic biology community, we have made it freely available on request.<br />
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<h3>Characterization of G.xylinus ATCC 53582 and KI strains</h3><br />
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<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
</p><br />
<p> Cellulose productivity of G.xylinus ATCC 53582 and KI on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the KI strain is approximately one fifth of that of ATCC 53582 strain (see Figure 2 for comparison of ATCC53582 and KI strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for KI cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus KI). <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 2. Cellulose production of G.xylinus ATCC 53582 and KI strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
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<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure 3.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, KI strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 3 for comparison of ATCC and KI strains and details of he experiment). This could be due to the niche KI strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 3. Cellulose productivity of G.xylinum ATCC 53582 and igem (KI) strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
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<p> Natural antibiotics resistance of ATCC 53582 and KI strains </p><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 4. Natural antibiotics resistance of G.xylinus ATCC 53582 and igem (KI) strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and KI seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
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<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 4 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and KI strain). However, when using high cell densities for plating (after transformations), antibiotic-resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus igem is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the igem strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
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<h3>Plasmid backbones</h3><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 5. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus igem after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
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<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and igem strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus KI and ATCC 53583 (See Figure 5). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
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<h3>Anderson promoters</h3><br />
<div><br />
<p> We inserted 15 Anderson promoter-RFP coding devices into pSEVA331-Bb backbone, transformed them into G.xylinus igem strain in order to determine their relative strengths in Gluconacetobacter (see Figure 6 for some of them: <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/1/1a/IC14_Composite_image_correct.jpg"><br />
<figcaption>Figure 6. Different Anderson promoter-RFP constructs expressed in G.xylinus. Left- wild-type negative control. Right- G.xylinus igem transformed with pSEVA331Bb-Anderson promoter constructs. Plates grown at 30C for 7 days before imageing under blue light. </figcaption><br />
</figure><br />
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<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p><i>G. xylinus</i> is obligatively aerobic because of which cellulose production is reduced below the medium surface in static culture[1] due to reduced oxygen concentrations, reducing the overall volume of the pellicle that is biologically active. Transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i> (another obligate aerobe that expresses this protein in oxygen-poor environments) has been shown to result in increased the metabolic activity and cellulose production of G.xylinus [2].<br />
Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
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<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure 7. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem wild type cells and cells transformed with pSEVA331-BBa_K1321200 plasmid were cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days, after which OD600 was measured. Samples were diluted 1:1 in HS-cellulase medium before measurement. Negative controls (HS-cellulase without inoculations) showed no growth. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
</figure><br />
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<p>We cloned the Vhb gene behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS (part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a>) in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure 7) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
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<p>We also made a hypothesis of the metabolic pathway involving the interaction of vHb, which could potentially explain its effect on enhancing bacterial cellulose yield. As described in the diagram above, vHb plays a role as oxygen storage: it will actively capture oxygen molecules when when the oxygen concentration is high in the cell whereas release oxygen molecules when it is low. As such, the presence of vHb will smooth out the oxygen level within the cell and therefore provides it with a relatively constant oxygen supply. Considering the fact that oxygen also bind to protein complex cG PDE (cyclic GMP phosphodiesterase), where the binding will impose an upregulating effect on bacterial cellulose production, a constant oxygen level maintained by vHb will inevitably contribute to enhance bacterial cellulose yield.</p><br />
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<figcaption>Figure Z. Effects of vHb interaction on cellular oxygen level.</figcaption><br />
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<section id="results"><br />
<h2>Results</h2><br />
<p>We have isolated a new strain of Acetobacter from kombucha, and determined its growth rate, cellulose productivity, and natural antibiotic resistance. We have also developed a set of methods for genetic engineering of this strain and have determined five new plasmids capable of replication in this strain. Thus we are the first to genetically engineer it.</p><br />
<p>We have discovered four new plasmids capable of replication in Acetobacter species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K - both in G.xylinus ATCC 53582 strain, and in the strain we isolated from Kombucha. We have biobricked these plasmids, with the exception of pBAV1K, which had been originally engineered to be biobrick compatible (however, pBAV1K can not be uploaded to parts registry due to binding materials-transfer agreements).<br />
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<h2>References</h2><br />
<ul><br />
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<li>[1] https://www.researchgate.net/ publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/File:IC14Flow_rates.pngFile:IC14Flow rates.png2014-10-18T03:37:32Z<p>Mf1512: </p>
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<div></div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2014-10-18T03:36:23Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
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<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
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<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
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<li><a data-scroll href="#engineering">Engineering</a><br />
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<li><a data-scroll href="#results">Results</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
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<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus KI</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and KI, creating a genetic toolbox of consisting of five new plasmid backbones and around 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
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<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em></li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem</li> <br />
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<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em></li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a powerful platform for the synthesis of new cellulose-based biomaterials and water filters</li><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
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<h2>Introduction</h2><br />
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<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
In the middle of the summer, we isolated a new strain of Gluconacetobacter (named G. xylinus igem, previously named G. xylinus KI for Kombucha isolate ) from Kombucha tea and characterized its properties fully. We found it to be more amenable for genetic engineering than <em>G. xylinus</em> ATCC 53582 strain (see below), so we continued work on this strain, including sequencing its genome due to potential economic importance (it is a key component in the popular Kombucha tea). <br />
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<section id="aim"><br />
<h2>Aims</h2><br />
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<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and Kombucha-isolated strain and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
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<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
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<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.<br />
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In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. We are currently in the process of completing the bioinformatics analyses of the genomes, and will publish the full genome sequences soon.<br />
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<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and KI strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and KI strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus KI strains to increase cellulose productivity and decrease its cost. In total, by cloning widely used parts into our <em>G. xylinus</em> shuttle vectors we created a toolkit consisting of:<br />
<ul><br />
<li> 5 new shuttle vectors </li> <br />
<li> 15 Anderson promoters </li><br />
<li> Fluorescent proteins RFP and sfGFP </li><br />
<li> 5 chromoproteins </li><br />
<li> 11 CBD fusion proteins </li> <br />
<li> Vitreoscilla hemoglobin gene </li> <br />
<li> TetR and Arabinose inducible promoters </li><br />
</ul><br />
</p><br />
<p> Because the toolkit is in the non-standard pSEVA331-Bb backbone, it can not be housed in the Parts Registry, however in order to make it accessible for the synthetic biology community, we have made it freely available on request.<br />
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<div class="accordion"><br />
<h3>Characterization of G.xylinus ATCC 53582 and KI strains</h3><br />
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<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
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<p> Cellulose productivity of G.xylinus ATCC 53582 and KI on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the KI strain is approximately one fifth of that of ATCC 53582 strain (see Figure 2 for comparison of ATCC53582 and KI strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for KI cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus KI). <br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 2. Cellulose production of G.xylinus ATCC 53582 and KI strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
</figure><br />
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<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure 3.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, KI strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 3 for comparison of ATCC and KI strains and details of he experiment). This could be due to the niche KI strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
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<figcaption> Figure 3. Cellulose productivity of G.xylinum ATCC 53582 and igem (KI) strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
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<p> Natural antibiotics resistance of ATCC 53582 and KI strains </p><br />
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<figcaption> Figure 4. Natural antibiotics resistance of G.xylinus ATCC 53582 and igem (KI) strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and KI seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
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<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 4 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and KI strain). However, when using high cell densities for plating (after transformations), antibiotic-resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus igem is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the igem strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
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<h3>Plasmid backbones</h3><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 5. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus igem after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
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<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and igem strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus KI and ATCC 53583 (See Figure 5). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
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<h3>Anderson promoters</h3><br />
<div><br />
<p> We inserted 15 Anderson promoter-RFP coding devices into pSEVA331-Bb backbone, transformed them into G.xylinus igem strain in order to determine their relative strengths in Gluconacetobacter (see Figure 6 for some of them: <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/1/1a/IC14_Composite_image_correct.jpg"><br />
<figcaption>Figure 6. Different Anderson promoter-RFP constructs expressed in G.xylinus. Left- wild-type negative control. Right- G.xylinus igem transformed with pSEVA331Bb-Anderson promoter constructs. Plates grown at 30C for 7 days before imageing under blue light. </figcaption><br />
</figure><br />
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<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p><i>G. xylinus</i> is obligatively aerobic because of which cellulose production is reduced below the medium surface in static culture[1] due to reduced oxygen concentrations, reducing the overall volume of the pellicle that is biologically active. Transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i> (another obligate aerobe that expresses this protein in oxygen-poor environments) has been shown to result in increased the metabolic activity and cellulose production of G.xylinus [2].<br />
Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
<br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure 7. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem wild type cells and cells transformed with pSEVA331-BBa_K1321200 plasmid were cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days, after which OD600 was measured. Samples were diluted 1:1 in HS-cellulase medium before measurement. Negative controls (HS-cellulase without inoculations) showed no growth. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
</figure><br />
<br />
<p>We cloned the Vhb gene behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS (part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a>) in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure 7) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
<br />
<p>We also made a hypothesis of the metabolic pathway involving the interaction of vHb, which could potentially explain its effect on enhancing bacterial cellulose yield. As described in the diagram above, vHb plays a role as oxygen storage: it will actively capture oxygen molecules when when the oxygen concentration is high in the cell whereas release oxygen molecules when it is low. As such, the presence of vHb will smooth out the oxygen level within the cell and therefore provides it with a relatively constant oxygen supply. Considering the fact that oxygen also bind to protein complex cG PDE (cyclic GMP phosphodiesterase), where the binding will impose an upregulating effect on bacterial cellulose production, a constant oxygen level maintained by vHb will inevitably contribute to enhance bacterial cellulose yield.</p><br />
<br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://lh5.googleusercontent.com/OkLBTdjMaO6PjVewhOFchlpEd3x4REDL53KYLp_sSxkD35RU4U8Gh21PJKUSfTA1GZ7ShEayn0Y=w751-h608"><br />
<figcaption>Figure Z. Effects of vHb interaction on cellular oxygen level.</figcaption><br />
</figure> <br />
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</div><br />
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</div><br />
</section><br />
<section id="engineering"><br />
<h2>Engineering</h2><br />
<br />
<div class="accordion"><br />
<h3>Sequencing the genomes of <em>G. xylinus</em> ATCC 53582 and Kombucha-isolated <em>G. xylinus</em> strains</h3><br />
<div><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of <em>G. xylinus</em> is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.</p><br />
<p>In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. <br />
We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction).<br />
We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq present at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9063619 reads with 98.71% reads identified). We completed the genome assembly with the support of Imperial Bioinformatics Support service using their in-house assembly pipeline. </p><br />
</div><br />
<h3>Creating a toolbox for Gluconacetobacter genetic engineering</h3><br />
<div><br />
<p>In parallel to sequencing the genome, we began building a large toolbox for <em>G. xylinus genetic engineering</em>. Only a few vectors have been developed for <em>G. xylinus</em>, and most of them were not available for us for various reasons (including patents, materials-transfer agreements, and retired or deceased researchers). For this reason, we first tested out a set of new plasmids, and engineered them to be Biobrick compatible. As some <em>E. coli</em> promoters and proteins have been shown to work in <em>Gluconacetobacter</em> or <em>Acetobacter</em> species (Setyawati, 2007; Chien et al., 2006), and similarly to <em>E.coli</em>, <em>G. xylinus</em> is a Gram- species, we decided to test out and modify already existing Biobricks in the parts registry in <em>G. xylinus</em>. Therefore, the toolkit consists of:</p><br />
<br />
<br />
<br />
<ul><br />
<li>New, Biobrick compatible shuttle vectors for <em>Gluconacetobacter</em> and <em>E. coli</em><br />
<ul><br />
<br />
<li><em>pSEVA321</em><br />
</li><br />
<li><em>pSEVA331</em><br />
</li><br />
<li><em>pSEVA351</em><br />
</li><br />
<li><em>pBAV1K</em><br />
</li><br />
<br />
</ul><br />
</li>f<br />
<br />
<li>Characterization and optimization of parts from the Registry, including:<br />
<ul><br />
<li>Fluorescent proteins, GFP, RFP, YFP, CFP<br />
</li><br />
<li>Chromoproteins</li><br />
<li>Vitreoscilla haemoglobin<br />
</li><br />
<li>The entire Anderson promoter library<br />
</li><br />
<li>Widely used gene expression control proteins AraC and TetR.<br />
</li><br />
<br />
<br />
</ul><br />
<br />
</li><br />
</ul><br />
</div><br />
<h3>Engineering a high cellulose-producing ATCC 53582 strain</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
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<br />
</div><br />
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</div><br />
</section><br />
<br />
<br />
<section id="results"><br />
<h2>Results</h2><br />
<p>We have isolated a new strain of Acetobacter from kombucha, and determined its growth rate, cellulose productivity, and natural antibiotic resistance. We have also developed a set of methods for genetic engineering of this strain and have determined five new plasmids capable of replication in this strain. Thus we are the first to genetically engineer it.</p><br />
<p>We have discovered four new plasmids capable of replication in Acetobacter species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K - both in G.xylinus ATCC 53582 strain, and in the strain we isolated from Kombucha. We have biobricked these plasmids, with the exception of pBAV1K, which had been originally engineered to be biobrick compatible (however, pBAV1K can not be uploaded to parts registry due to binding materials-transfer agreements).<br />
</p><br />
</section><br />
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<h2>References</h2><br />
<ul><br />
<br />
<li>[1] https://www.researchgate.net/ publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/Mass_Production_and_ProcessingTeam:Imperial/Mass Production and Processing2014-10-18T03:36:09Z<p>Mf1512: </p>
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<h1>Mass Production and Processing</h1><br />
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<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<h2>Introduction</h2><br />
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<img class="image-full" src=https://static.igem.org/mediawiki/2014/7/7b/IC14-Post-prod-fig-1-blue.png ><br />
<figcaption>Figure 1: Blue dyed bacterial cellulose</figcaption><br />
</figure><br />
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<p> Bacterial cellulose (BC) exhibits a <a href="https://2014.igem.org/Team:Imperial/Project_Background">multitude of different properties</a> depending on the processing, growth conditions, functionalisation and strain used (Bismarck 2013) for production of the material. Acquiring large quantities of cellulose produced would allow testing of a broad variety of cellulose processing methods and functionalisation steps. </p><br />
<br />
<p> By mass producing cellulose this enables a better understanding of what material properties can be realistically produced during the short duration of iGEM. More importantly, it improves the likelihood of finding suitable processing candidates for the project’s aim of making a customisable ultrafiltration membrane, at the same time as allowing room for creativity and exploration of the remarkable properties of cellulose. </p><br />
<br />
<h3>Minimum requirements</h3><br />
<ol><br />
<li>Treatment of BC requires killing the cells, particularly if the cells are genetically engineered, which is the aim for putting the customisable in ultrafiltration membranes.</li><br />
<li>Based on brainstorming with Central Saint Martins student Zuzana, removing the colour of BC is required as it looks displeasing to the eye otherwise, and seems counter-intuituve to filter clean water with cellulose coloured like turbid water. </li><br />
<li>Removal of the smell of BC has also been raised as a requirement, particularly by producers who work in close contact with the processing facilities.</li><br />
</ol><br />
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<h3>Mass Production Methods</h3><br />
<br />
<figure class="content-image image-right image-small"> <br />
<img class="image-full" src=https://static.igem.org/mediawiki/2014/0/04/IC14-post-prod-fig-2-pellicle-comparison.png > <br />
<figcaption>Figure 2. left: A granular pellicle, right: even pellicle</figcaption> <br />
</figure> <br />
<br />
<p>Setting up the mass production of cellulose was done according to the <a href="https://2014.igem.org/Team:Imperial/Protocols#gluconacetobacter">Kombucha media protocol</a> , which involved setting up 61 trays with media and G. xylinus and yeast co-culture as shown in figure 3. The trays were left to grow up over 7 days, after which diminishing pellicle growth was detected. Upon harvesting, the pellicles were sorted according to granular pellicles (see figure 2 left) and even pellicles (figure 2 right). All pellicles were kept in distilled water in large plastic buckets or containers. </p><br />
<br />
<p>Below shows the general workflow we employed to mass produce our cellulose and illustrates the process of manufacturing biomaterials with significantly different properties despite originating from the same BC source.<br />
</p><br />
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<img class="content-image image-full" src="https://static.igem.org/mediawiki/2014/b/bf/IC14-mass_production2.png"> <br />
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<h2>References</h2><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2014-10-18T03:33:56Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
</li><br />
<li><a data-scroll href="#aim">Aims</a><br />
</li><br />
<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
</li><br />
<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
</li><br />
<li><a data-scroll href="#engineering">Engineering</a><br />
</li><br />
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<li><a data-scroll href="#results">Results</a><br />
</li><br />
<li><a data-scroll href="#references">References</a><br />
</li><br />
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<section id="overview"><br />
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<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus KI</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and KI, creating a genetic toolbox of consisting of five new plasmid backbones and around 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
</div><br />
<div class="pure-u-1-2"><br />
<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em></li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem</li> <br />
<br />
<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em></li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a powerful platform for the synthesis of new cellulose-based biomaterials and water filters</li><br />
<br />
<br />
<br />
<br />
<br />
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</section><br />
<section id="introduction"><br />
<br/><br />
<figure class="content-image image-full"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
</figure><br />
<h2>Introduction</h2><br />
<br />
<br />
<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
In the middle of the summer, we isolated a new strain of Gluconacetobacter (named G. xylinus igem, previously named G. xylinus KI for Kombucha isolate ) from Kombucha tea and characterized its properties fully. We found it to be more amenable for genetic engineering than <em>G. xylinus</em> ATCC 53582 strain (see below), so we continued work on this strain, including sequencing its genome due to potential economic importance (it is a key component in the popular Kombucha tea). <br />
<br />
</p><br />
<br />
</section><br />
<section id="aim"><br />
<h2>Aims</h2><br />
<br />
<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and Kombucha-isolated strain and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
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<br />
</section><br />
<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
</figure><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.<br />
<br />
In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. We are currently in the process of completing the bioinformatics analyses of the genomes, and will publish the full genome sequences soon.<br />
<br />
</p><br />
</section><br />
<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and KI strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and KI strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus KI strains to increase cellulose productivity and decrease its cost. In total, by cloning widely used parts into our <em>G. xylinus</em> shuttle vectors we created a toolkit consisting of:<br />
<ul><br />
<li> 5 new shuttle vectors </li> <br />
<li> 15 Anderson promoters </li><br />
<li> Fluorescent proteins RFP and sfGFP </li><br />
<li> 5 chromoproteins </li><br />
<li> 11 CBD fusion proteins </li> <br />
<li> Vitreoscilla hemoglobin gene </li> <br />
<li> TetR and Arabinose inducible promoters </li><br />
</ul><br />
</p><br />
<p> Because the toolkit is in the non-standard pSEVA331-Bb backbone, it can not be housed in the Parts Registry, however in order to make it accessible for the synthetic biology community, we have made it freely available on request.<br />
</p><br />
<div class="accordion"><br />
<h3>Characterization of G.xylinus ATCC 53582 and KI strains</h3><br />
<div><br />
<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
</p><br />
<p> Cellulose productivity of G.xylinus ATCC 53582 and KI on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the KI strain is approximately one fifth of that of ATCC 53582 strain (see Figure 2 for comparison of ATCC53582 and KI strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for KI cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus KI). <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 2. Cellulose production of G.xylinus ATCC 53582 and KI strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
</figure><br />
</p> <br />
<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure 3.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, KI strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 3 for comparison of ATCC and KI strains and details of he experiment). This could be due to the niche KI strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 3. Cellulose productivity of G.xylinum ATCC 53582 and igem (KI) strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
</figure><br />
</p> <br />
<p> Natural antibiotics resistance of ATCC 53582 and KI strains </p><br />
<p></p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 4. Natural antibiotics resistance of G.xylinus ATCC 53582 and igem (KI) strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and KI seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
</figure><br />
<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 4 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and KI strain). However, when using high cell densities for plating (after transformations), antibiotic-resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus igem is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the igem strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
<br />
<br />
</div><br />
<h3>Plasmid backbones</h3><br />
<div><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 5. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus igem after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
</figure><br />
<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and igem strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus KI and ATCC 53583 (See Figure 5). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
</ul><br />
</div><br />
<h3>Anderson promoters</h3><br />
<div><br />
<p> We inserted 15 Anderson promoter-RFP coding devices into pSEVA331-Bb backbone, transformed them into G.xylinus igem strain in order to determine their relative strengths in Gluconacetobacter (see Figure Y for some of them: <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/1/1a/IC14_Composite_image_correct.jpg"><br />
<figcaption>Figure Z. Different Anderson promoter-RFP constructs expressed in G.xylinus. Left- wild-type negative control. Right- G.xylinus igem transformed with pSEVA331Bb-Anderson promoter constructs. Plates grown at 30C for 7 days before imageing under blue light. </figcaption><br />
</figure><br />
</p><br />
</div><br />
<br />
<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p><i>G. xylinus</i> is obligatively aerobic because of which cellulose production is reduced below the medium surface in static culture[1] due to reduced oxygen concentrations, reducing the overall volume of the pellicle that is biologically active. Transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i> (another obligate aerobe that expresses this protein in oxygen-poor environments) has been shown to result in increased the metabolic activity and cellulose production of G.xylinus [2].<br />
Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
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<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure Z. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem wild type cells and cells transformed with pSEVA331-BBa_K1321200 plasmid were cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days, after which OD600 was measured. Samples were diluted 1:1 in HS-cellulase medium before measurement. Negative controls (HS-cellulase without inoculations) showed no growth. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
</figure><br />
<br />
<p>We cloned the Vhb gene behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS (part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a>) in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure Z) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
<br />
<p>We also made a hypothesis of the metabolic pathway involving the interaction of vHb, which could potentially explain its effect on enhancing bacterial cellulose yield. As described in the diagram above, vHb plays a role as oxygen storage: it will actively capture oxygen molecules when when the oxygen concentration is high in the cell whereas release oxygen molecules when it is low. As such, the presence of vHb will smooth out the oxygen level within the cell and therefore provides it with a relatively constant oxygen supply. Considering the fact that oxygen also bind to protein complex cG PDE (cyclic GMP phosphodiesterase), where the binding will impose an upregulating effect on bacterial cellulose production, a constant oxygen level maintained by vHb will inevitably contribute to enhance bacterial cellulose yield.</p><br />
<br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://lh5.googleusercontent.com/OkLBTdjMaO6PjVewhOFchlpEd3x4REDL53KYLp_sSxkD35RU4U8Gh21PJKUSfTA1GZ7ShEayn0Y=w751-h608"><br />
<figcaption>Figure Z. Effects of vHb interaction on cellular oxygen level.</figcaption><br />
</figure> <br />
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</div><br />
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</div><br />
</section><br />
<section id="engineering"><br />
<h2>Engineering</h2><br />
<br />
<div class="accordion"><br />
<h3>Sequencing the genomes of <em>G. xylinus</em> ATCC 53582 and Kombucha-isolated <em>G. xylinus</em> strains</h3><br />
<div><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of <em>G. xylinus</em> is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.</p><br />
<p>In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. <br />
We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction).<br />
We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq present at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9063619 reads with 98.71% reads identified). We completed the genome assembly with the support of Imperial Bioinformatics Support service using their in-house assembly pipeline. </p><br />
</div><br />
<h3>Creating a toolbox for Gluconacetobacter genetic engineering</h3><br />
<div><br />
<p>In parallel to sequencing the genome, we began building a large toolbox for <em>G. xylinus genetic engineering</em>. Only a few vectors have been developed for <em>G. xylinus</em>, and most of them were not available for us for various reasons (including patents, materials-transfer agreements, and retired or deceased researchers). For this reason, we first tested out a set of new plasmids, and engineered them to be Biobrick compatible. As some <em>E. coli</em> promoters and proteins have been shown to work in <em>Gluconacetobacter</em> or <em>Acetobacter</em> species (Setyawati, 2007; Chien et al., 2006), and similarly to <em>E.coli</em>, <em>G. xylinus</em> is a Gram- species, we decided to test out and modify already existing Biobricks in the parts registry in <em>G. xylinus</em>. Therefore, the toolkit consists of:</p><br />
<br />
<br />
<br />
<ul><br />
<li>New, Biobrick compatible shuttle vectors for <em>Gluconacetobacter</em> and <em>E. coli</em><br />
<ul><br />
<br />
<li><em>pSEVA321</em><br />
</li><br />
<li><em>pSEVA331</em><br />
</li><br />
<li><em>pSEVA351</em><br />
</li><br />
<li><em>pBAV1K</em><br />
</li><br />
<br />
</ul><br />
</li>f<br />
<br />
<li>Characterization and optimization of parts from the Registry, including:<br />
<ul><br />
<li>Fluorescent proteins, GFP, RFP, YFP, CFP<br />
</li><br />
<li>Chromoproteins</li><br />
<li>Vitreoscilla haemoglobin<br />
</li><br />
<li>The entire Anderson promoter library<br />
</li><br />
<li>Widely used gene expression control proteins AraC and TetR.<br />
</li><br />
<br />
<br />
</ul><br />
<br />
</li><br />
</ul><br />
</div><br />
<h3>Engineering a high cellulose-producing ATCC 53582 strain</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
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</div><br />
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</div><br />
</section><br />
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<br />
<section id="results"><br />
<h2>Results</h2><br />
<p>We have isolated a new strain of Acetobacter from kombucha, and determined its growth rate, cellulose productivity, and natural antibiotic resistance. We have also developed a set of methods for genetic engineering of this strain and have determined five new plasmids capable of replication in this strain. Thus we are the first to genetically engineer it.</p><br />
<p>We have discovered four new plasmids capable of replication in Acetobacter species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K - both in G.xylinus ATCC 53582 strain, and in the strain we isolated from Kombucha. We have biobricked these plasmids, with the exception of pBAV1K, which had been originally engineered to be biobrick compatible (however, pBAV1K can not be uploaded to parts registry due to binding materials-transfer agreements).<br />
</p><br />
</section><br />
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<section id="references"><br />
<h2>References</h2><br />
<ul><br />
<br />
<li>[1] https://www.researchgate.net/ publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2014-10-18T03:29:31Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
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<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
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<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
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<li><a data-scroll href="#engineering">Engineering</a><br />
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<li><a data-scroll href="#results">Results</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
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<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus KI</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and KI, creating a genetic toolbox of consisting of five new plasmid backbones and around 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
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<div class="pure-u-1-2"><br />
<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em></li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem</li> <br />
<br />
<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em></li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a powerful platform for the synthesis of new cellulose-based biomaterials and water filters</li><br />
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<section id="introduction"><br />
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<figure class="content-image image-full"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
</figure><br />
<h2>Introduction</h2><br />
<br />
<br />
<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
In the middle of the summer, we isolated a new strain of Gluconacetobacter (named G. xylinus igem, previously named G. xylinus KI for Kombucha isolate ) from Kombucha tea and characterized its properties fully. We found it to be more amenable for genetic engineering than <em>G. xylinus</em> ATCC 53582 strain (see below), so we continued work on this strain, including sequencing its genome due to potential economic importance (it is a key component in the popular Kombucha tea). <br />
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</p><br />
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</section><br />
<section id="aim"><br />
<h2>Aims</h2><br />
<br />
<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and Kombucha-isolated strain and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
<br />
<br />
</section><br />
<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
</figure><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.<br />
<br />
In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. We are currently in the process of completing the bioinformatics analyses of the genomes, and will publish the full genome sequences soon.<br />
<br />
</p><br />
</section><br />
<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and KI strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and KI strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus KI strains to increase cellulose productivity and decrease its cost. In total, by cloning widely used parts into our <em>G. xylinus</em> shuttle vectors we created a toolkit consisting of:<br />
<ul><br />
<li> 5 new shuttle vectors </li> <br />
<li> 15 Anderson promoters </li><br />
<li> Fluorescent proteins RFP and sfGFP </li><br />
<li> 5 chromoproteins </li><br />
<li> 11 CBD fusion proteins </li> <br />
<li> Vitreoscilla hemoglobin gene </li> <br />
<li> TetR and Arabinose inducible promoters </li><br />
</ul><br />
</p><br />
<p> Because the toolkit is in the non-standard pSEVA331-Bb backbone, it can not be housed in the Parts Registry, however in order to make it accessible for the synthetic biology community, we have made it freely available on request.<br />
</p><br />
<div class="accordion"><br />
<h3>Characterization of G.xylinus ATCC 53582 and KI strains</h3><br />
<div><br />
<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
</p><br />
<p> Cellulose productivity of G.xylinus ATCC 53582 and KI on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the KI strain is approximately one fifth of that of ATCC 53582 strain (see Figure 2 for comparison of ATCC53582 and KI strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for KI cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus KI). <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 2. Cellulose production of G.xylinus ATCC 53582 and KI strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
</figure><br />
</p> <br />
<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure Y.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, KI strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 2 for comparison of ATCC and KI strains and details of he experiment). This could be due to the niche KI strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 4. Cellulose productivity of G.xylinum ATCC 53582 and KI strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
</figure><br />
</p> <br />
<p> Natural antibiotics resistance of ATCC 53582 and KI strains </p><br />
<p></p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 5. Natural antibiotics resistance of G.xylinus ATCC 53582 and KI strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and KI seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
</figure><br />
<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 5 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and KI strain). However, when using high cell densities for plating (after transformations), antibiotic -resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus KI is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the KI strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
<br />
<br />
</div><br />
<h3>Plasmid backbones</h3><br />
<div><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 1. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus KI after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
</figure><br />
<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and Ki strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus KI and ATCC 53583 (See figure X). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
</ul><br />
</div><br />
<h3>Anderson promoters</h3><br />
<div><br />
<p> We inserted 15 Anderson promoter-RFP coding devices into pSEVA331-Bb backbone, transformed them into G.xylinus igem strain in order to determine their relative strengths in Gluconacetobacter (see Figure Y for some of them: <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/1/1a/IC14_Composite_image_correct.jpg"><br />
<figcaption>Figure Z. Different Anderson promoter-RFP constructs expressed in G.xylinus. Left- wild-type negative control. Right- G.xylinus igem transformed with pSEVA331Bb-Anderson promoter constructs. Plates grown at 30C for 7 days before imageing under blue light. </figcaption><br />
</figure><br />
</p><br />
</div><br />
<br />
<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p><i>G. xylinus</i> is obligatively aerobic because of which cellulose production is reduced below the medium surface in static culture[1] due to reduced oxygen concentrations, reducing the overall volume of the pellicle that is biologically active. Transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i> (another obligate aerobe that expresses this protein in oxygen-poor environments) has been shown to result in increased the metabolic activity and cellulose production of G.xylinus [2].<br />
Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
<br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure Z. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem wild type cells and cells transformed with pSEVA331-BBa_K1321200 plasmid were cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days, after which OD600 was measured. Samples were diluted 1:1 in HS-cellulase medium before measurement. Negative controls (HS-cellulase without inoculations) showed no growth. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
</figure><br />
<br />
<p>We cloned the Vhb gene behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS (part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a>) in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure Z) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
<br />
<p>We also made a hypothesis of the metabolic pathway involving the interaction of vHb, which could potentially explain its effect on enhancing bacterial cellulose yield. As described in the diagram above, vHb plays a role as oxygen storage: it will actively capture oxygen molecules when when the oxygen concentration is high in the cell whereas release oxygen molecules when it is low. As such, the presence of vHb will smooth out the oxygen level within the cell and therefore provides it with a relatively constant oxygen supply. Considering the fact that oxygen also bind to protein complex cG PDE (cyclic GMP phosphodiesterase), where the binding will impose an upregulating effect on bacterial cellulose production, a constant oxygen level maintained by vHb will inevitably contribute to enhance bacterial cellulose yield.</p><br />
<br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://lh5.googleusercontent.com/OkLBTdjMaO6PjVewhOFchlpEd3x4REDL53KYLp_sSxkD35RU4U8Gh21PJKUSfTA1GZ7ShEayn0Y=w751-h608"><br />
<figcaption>Figure Z. Effects of vHb interaction on cellular oxygen level.</figcaption><br />
</figure> <br />
<br />
</div><br />
<br />
<br />
</div><br />
</section><br />
<section id="engineering"><br />
<h2>Engineering</h2><br />
<br />
<div class="accordion"><br />
<h3>Sequencing the genomes of <em>G. xylinus</em> ATCC 53582 and Kombucha-isolated <em>G. xylinus</em> strains</h3><br />
<div><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of <em>G. xylinus</em> is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.</p><br />
<p>In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. <br />
We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction).<br />
We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq present at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9063619 reads with 98.71% reads identified). We completed the genome assembly with the support of Imperial Bioinformatics Support service using their in-house assembly pipeline. </p><br />
</div><br />
<h3>Creating a toolbox for Gluconacetobacter genetic engineering</h3><br />
<div><br />
<p>In parallel to sequencing the genome, we began building a large toolbox for <em>G. xylinus genetic engineering</em>. Only a few vectors have been developed for <em>G. xylinus</em>, and most of them were not available for us for various reasons (including patents, materials-transfer agreements, and retired or deceased researchers). For this reason, we first tested out a set of new plasmids, and engineered them to be Biobrick compatible. As some <em>E. coli</em> promoters and proteins have been shown to work in <em>Gluconacetobacter</em> or <em>Acetobacter</em> species (Setyawati, 2007; Chien et al., 2006), and similarly to <em>E.coli</em>, <em>G. xylinus</em> is a Gram- species, we decided to test out and modify already existing Biobricks in the parts registry in <em>G. xylinus</em>. Therefore, the toolkit consists of:</p><br />
<br />
<br />
<br />
<ul><br />
<li>New, Biobrick compatible shuttle vectors for <em>Gluconacetobacter</em> and <em>E. coli</em><br />
<ul><br />
<br />
<li><em>pSEVA321</em><br />
</li><br />
<li><em>pSEVA331</em><br />
</li><br />
<li><em>pSEVA351</em><br />
</li><br />
<li><em>pBAV1K</em><br />
</li><br />
<br />
</ul><br />
</li>f<br />
<br />
<li>Characterization and optimization of parts from the Registry, including:<br />
<ul><br />
<li>Fluorescent proteins, GFP, RFP, YFP, CFP<br />
</li><br />
<li>Chromoproteins</li><br />
<li>Vitreoscilla haemoglobin<br />
</li><br />
<li>The entire Anderson promoter library<br />
</li><br />
<li>Widely used gene expression control proteins AraC and TetR.<br />
</li><br />
<br />
<br />
</ul><br />
<br />
</li><br />
</ul><br />
</div><br />
<h3>Engineering a high cellulose-producing ATCC 53582 strain</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
<br />
<br />
</div><br />
<br />
</div><br />
</section><br />
<br />
<br />
<section id="results"><br />
<h2>Results</h2><br />
<p>We have isolated a new strain of Acetobacter from kombucha, and determined its growth rate, cellulose productivity, and natural antibiotic resistance. We have also developed a set of methods for genetic engineering of this strain and have determined five new plasmids capable of replication in this strain. Thus we are the first to genetically engineer it.</p><br />
<p>We have discovered four new plasmids capable of replication in Acetobacter species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K - both in G.xylinus ATCC 53582 strain, and in the strain we isolated from Kombucha. We have biobricked these plasmids, with the exception of pBAV1K, which had been originally engineered to be biobrick compatible (however, pBAV1K can not be uploaded to parts registry due to binding materials-transfer agreements).<br />
</p><br />
</section><br />
<br />
<section id="references"><br />
<h2>References</h2><br />
<ul><br />
<br />
<li>[1] https://www.researchgate.net/ publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2014-10-18T03:24:45Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<li><a data-scroll href="#introduction">Introduction</a><br />
</li><br />
<li><a data-scroll href="#aim">Aims</a><br />
</li><br />
<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
</li><br />
<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
</li><br />
<li><a data-scroll href="#engineering">Engineering</a><br />
</li><br />
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<li><a data-scroll href="#results">Results</a><br />
</li><br />
<li><a data-scroll href="#references">References</a><br />
</li><br />
</ul><br />
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</div><br />
<section id="overview"><br />
<div class="pure-g"><br />
<div class="pure-u-1-2"><br />
<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus KI</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and KI, creating a genetic toolbox of consisting of five new plasmid backbones and around 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
</div><br />
<div class="pure-u-1-2"><br />
<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em></li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem</li> <br />
<br />
<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em></li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a powerful platform for the synthesis of new cellulose-based biomaterials and water filters</li><br />
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</ul><br />
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</div><br />
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</section><br />
<section id="introduction"><br />
<br/><br />
<figure class="content-image image-full"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
</figure><br />
<h2>Introduction</h2><br />
<br />
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<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
</p><br />
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</section><br />
<section id="aim"><br />
<h2>Aims</h2><br />
<br />
<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and Kombucha-isolated strain and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
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</section><br />
<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
</figure><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.<br />
<br />
In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. We are currently in the process of completing the bioinformatics analyses of the genomes, and will publish the full genome sequences soon.<br />
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</p><br />
</section><br />
<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and KI strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and KI strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus KI strains to increase cellulose productivity and decrease its cost. In total, by cloning widely used parts into our <em>G. xylinus</em> shuttle vectors we created a toolkit consisting of:<br />
<ul><br />
<li> 5 new shuttle vectors </li> <br />
<li> 15 Anderson promoters </li><br />
<li> Fluorescent proteins RFP and sfGFP </li><br />
<li> 5 chromoproteins </li><br />
<li> 11 CBD fusion proteins </li> <br />
<li> Vitreoscilla hemoglobin gene </li> <br />
<li> TetR and Arabinose inducible promoters </li><br />
</ul><br />
</p><br />
<p> Because the toolkit is in the non-standard pSEVA331-Bb backbone, it can not be housed in the Parts Registry, however in order to make it accessible for the synthetic biology community, we have made it freely available on request.<br />
</p><br />
<div class="accordion"><br />
<h3>Characterization of G.xylinus ATCC 53582 and KI strains</h3><br />
<div><br />
<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
</p><br />
<p> Cellulose productivity of G.xylinus ATCC 53582 and KI on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the KI strain is approximately one fifth of that of ATCC 53582 strain (see Figure 2 for comparison of ATCC53582 and KI strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for KI cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus KI). <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 2. Cellulose production of G.xylinus ATCC 53582 and KI strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
</figure><br />
</p> <br />
<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure Y.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, KI strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 2 for comparison of ATCC and KI strains and details of he experiment). This could be due to the niche KI strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 4. Cellulose productivity of G.xylinum ATCC 53582 and KI strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
</figure><br />
</p> <br />
<p> Natural antibiotics resistance of ATCC 53582 and KI strains </p><br />
<p></p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 5. Natural antibiotics resistance of G.xylinus ATCC 53582 and KI strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and KI seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
</figure><br />
<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 5 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and KI strain). However, when using high cell densities for plating (after transformations), antibiotic -resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus KI is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the KI strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
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</div><br />
<h3>Plasmid backbones</h3><br />
<div><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 1. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus KI after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
</figure><br />
<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and Ki strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus KI and ATCC 53583 (See figure X). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
</ul><br />
</div><br />
<h3>Anderson promoters</h3><br />
<div><br />
<p> We inserted 15 Anderson promoter-RFP coding devices into pSEVA331-Bb backbone, transformed them into G.xylinus igem strain in order to determine their relative strengths in Gluconacetobacter (see Figure Y for some of them: <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/1/1a/IC14_Composite_image_correct.jpg"><br />
<figcaption>Figure Z. Different Anderson promoter-RFP constructs expressed in G.xylinus. Left- wild-type negative control. Right- G.xylinus igem transformed with pSEVA331Bb-Anderson promoter constructs. Plates grown at 30C for 7 days before imageing under blue light. </figcaption><br />
</figure><br />
</p><br />
</div><br />
<br />
<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p><i>G. xylinus</i> is obligatively aerobic because of which cellulose production is reduced below the medium surface in static culture[1] due to reduced oxygen concentrations, reducing the overall volume of the pellicle that is biologically active. Transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i> (another obligate aerobe that expresses this protein in oxygen-poor environments) has been shown to result in increased the metabolic activity and cellulose production of G.xylinus [2].<br />
Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
<br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure Z. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem wild type cells and cells transformed with pSEVA331-BBa_K1321200 plasmid were cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days, after which OD600 was measured. Samples were diluted 1:1 in HS-cellulase medium before measurement. Negative controls (HS-cellulase without inoculations) showed no growth. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
</figure><br />
<br />
<p>We cloned the Vhb gene behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS (part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a>) in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure Z) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
<br />
<p>We also made a hypothesis of the metabolic pathway involving the interaction of vHb, which could potentially explain its effect on enhancing bacterial cellulose yield. As described in the diagram above, vHb plays a role as oxygen storage: it will actively capture oxygen molecules when when the oxygen concentration is high in the cell whereas release oxygen molecules when it is low. As such, the presence of vHb will smooth out the oxygen level within the cell and therefore provides it with a relatively constant oxygen supply. Considering the fact that oxygen also bind to protein complex cG PDE (cyclic GMP phosphodiesterase), where the binding will impose an upregulating effect on bacterial cellulose production, a constant oxygen level maintained by vHb will inevitably contribute to enhance bacterial cellulose yield.</p><br />
<br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://lh5.googleusercontent.com/OkLBTdjMaO6PjVewhOFchlpEd3x4REDL53KYLp_sSxkD35RU4U8Gh21PJKUSfTA1GZ7ShEayn0Y=w751-h608"><br />
<figcaption>Figure Z. Effects of vHb interaction on cellular oxygen level.</figcaption><br />
</figure> <br />
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</div><br />
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</div><br />
</section><br />
<section id="engineering"><br />
<h2>Engineering</h2><br />
<br />
<div class="accordion"><br />
<h3>Sequencing the genomes of <em>G. xylinus</em> ATCC 53582 and Kombucha-isolated <em>G. xylinus</em> strains</h3><br />
<div><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of <em>G. xylinus</em> is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.</p><br />
<p>In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. <br />
We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction).<br />
We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq present at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9063619 reads with 98.71% reads identified). We completed the genome assembly with the support of Imperial Bioinformatics Support service using their in-house assembly pipeline. </p><br />
</div><br />
<h3>Creating a toolbox for Gluconacetobacter genetic engineering</h3><br />
<div><br />
<p>In parallel to sequencing the genome, we began building a large toolbox for <em>G. xylinus genetic engineering</em>. Only a few vectors have been developed for <em>G. xylinus</em>, and most of them were not available for us for various reasons (including patents, materials-transfer agreements, and retired or deceased researchers). For this reason, we first tested out a set of new plasmids, and engineered them to be Biobrick compatible. As some <em>E. coli</em> promoters and proteins have been shown to work in <em>Gluconacetobacter</em> or <em>Acetobacter</em> species (Setyawati, 2007; Chien et al., 2006), and similarly to <em>E.coli</em>, <em>G. xylinus</em> is a Gram- species, we decided to test out and modify already existing Biobricks in the parts registry in <em>G. xylinus</em>. Therefore, the toolkit consists of:</p><br />
<br />
<br />
<br />
<ul><br />
<li>New, Biobrick compatible shuttle vectors for <em>Gluconacetobacter</em> and <em>E. coli</em><br />
<ul><br />
<br />
<li><em>pSEVA321</em><br />
</li><br />
<li><em>pSEVA331</em><br />
</li><br />
<li><em>pSEVA351</em><br />
</li><br />
<li><em>pBAV1K</em><br />
</li><br />
<br />
</ul><br />
</li>f<br />
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<li>Characterization and optimization of parts from the Registry, including:<br />
<ul><br />
<li>Fluorescent proteins, GFP, RFP, YFP, CFP<br />
</li><br />
<li>Chromoproteins</li><br />
<li>Vitreoscilla haemoglobin<br />
</li><br />
<li>The entire Anderson promoter library<br />
</li><br />
<li>Widely used gene expression control proteins AraC and TetR.<br />
</li><br />
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<br />
</ul><br />
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</li><br />
</ul><br />
</div><br />
<h3>Engineering a high cellulose-producing ATCC 53582 strain</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
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</section><br />
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<section id="results"><br />
<h2>Results</h2><br />
<p>We have isolated a new strain of Acetobacter from kombucha, and determined its growth rate, cellulose productivity, and natural antibiotic resistance. We have also developed a set of methods for genetic engineering of this strain and have determined five new plasmids capable of replication in this strain. Thus we are the first to genetically engineer it.</p><br />
<p>We have discovered four new plasmids capable of replication in Acetobacter species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K - both in G.xylinus ATCC 53582 strain, and in the strain we isolated from Kombucha. We have biobricked these plasmids, with the exception of pBAV1K, which had been originally engineered to be biobrick compatible (however, pBAV1K can not be uploaded to parts registry due to binding materials-transfer agreements).<br />
</p><br />
</section><br />
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<h2>References</h2><br />
<ul><br />
<br />
<li>[1] https://www.researchgate.net/ publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2014-10-18T03:21:46Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
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<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
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<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
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<li><a data-scroll href="#engineering">Engineering</a><br />
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<li><a data-scroll href="#results">Results</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
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<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus KI</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and KI, creating a genetic toolbox of consisting of five new plasmid backbones and around 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
</div><br />
<div class="pure-u-1-2"><br />
<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em></li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem</li> <br />
<br />
<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em></li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a powerful platform for the synthesis of new cellulose-based biomaterials and water filters</li><br />
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<section id="introduction"><br />
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<figure class="content-image image-full"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
</figure><br />
<h2>Introduction</h2><br />
<br />
<br />
<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
</p><br />
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</section><br />
<section id="aim"><br />
<h2>Aims</h2><br />
<br />
<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and Kombucha-isolated strain and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
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</section><br />
<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
</figure><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.<br />
<br />
In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. We are currently in the process of completing the bioinformatics analyses of the genomes, and will publish the full genome sequences soon.<br />
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</p><br />
</section><br />
<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and KI strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and KI strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus KI strains to increase cellulose productivity and decrease its cost. In total, by cloning widely used parts into our <em>G. xylinus</em> shuttle vectors we created a toolkit consisting of:<br />
<ul><br />
<li> 5 new shuttle vectors </li> <br />
<li> 15 Anderson promoters </li><br />
<li> Fluorescent proteins RFP and sfGFP </li><br />
<li> 5 chromoproteins </li><br />
<li> 11 CBD fusion proteins </li> <br />
<li> Vitreoscilla hemoglobin gene </li> <br />
<li> TetR and Arabinose inducible promoters </li><br />
</ul><br />
</p><br />
<p> Because the toolkit is in the non-standard pSEVA331-Bb backbone, it can not be housed in the Parts Registry, however in order to make it accessible for the synthetic biology community, we have made it freely available on request.<br />
</p><br />
<div class="accordion"><br />
<h3>Characterization of G.xylinus ATCC 53582 and KI strains</h3><br />
<div><br />
<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
</p><br />
<p> Cellulose productivity of G.xylinus ATCC 53582 and KI on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the KI strain is approximately one fifth of that of ATCC 53582 strain (see Figure X for comparison of ATCC53582 and KI strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for KI cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus KI). <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 3. Cellulose production of G.xylinus ATCC 53582 and KI strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
</figure><br />
</p> <br />
<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure Y.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, KI strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 2 for comparison of ATCC and KI strains and details of he experiment). This could be due to the niche KI strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 4. Cellulose productivity of G.xylinum ATCC 53582 and KI strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
</figure><br />
</p> <br />
<p> Natural antibiotics resistance of ATCC 53582 and KI strains </p><br />
<p></p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 5. Natural antibiotics resistance of G.xylinus ATCC 53582 and KI strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and KI seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
</figure><br />
<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 5 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and KI strain). However, when using high cell densities for plating (after transformations), antibiotic -resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus KI is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the KI strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
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</div><br />
<h3>Plasmid backbones</h3><br />
<div><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 1. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus KI after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
</figure><br />
<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and Ki strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus KI and ATCC 53583 (See figure X). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
</ul><br />
</div><br />
<h3>Anderson promoters</h3><br />
<div><br />
<p> We inserted 15 Anderson promoter-RFP coding devices into pSEVA331-Bb backbone, transformed them into G.xylinus igem strain in order to determine their relative strengths in Gluconacetobacter (see Figure Y for some of them: <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/1/1a/IC14_Composite_image_correct.jpg"><br />
<figcaption>Figure Z. Different Anderson promoter-RFP constructs expressed in G.xylinus. Left- wild-type negative control. Right- G.xylinus igem transformed with pSEVA331Bb-Anderson promoter constructs. Plates grown at 30C for 7 days before imageing under blue light. </figcaption><br />
</figure><br />
</p><br />
</div><br />
<br />
<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p><i>G. xylinus</i> is obligatively aerobic because of which cellulose production is reduced below the medium surface in static culture[1] due to reduced oxygen concentrations, reducing the overall volume of the pellicle that is biologically active. Transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i> (another obligate aerobe that expresses this protein in oxygen-poor environments) has been shown to result in increased the metabolic activity and cellulose production of G.xylinus [2].<br />
Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
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<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure Z. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem wild type cells and cells transformed with pSEVA331-BBa_K1321200 plasmid were cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days, after which OD600 was measured. Samples were diluted 1:1 in HS-cellulase medium before measurement. Negative controls (HS-cellulase without inoculations) showed no growth. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
</figure><br />
<br />
<p>We cloned the Vhb gene behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS (part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a>) in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure Z) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
<br />
<p>We also made a hypothesis of the metabolic pathway involving the interaction of vHb, which could potentially explain its effect on enhancing bacterial cellulose yield. As described in the diagram above, vHb plays a role as oxygen storage: it will actively capture oxygen molecules when when the oxygen concentration is high in the cell whereas release oxygen molecules when it is low. As such, the presence of vHb will smooth out the oxygen level within the cell and therefore provides it with a relatively constant oxygen supply. Considering the fact that oxygen also bind to protein complex cG PDE (cyclic GMP phosphodiesterase), where the binding will impose an upregulating effect on bacterial cellulose production, a constant oxygen level maintained by vHb will inevitably contribute to enhance bacterial cellulose yield.</p><br />
<br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://lh5.googleusercontent.com/OkLBTdjMaO6PjVewhOFchlpEd3x4REDL53KYLp_sSxkD35RU4U8Gh21PJKUSfTA1GZ7ShEayn0Y=w751-h608"><br />
<figcaption>Figure Z. Effects of vHb interaction on cellular oxygen level.</figcaption><br />
</figure> <br />
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</div><br />
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</div><br />
</section><br />
<section id="engineering"><br />
<h2>Engineering</h2><br />
<br />
<div class="accordion"><br />
<h3>Sequencing the genomes of <em>G. xylinus</em> ATCC 53582 and Kombucha-isolated <em>G. xylinus</em> strains</h3><br />
<div><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of <em>G. xylinus</em> is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.</p><br />
<p>In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. <br />
We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction).<br />
We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq present at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9063619 reads with 98.71% reads identified). We completed the genome assembly with the support of Imperial Bioinformatics Support service using their in-house assembly pipeline. </p><br />
</div><br />
<h3>Creating a toolbox for Gluconacetobacter genetic engineering</h3><br />
<div><br />
<p>In parallel to sequencing the genome, we began building a large toolbox for <em>G. xylinus genetic engineering</em>. Only a few vectors have been developed for <em>G. xylinus</em>, and most of them were not available for us for various reasons (including patents, materials-transfer agreements, and retired or deceased researchers). For this reason, we first tested out a set of new plasmids, and engineered them to be Biobrick compatible. As some <em>E. coli</em> promoters and proteins have been shown to work in <em>Gluconacetobacter</em> or <em>Acetobacter</em> species (Setyawati, 2007; Chien et al., 2006), and similarly to <em>E.coli</em>, <em>G. xylinus</em> is a Gram- species, we decided to test out and modify already existing Biobricks in the parts registry in <em>G. xylinus</em>. Therefore, the toolkit consists of:</p><br />
<br />
<br />
<br />
<ul><br />
<li>New, Biobrick compatible shuttle vectors for <em>Gluconacetobacter</em> and <em>E. coli</em><br />
<ul><br />
<br />
<li><em>pSEVA321</em><br />
</li><br />
<li><em>pSEVA331</em><br />
</li><br />
<li><em>pSEVA351</em><br />
</li><br />
<li><em>pBAV1K</em><br />
</li><br />
<br />
</ul><br />
</li>f<br />
<br />
<li>Characterization and optimization of parts from the Registry, including:<br />
<ul><br />
<li>Fluorescent proteins, GFP, RFP, YFP, CFP<br />
</li><br />
<li>Chromoproteins</li><br />
<li>Vitreoscilla haemoglobin<br />
</li><br />
<li>The entire Anderson promoter library<br />
</li><br />
<li>Widely used gene expression control proteins AraC and TetR.<br />
</li><br />
<br />
<br />
</ul><br />
<br />
</li><br />
</ul><br />
</div><br />
<h3>Engineering a high cellulose-producing ATCC 53582 strain</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
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</div><br />
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</div><br />
</section><br />
<br />
<br />
<section id="results"><br />
<h2>Results</h2><br />
<p>We have isolated a new strain of Acetobacter from kombucha, and determined its growth rate, cellulose productivity, and natural antibiotic resistance. We have also developed a set of methods for genetic engineering of this strain and have determined five new plasmids capable of replication in this strain. Thus we are the first to genetically engineer it.</p><br />
<p>We have discovered four new plasmids capable of replication in Acetobacter species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K - both in G.xylinus ATCC 53582 strain, and in the strain we isolated from Kombucha. We have biobricked these plasmids, with the exception of pBAV1K, which had been originally engineered to be biobrick compatible (however, pBAV1K can not be uploaded to parts registry due to binding materials-transfer agreements).<br />
</p><br />
</section><br />
<br />
<section id="references"><br />
<h2>References</h2><br />
<ul><br />
<br />
<li>[1] https://www.researchgate.net/ publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/File:IC14_Composite_image_correct.jpgFile:IC14 Composite image correct.jpg2014-10-18T03:19:07Z<p>Mf1512: </p>
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<div></div>Mf1512http://2014.igem.org/File:IC14-MechTest-6.pngFile:IC14-MechTest-6.png2014-10-18T02:52:06Z<p>Mf1512: uploaded a new version of &quot;File:IC14-MechTest-6.png&quot;: Added blended to title</p>
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<div></div>Mf1512http://2014.igem.org/Team:Imperial/Mechanical_TestingTeam:Imperial/Mechanical Testing2014-10-18T02:47:41Z<p>Mf1512: </p>
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<h1>Mechanical Testing</h1><br />
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<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
</li><br />
<li><a data-scroll href="#methods">Methods</a><br />
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<li><a data-scroll href="#results">Results</a><br />
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<li><a data-scroll href="#discussion">Discussion</a><br />
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<li><a data-scroll href="#limitations">Limitations</a><br />
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<li><a data-scroll href="#manufacturing">Manufacturing Relevance</a><br />
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<li><a data-scroll href="#appendix">Appendix</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
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<h2>Overview</h2><br />
<p>To check the feasibility of using our bacterial cellulose as a customisable ultrafiltration membrane, the mechanical properties were tested. For this to work, we checked the tensile strength of our filter, calculated the shear stress and estimated the processing method that produces membrane with the longest life-time. Our ultimate tensile stress calculated matched with literature values of bacterial cellulose within standard deviation, and the data analysis provided the insight to use non-blended cellulose rather than blended..</p><br />
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<div class="pure-u-1-2"><br />
<h2>Key Achievements </h2><br />
<ul><br />
<li>Introduced a new aspect to the manufacturing track in terms of quantitatively characterising the mechanical properties of our product, bacterial cellulose<br />
</li><br />
<li>Quantified the tensile stress-strain properties of our bacterial cellulose.<br />
</li><br />
<li>Discovered that our bacterial cellulose can sustain an order of magnitude higher water pressures than those typically used for ultrafiltration membranes.<br />
</li><br />
<li>Established that non-blended compared to blended bacterial cellulose was significantly more ductile, and would have longer life time as a water filter, which informed the choice of cellulose for the final product.<br />
</li><br />
<li>Tested and analysed 20 samples of bacterial cellulose test pieces.<br />
</li><br />
<li>Created a size and quality optimised gif for visualisation of the experiment and written a protocol for future iGEM teams to do the same.<br />
</li><br />
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<section id="introduction"><br />
<h2>Introduction</h2><br />
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<img class="content-image image-full" src="https://static.igem.org/mediawiki/2014/6/60/IC14-MechTest-CelluloseTest.jpg"><br />
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<img class="content-image" src="https://static.igem.org/mediawiki/2014/e/e3/IC14-MechTest-CBIS1.jpg" width=150><br />
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<img class="content-image" src="https://static.igem.org/mediawiki/2014/f/f7/IC14-MechTest-CBIS2.jpg" width=100><br />
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<br/><br />
<p><em>“How long would the bacterial cellulose water filter last? What will be the pressure we can apply on to it?”</em> - Dr. Chipps (Principal Research Scientist at Thames Water)</p><br />
<p>The most accessible method of finding an answer to Dr. Chipps’ questions is to asses the quality of our material by pulling a test piece until it breaks, a tensile test. The quality of our bacterial cellulose will determine the water pressure that can be applied to it in when used as a customisable ultrafiltration membrane. Since the water pressure is a major limiting factor of the flow rates produced in such a setting, a key step to establishing the feasibility of our project is to perform a tensile test.</p><br />
<p>Tensile testing of bacterial cellulose is a common way of characterising and comparing its mechanical properties (Khesk 2006, Cheng 2009, Shezad 2010) and provides an indication of the orientation of the fibres in the bacterial cellulose pellicle. Another advantage is that for a polycrystalline material like bacterial cellulose grown in static media, the shear stress, directly related to hydrostatic water pressure, can be interpolated based on Von Mises Criterion.</p><br />
<p>Therefore, we contacted Dr. Angelo Karunaratne of the Royal British Legion Centre of Blast Injury Studies at Imperial College, requesting access to test samples in an Instron 5866 materials testing machine (Instron Inc., Norwood, USA). For this test our aim was to calculate the young’s modulus, ultimate tensile stress, yield stress and strain at failure.</p><br />
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</section><br />
</div><br />
<div class="pure-u-1-1"><br />
<section id="methods"><br />
<h2>Methods</h2><br />
<br />
<br />
<br />
<br />
<br />
<br />
<figure class="content-image image-small"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/6/60/IC14-MechTest-Instron.jpg"><br />
<figcaption>Figure 1: Instron 5866 materials testing machine</figcaption><br />
</figure><br />
<br />
<figure class="content-image image-right image-small"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/9/92/IC14-MechTest-Test2b.jpg"><br />
<figcaption>Figure 2b: Actual test state: tensile testing state of thin bacterial cellulose pellicle<br />
</figcaption><br />
</figure><br />
<br />
<figure class="content-image image-right image-small"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/0/03/IC14-MechTest-Test2a.jpg"><br />
<figcaption>Figure 2a: Dumbbell shape used as the shape for testing</figcaption><br />
<br />
</figure><br />
<br />
<br />
<p>Samples were prepared by oven drying at 60 C for 6 hours between VWR 413 filter paper, without pressure. Subsequently, samples were kept at room temperature (20 C) before testing. Cellulose was cut into 0.1 mm x 11 mm x 32 mm dumbbell shapes as shown in figure 2 a, following the shape recommended by ISO 527-3 guidelines for determination of tensile properties of plastics. Dumbbell shapes have the advantage of avoiding stress concentration at the clamps, and the dimensions chosen had sufficiently similar small width to large width ratio to literature dimensions (0.16mm × 100mm × 150mm) to allow for comparison across studies (Sherif 2006). A digital vernier calliper was used to measure the thickness of each of the samples. 9 samples were used for the main cellulose fabric intended for water filtration, 2 were used for blended cellulose samples due to limited availability.</p><br />
<br />
<figure style="float:left;margin:10px;"><br />
<img src="https://static.igem.org/mediawiki/2014/5/53/IC14-BC_tensile_test.gif" width="200"><br />
<figcaption>Figure 3: Cellulose failing under stress</figcaption><br />
</figure><br />
<p>The test involves clamping test pieces into the instron machine, moving the upper clamp upwards at a specific elongation time, set to 20 mm/min to match that of existing literature (Sherif, 2006) and tracking the tensile force of the BC sample until the sample fails. Stress() was calculated as the tensile force divided by the cross sectional area measured as width x thickness. Young’s modulus (E) was calculated as stress/strain in the linear section of the average graph. Strain () was calculated as L/L0where L is the elongation from the initial length L0. Samples were tested until visually confirming failure as shown in figure 3.</p><br />
<br />
<br />
<p>Two main sample types were tested. The BC pellicles intended for water filtration were immersed in a solution of Wizz Active Oxygen Fabric Stain Remover dissolved in distilled water. After 4 days, the pellicles were removed from the container, cut into squares of 5cm by 5cm, and allowed to dry between VWR 413 filter paper in an oven at 60 ⁰C for 3 hours. Then, the BC layered in filter paper was moved to room temperature, folded in Blue Roll, clamped between heatproof mats with mechanical clamps and left for 24 hours. This type of cellulose will be referred to as non-blended BC.</p><br />
<p>Alternative BC samples were treated in a 0.1 M NaOH solution in 80C for 3 hours, upon which it was dried off with blue roll and immersed in distilled water. After being in the distilled water solution for 12 hours, the BC was dried of with blue roll and blended in a Jamie Oliver food processor for 12 min at speed 2.</p><br />
<figure class="content-image image-half "><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/2e/IC14-MechTest-ActiveOxygen.jpg"><br />
<figcaption>Figure 4: Active Oxygen Fabric Stain Remover treated BC for 4 days</figcaption><br />
</figure><br />
<br />
<figure class="content-image image-half "><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/d/d2/IC14-MechTest-Blended.png"><br />
<figcaption>Figure 5: a) 0.1 M NaOH treated BC, blended and spread onto VWR 413 filter paper to dry b) Phillips Jamie Oliver Blender that produced the BC of part a.</figcaption><br />
</figure><br />
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</section><br />
</div><br />
<div class="pure-u-1-1"><br />
<section id="results"><br />
<h2>Results</h2><br />
<figure class="content-image"><br />
<table class="tg image-full"><br />
<tr><br />
<th class="tg-e3zv">Sample processing</th><br />
<th class="tg-e3zv">Ultimate tensile stress (MPa)</th><br />
<th class="tg-e3zv">Yield stress (Mpa)</th><br />
<th class="tg-e3zv">Young's modulus (Mpa)</th><br />
<th class="tg-e3zv">Strain at break (%)</th><br />
</tr><br />
<tr><br />
<td class="tg-e3zv">Non-blended bacterial cellulose</td><br />
<td class="tg-031e">29.9 ± 6.4</td><br />
<td class="tg-031e">25.2 ± 7.2</td><br />
<td class="tg-031e">1.5 ± 0.1</td><br />
<td class="tg-031e">21 ± 3</td><br />
</tr><br />
<tr><br />
<td class="tg-e3zv">Blended bacterial cellulose</td><br />
<td class="tg-031e">208 ± 80</td><br />
<td class="tg-031e">155 ± 11</td><br />
<td class="tg-031e">50.5 ± 1</td><br />
<td class="tg-031e">4.1 ± 0.9</td><br />
</tr><br />
</table><br />
<figcaption>Table 1</figcaption><br />
</figure><br />
<div class="pure-g"><br />
<div class="pure-u-1-2"><br />
<figure class="content-image image-full "><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/1/15/IC14-MechTest-6.png"><br />
<figcaption>Figure 6: Tensile Stress-Strain Graph of Bacterial Cellulose</figcaption><br />
</figure><br />
</div><br />
<div class="pure-u-1-2"><br />
<figure class="content-image image-full "><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4b/IC14-MechTest-7.png"><br />
<figcaption>Figure 7: Average Tensile Stress-Strain Graph of Bacterial Cellulose</figcaption><br />
</figure><br />
</div><br />
<div class="pure-u-1-2"><br />
<figure class="content-image image-full "><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/9/9b/IC14-MechTest-8.png"><br />
<figcaption>Figure 8: Tensile Stress-Strain Graph of Blended Bacterial Cellulose</figcaption><br />
</figure><br />
</div><br />
<div class="pure-u-1-2"><br />
<figure class="content-image image-full "><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/0/03/IC14-MechTest-9.png"><br />
<figcaption>Figure 9: Tensile Stress Average of Blended Cellulose</figcaption><br />
</figure><br />
</div><br />
<div class="pure-u-1-2"><br />
<figure class="content-image image-full "><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/e/eb/IC14-MechTest-9.5.png"><br />
<figcaption>Figure 10: Mechanical Properties of Blended vs. Non-Blended Cellulose</figcaption><br />
</figure><br />
</div><br />
<div class="pure-u-1-2"><br />
<figure class="content-image image-full "><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/56/IC14-MechTest-10.png"><br />
<figcaption>Figure 11: Average Tensile Stress of Blended and Non-Blended Cellulose</figcaption><br />
</figure><br />
</div><br />
<div class="pure-u-1-1"><br />
<p>The results of the mechanical tests of blended BC and non-blended BC are shown in table 1. The corresponding measurement data are shown in figure 3 and 4. As shown in table 1, the E, and of non-blended BC was 1.52 MPa, 30 MPa and 35%, respectively. 9 samples were tested to account for variability in cellulose samples produced as graphed in figure 5. The tensile stress is equal within standard deviation to that of 34 MPa found by (Chen 2009), indicating literature levels of tensile strength of the material produced.</p><br />
<p>As shown in table 1, the yield stress of blended cellulose is 178 MPa higher than that of non-blended cellulose whilst the strain at break was 4% for blended compared to 21% for non-blended cellulose. This indicates that blended cellulose is more brittle than non-blended, making non-blended cellulose a more promising candidate for water filtration. This is because brittle materials are more prone to fatigue-crack propagation (Ritchie 1998) when exposed to cyclic loading characteristic to the use of ultrafiltration membranes, giving brittle materials a shorter expected life time.</p><br />
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</div><br />
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</section><br />
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<div class="pure-u-1-1"><br />
<section id="discussion"><br />
<h2>Discussion</h2><br />
<p>What is the maximum water pressure that can be applied?</p><br />
<p>For the cellulose tested with an ultimate tensile stress of 30 MPa and a yield stress of 27 MPa, it is possible to estimate the maximum pressure that our bacterial cellulose filter can sustain by calculating the maximum shear stress (τ). Based on the work of (Jajuee 2006) on membrane filtration, it can be assume that shear stress is the most considerable force acting on the customised cellulose membrane. Combining this with the assumption that non-blended bacterial cellulose is ductile due to being polycrystalline (Tischer 2010), as can be seen in the gif of figure 3 and by the large plastic region shown in figure 7, the Von Mises Criterion can be used to calculate the shear stress. This relates shear stress to yield stress by: σ=√3 τ.Hence, the maximum shear stress is 15.5 MPa. Now, a safety factor of 2 would ensure safe usage of the cellulose membrane, thus a shear stress of half the theoretical maximum shear stress i.e. 7.5 MPa is the advisable working shear stress. This correlates to a hydrostatic pressure of 75 bar, which more than suffices for the pressure ranges of 0.3-3 bar used in ultrafiltration membranes (Reclamation Fact Sheet)</p><br />
</section><br />
<br />
<section id="limitations"><br />
<h2>Limitations</h2><br />
<p>In addition to tensile testing, it would be ideal to perform a fatigue stress test as a material like a water filter loaded over many cycles is more likely to fail due to fatigue than tensile loading, which expectedly occur at lower rates than the ultimate tensile stress predicted. To accommodate for this limitation, the cellulose we have produced is being tested by Professor Alexander Bismarck’s group at University of Vienna, which will also provide measurements of its permeability i.e. flow rates at different water pressures. These results are on their way as we speak.</p><br />
</section><br />
<section id="manufacturing"><br />
<h2>Relevance to Manufacturing track</h2><br />
<p>To the best of our knowledge, characterising the mechanical properties of the various products produced in the iGEM Manufacturing Track has been rather overlooked by previous teams, despite these being key determinants of the feasibility of a product such as biomaterials, glue or polymers for real life implementation. Based on previous year’s lack of tensile test or other more advanced material property tests, we hypothesise that this may be due to limited time and lack of templates for data processing. To accommodate for this lack, we have included a template for analysis of tensile stress-strain data in the appendix, which should reduce the time spent on such endeavours and encourage others to explore the quality of their product.</p><br />
</section><br />
<section id="appendix"><br />
<h2>Appendix</h2><br />
<p>Raw and processed data included for future iGEM teams to use as template for data processing: <a href="https://static.igem.org/mediawiki/2014/8/82/IC14-Mechanical_Properties_Tensile.xls">IC14-Mechanical_Properties_Tensile.xls</a><br />
</p><br />
</section><br />
<br />
<br />
<section id="references"><br />
<h2>References</h2><br />
<ul><br />
<li>Anon, ISO 527-3 official guidelines. Available at: http://211.67.52.20:8088/xitong/BZ\9524171.pdf [Accessed October 12, 2014].</li><br />
<li>Anon, Von Mises Stress. Available at: http://www.continuummechanics.org/cm/vonmisesstress.html [Accessed October 12, 2014].</li><br />
<li>Cheng, K.-C., Catchmark, J.M. & Demirci, A., 2009. Effect of different additives on bacterial cellulose production by Acetobacter xylinum and analysis of material property. Cellulose, 16(6), pp.1033–1045. Available at: http://www.scopus.com/inward/record.url?eid=2-s2.0-72849151190&partnerID=tZOtx3y1 [Accessed September 12, 2014].</li><br />
<li>Con-Serv Water, Operation and Maintenance Manual. Available at: [http://www.con-servwater.com/uploads/CON-SERV_6_GPM_UF_SYSTEM_OPERATING_MANUAL_7-28-11_Rev.pdf] [Accessed October 12, 2014].</li><br />
<li>Jajuee, B. et al., 2006. Measurements and CFD simulations of gas holdup and liquid velocity in novel airlift membrane contactor. AIChE Journal, 52(12), pp.4079–4089. Available at: http://doi.wiley.com/10.1002/aic.11010 [Accessed October 16, 2014].</li><br />
<li>Keshk, S., 2006. Physical properties of bacterial cellulose sheets produced in presence of lignosulfonate. Enzyme and Microbial Technology, 40(1), pp.9–12. Available at: http://www.sciencedirect.com/science/article/pii/S0141022906004078 [Accessed October 13, 2014].</li><br />
<li>Shezad, O. et al., 2010. Physicochemical and mechanical characterization of bacterial cellulose produced with an excellent productivity in static conditions using a simple fed-batch cultivation strategy. Carbohydrate Polymers, 82(1), pp.173–180. Available at: http://www.sciencedirect.com/science/article/pii/S0144861710003334 [Accessed October 12, 2014].</li><br />
<li>Tischer, P.C.S.F. et al., 2010. Nanostructural reorganization of bacterial cellulose by ultrasonic treatment. Biomacromolecules, 11(5), pp.1217–24. Available at: http://dx.doi.org/10.1021/bm901383a [Accessed October 17, 2014].</li><br />
<br />
<br />
<br />
<br />
<br />
</ul><br />
</section><br />
</div><br />
</div><br />
</body><br />
</html><br />
{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/ImplementationTeam:Imperial/Implementation2014-10-18T02:39:08Z<p>Mf1512: </p>
<hr />
<div>{{:Team:Imperial/Templates:header}}<br />
<html><br />
<body><br />
<div class="content-wrapper"><br />
<br />
<div class="pure-g"><br />
<br />
<div class="pure-u-1-1 main"><br />
<h1>Implementation</h1><br />
<div id="navWrap"><br />
<div id="subNav"><br />
<ul><br />
<li><a data-scroll href="#Ultrafiltration">Ultrafiltration</a><br />
</li><br />
<li><a data-scroll href="#Aqualose">Aqualose</a><br />
</li><br />
<li><a data-scroll href="#Future Functionalisation">Future Functionalisation</a><br />
</li><br />
<li><a data-scroll href="#Process Engineering">Process Engineering</a><br />
</li><br />
<li><a data-scroll href="#References">References</a><br />
</li><br />
<br />
</ul><br />
<br class="clearLeft" /><br />
</div><br />
</div><br />
</div><br />
</div><br />
<section id="overview"><br />
<div class="pure-g"><br />
<div class="pure-u-1-2"><br />
<h2> </h2><br />
<p><em><strong>“To meet new environmental regulations, a step change is required in how we process water. Functionalised cellulose filtration could be a viable part of the solution.”</strong></em> - Dr. Michael Chipps, Principal Research Scientist at Thames Water<br />
</p><br />
</div><br />
<div class="pure-u-1-2"><br />
<h2>At a Glance</h2><br />
<ul><br />
<li>Ultrafiltration has many advantages for <a href="https://2014.igem.org/Team:Imperial/Water_Report#wastewate"> wastewater recycling </a></li><br />
<li>Limitations in current ultrafiltration technologies can be addressed by functionalised membranes for specific contaminant binding</li><br />
<li>The modularity underlying membrane fuctionalisation allows future targeting of any current or emerging contaminant</li><br />
<li>Our aqualose water treatment process offers a cost effective alternative to traditional ultrafiltration</li><br />
<li>The process is adaptable functioning equally as a bolt on to existing treatment facilities or as a standalone purification solution</li><br />
<br />
<br />
</ul><br />
</div><br />
<br />
<br />
</div><br />
<br />
</section><br />
<div class="pure-g"><br />
<div class="pure-u-1-1"><br />
<section id="Introduction"><br />
<h2>Ultrafiltration</h2><br />
<figure class="content-image image-right"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/47/IC-14_Water_Purification_Spectrum1.JPG"><br />
<figcaption>Figure 1: Size exclusion for different grades of filter (from http://www.edstrom.com/)</figcaption><br />
</figure> <br />
<p><br />
Ultrafiltration (UF) membranes have a pore size of 0.1 to 0.01um (10 to 100nm) and are capable of removing particulates, bacteria and viruses. Microbial cellulose sheets naturally have pore sizes in this range (Gatenholm, P., & Klemm, D. (2010), Mautner et al 2014). Current ultrafiltration cannot remove small molecule contaminants such as pesticides and heavy metals however. Whilst nanofiltration and reverse osmosis membranes can exclude these small molecules they are expensive and energy intensive to use. Flow rates are low, they require very high pressures and the input water must be already purified by primary and secondary processes to avoid damaging the membranes.</p><br />
<br />
<br />
<p>Depending on input water quality UF systems may replace or complement existing secondary (coagulation, flocculation, sedimentation) and tertiary filtration (sand filtration and chlorination) systems in water treatment plants. Pretreatment of feed water is usually required to prevent reduce damage to the membrane units though ultrafiltration may be used in standalone systems for isolated regions. UF processes have the following advantages over traditional treatment methods:<br />
</p><br />
<p><br />
<ul><br />
<li><br />
Chemical free (aside from cleaning)</li><br />
<li><br />
Constant output quality regardless of feed quality (excluding small molecule contaminants, changes in input water quality affect only the life of the membrane, not the quality of the flow through)</li><br />
<li><br />
Compact plant size, efficient for small scale, <a href="https://2014.igem.org/Team:Imperial/Water_Report#decentralisation"> decentralised</a> purification</li><br />
<li><br />
High quality of output water particularly with regards to pathogen removal</li><br />
</ul><br />
</p><br />
<p><br />
UF processes are currently limited by the high cost of membranes, inevitable membrane fouling means they must be regularly replaced. There use is also restricted by limitations in removal of small molecule contaminants. They can only be employed where feed water is free of these contaminants or in tandem with other (often slow or energy intensive) treatment methods for removing them. </p><br />
</section><br />
<section id="Aqualose"><br />
<h2>Aqualose</h2><br />
<br />
<table class="tg image-full"><br />
<tr><br />
<th class="tg-e3zv">Component</th><br />
<th class="tg-e3zv">Quantity</th><br />
<th class="tg-e3zv">Source</th><br />
<th class="tg-e3zv">Cost breakdown (£)</th><br />
<th class="tg-e3zv">Cost (£)</th><br />
</tr><br />
<tr><br />
<td class="tg-031e">Water</td><br />
<td class="tg-031e">4l</td><br />
<td class="tg-031e">London South West Water</td><br />
<td class="tg-031e">4 liters of £5.5195 per m3</td><br />
<td class="tg-031e">0.02</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">400 g granulated sugar</td><br />
<td class="tg-031e">400g</td><br />
<td class="tg-031e">Tesco's</td><br />
<td class="tg-031e">79p per 1 kg</td><br />
<td class="tg-031e">0.32</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Clipper green tea tea bags</td><br />
<td class="tg-031e">4</td><br />
<td class="tg-031e">Clipper tea</td><br />
<td class="tg-031e">300 teabags for £9.99</td><br />
<td class="tg-031e">0.13</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Aspall organic cider vinegar</td><br />
<td class="tg-031e">2</td><br />
<td class="tg-031e">Aspall Suffolk</td><br />
<td class="tg-031e">400 ml of a £2.25 500 ml bottle</td><br />
<td class="tg-031e">1.80</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Total</td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv">2.27</td><br />
</tr><br />
<tr><br />
<td class="tg-e3zv">Product</td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
</tr><br />
<tr><br />
<td class="tg-e3zv">Component</td><br />
<td class="tg-e3zv">Quantity</td><br />
<td class="tg-e3zv">Source</td><br />
<td class="tg-e3zv">Price breakdown (£)</td><br />
<td class="tg-e3zv">Price per g (£)</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Bacterial cellulose yield</td><br />
<td class="tg-031e">60 cm by 40 cm = 0.24 m2</td><br />
<td class="tg-031e">production from single tray</td><br />
<td class="tg-031e">110 g/m2 x 0.24 m2 = 26.4g</td><br />
<td class="tg-e3zv">0.09</td><br />
</tr><br />
</table><br />
<figcaption>Figure 2: Cost analysis of Aqualose </figcaption><br />
<br />
<p>Microbial cellulose membranes are considerably cheaper than competitors. Whilst Polyvinylidene fluoride (PVDF) ultrafiltration membranes typically sell to the wastewater industry at upwards of $100 /m<sup>2</sup> (Shenzhen Youber Technology Co., Ltd. no date). Current bacterial cellulose production for nata de coco costs around $1.6 /gram (Lee et al. 2014) and our cellulose with <em>G. xylinus</em> igem cost only £0.09 ($0.14) /g. Our thin cellulose membranes for filtration weighed around 20 g/m<sup>2</sup> so would cost just $2.80 /m<sup>2</sup>. Whilst <a href="https://2014.igem.org/Team:Imperial/Mechanical_Testing"> Our studies</a> and existing literature (Fifield 2012) suggest bacterial cellulose is a strong and durable material however we would expect a shorter membrane life than the highly durable PVDF. Being considerably cheaper and easier to dispose of however, cellulose membranes could be more regularly replaced and still provide a more cost effective solution. This would also save on chemical washes regularly required to sustain the extended life of PVDF membranes. <br />
</p><br />
</section><br />
<section id="Future Functionalisation"><br />
<h2>Future Functionalisation</h2><br />
<p>Our system for functionalising cellulose membranes is inherently modular. Proteins to bind or degrade a limitless range of contaminants can be bound to cellulose membranes. Filtration can then be adapted to the particular contaminants, altering seasonally, geographically and adapting over time to emerging threats. Metaldehyde contamination in the English home counties (Mathiesen 2013) could be addressed with specific degradation enzymes and microcystin could be removed from supplies in Ohio contaminated from algal blooms (Queally 2014). <br />
</p><br />
</section><br />
<section id="Process Engineering"><br />
<h2>Process Engineering</h2><br />
<figure class="content-image image-right"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/3f/IC-14_Membrane_backwash.gif"><br />
<figcaption>Figure 3: Schematic for dead end membrane filtration with backwash (from www.meco.com)</figcaption><br />
</figure><br />
<p><br />
We envisage functionalised bacterial cellulose membranes utilised in a plate and frame, dead end filtration setup for easy backflush cleaning and replacement when fouled.<br />
<br />
<br />
This process is particularly applicable for water recycling. Ultrafiltration combines well with activated sludge for purification when organic solids content is high and the additional membrane functionalisation can target removal of recalcitrant small molecules that can accumulate in recycled water. Current wastewater treatment plants could be easily retrofitted with the technology with dedicated facilities constructed in the longer term. </p><br />
<br />
<h3>Disposal of Contaminants</h3><br />
<p>Heavy metal contaminants pose a serious problem for all water processing solutions as they ultimately cannot be destroyed. To prevent escape into the environment they must be recovered and disposed of into controlled landfill (European Commission 2002). Trapping contaminants on filtration membranes concentrates them for easy recovery. To prolong filter life, contaminants could be regularly eluted and collected. When membranes are spent they could either be disposed of directly into controlled landfill or to reduce volume - incinerated and the flue scrubbed. Incineration is the most effective solution to destroy recalcitrant organic pollutants and unlike for other materials, incineration of cellulose membranes produces no harmful by products.</p><br />
<br />
</p><br />
</br><br />
<br />
</section><br />
<div class="pure-u-1-1"><br />
<section id="References"><br />
<h2>References</h2><br />
<p>European Commission (2002) “Heavy Metals in Waste - Final Report” Accessed 17th October 2014 http://ec.europa.eu/environment/waste/studies/pdf/heavy_metalsreport.pdf<br />
<br />
Fifield, Leonard, “Bacterial Cellulose Composites Opportunities and Challenges” PNNL Applied Materials Science Group (2012). Accessed 17th October 2014<br />
http://www1.eere.energy.gov/manufacturing/rd/pdfs/sustainable_nanomaterials_workshop_pnnl.pdf<br />
<br />
<br />
Gatenholm, P., & Klemm, D. (2010). Bacterial nanocellulose as a renewable material for biomedical applications. MRS bulletin, 35(03), 208-213.<br />
<br />
James Queally “Toledo's tap water undrinkable for a second day; test results delayed” The LA times 3 August 2014. Accessed 17th October 2014<br />
http://www.latimes.com/nation/nationnow/la-na-nn-toledo-ohio-toxins-water-20140802-story.html#page=1<br />
Karl Mathiesen “Slug poison found in one in eight of England's drinking water sources” the Guardian, Wednesday 10 July 2013. Accessed 17th October 2014<br />
http://www.theguardian.com/environment/2013/jul/10/slug-poison-drinking-water-metaldehyde<br />
<br />
<br />
Lee, Koon‐Yang, Gizem Buldum, Athanasios Mantalaris, and Alexander Bismarck. "More than meets the eye in bacterial cellulose: Biosynthesis, bioprocessing, and applications in advanced fiber composites." Macromolecular bioscience 14, no. 1 (2014): 10-32.<br />
<br />
Mautner, A., Lee, K. Y., Lahtinen, P., Hakalahti, M., Tammelin, T., Li, K., & Bismarck, A. (2014). Nanopapers for organic solvent nanofiltration. Chemical Communications, 50(43), 5778-5781.<br />
<br />
Shenzhen Youber Technology Co., Ltd. Accessed 17th October 2014 http://water-treatment.en.alibaba.com/<br />
<br />
Figure references:<br />
http://www.edstrom.com/assets/1/7/Water_Purification_Spectrum1.JPG<br />
http://upload.wikimedia.org/wikipedia/en/c/c0/MBRvsASP_Schematic.jpg<br />
http://www.meco.com/meco-biopharmaceutical/products/membrane-filtration</p><br />
<br />
</section><br />
</div><br />
</div><br />
<br />
</div><br />
</body><br />
</html><br />
{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/ImplementationTeam:Imperial/Implementation2014-10-18T02:37:24Z<p>Mf1512: </p>
<hr />
<div>{{:Team:Imperial/Templates:header}}<br />
<html><br />
<body><br />
<div class="content-wrapper"><br />
<br />
<div class="pure-g"><br />
<br />
<div class="pure-u-1-1 main"><br />
<h1>Implementation</h1><br />
<div id="navWrap"><br />
<div id="subNav"><br />
<ul><br />
<li><a data-scroll href="#Ultrafiltration">Ultrafiltration</a><br />
</li><br />
<li><a data-scroll href="#Aqualose">Aqualose</a><br />
</li><br />
<li><a data-scroll href="#Future Functionalisation">Future Functionalisation</a><br />
</li><br />
<li><a data-scroll href="#Process Engineering">Process Engineering</a><br />
</li><br />
<li><a data-scroll href="#References">References</a><br />
</li><br />
<br />
</ul><br />
<br class="clearLeft" /><br />
</div><br />
</div><br />
</div><br />
</div><br />
<section id="overview"><br />
<div class="pure-g"><br />
<div class="pure-u-1-2"><br />
<h2> </h2><br />
<p><em><strong>“To meet new environmental regulations, a step change is required in how we process water. Functionalised cellulose filtration could be a viable part of the solution.”</strong></em> - Dr. Michael Chipps, Principal Research Scientist at Thames Water<br />
</p><br />
</div><br />
<div class="pure-u-1-2"><br />
<h2>At a Glance</h2><br />
<ul><br />
<li>Ultrafiltration has many advantages for <a href="https://2014.igem.org/Team:Imperial/Water_Report#wastewate"> wastewater recycling </a></li><br />
<li>Limitations in current ultrafiltration technologies can be addressed by functionalised membranes for specific contaminant binding</li><br />
<li>The modularity underlying membrane fuctionalisation allows future targeting of any current or emerging contaminant</li><br />
<li>Our aqualose water treatment process offers a cost effective alternative to traditional ultrafiltration</li><br />
<li>The process is adaptable functioning equally as a bolt on to existing treatment facilities or as a standalone purification solution</li><br />
<br />
<br />
</ul><br />
</div><br />
<br />
<br />
</div><br />
<br />
</section><br />
<div class="pure-g"><br />
<div class="pure-u-1-1"><br />
<section id="Introduction"><br />
<h2>Ultrafiltration</h2><br />
<figure class="content-image image-right"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/47/IC-14_Water_Purification_Spectrum1.JPG"><br />
<figcaption>Figure 1: Size exclusion for different grades of filter (from http://www.edstrom.com/)</figcaption><br />
</figure> <br />
<p><br />
Ultrafiltration (UF) membranes have a pore size of 0.1 to 0.01um (10 to 100nm) and are capable of removing particulates, bacteria and viruses. Microbial cellulose sheets naturally have pore sizes in this range (Gatenholm, P., & Klemm, D. (2010), Mautner et al 2014). Current ultrafiltration cannot remove small molecule contaminants such as pesticides and heavy metals however. Whilst nanofiltration and reverse osmosis membranes can exclude these small molecules they are expensive and energy intensive to use. Flow rates are low, they require very high pressures and the input water must be already purified by primary and secondary processes to avoid damaging the membranes.</p><br />
<br />
<br />
<p>Depending on input water quality UF systems may replace or complement existing secondary (coagulation, flocculation, sedimentation) and tertiary filtration (sand filtration and chlorination) systems in water treatment plants. Pretreatment of feed water is usually required to prevent reduce damage to the membrane units though ultrafiltration may be used in standalone systems for isolated regions. UF processes have the following advantages over traditional treatment methods:<br />
</p><br />
<p><br />
<ul><br />
<li><br />
Chemical free (aside from cleaning)</li><br />
<li><br />
Constant output quality regardless of feed quality (excluding small molecule contaminants, changes in input water quality affect only the life of the membrane, not the quality of the flow through)</li><br />
<li><br />
Compact plant size, efficient for small scale, <a href="https://2014.igem.org/Team:Imperial/Water_Report#decentralisation"> decentralised</a> purification</li><br />
<li><br />
High quality of output water particularly with regards to pathogen removal</li><br />
</ul><br />
</p><br />
<p><br />
UF processes are currently limited by the high cost of membranes, inevitable membrane fouling means they must be regularly replaced. There use is also restricted by limitations in removal of small molecule contaminants. They can only be employed where feed water is free of these contaminants or in tandem with other (often slow or energy intensive) treatment methods for removing them. </p><br />
</section><br />
<section id="Aqualose"><br />
<h2>Aqualose</h2><br />
<br />
<table class="tg image-full"><br />
<tr><br />
<th class="tg-e3zv">Component</th><br />
<th class="tg-e3zv">Quantity</th><br />
<th class="tg-e3zv">Source</th><br />
<th class="tg-e3zv">Cost breakdown (£)</th><br />
<th class="tg-e3zv">Cost (£)</th><br />
</tr><br />
<tr><br />
<td class="tg-031e">Water</td><br />
<td class="tg-031e">4l</td><br />
<td class="tg-031e">London South West Water</td><br />
<td class="tg-031e">4 liters of £5.5195 per m3</td><br />
<td class="tg-031e">0.02</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">400 g granulated sugar</td><br />
<td class="tg-031e">400g</td><br />
<td class="tg-031e">Tesco's</td><br />
<td class="tg-031e">79p per 1 kg</td><br />
<td class="tg-031e">0.32</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Clipper green tea tea bags</td><br />
<td class="tg-031e">4</td><br />
<td class="tg-031e">Clipper tea</td><br />
<td class="tg-031e">300 teabags for £9.99</td><br />
<td class="tg-031e">0.13</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Aspall organic cider vinegar</td><br />
<td class="tg-031e">2</td><br />
<td class="tg-031e">Aspall Suffolk</td><br />
<td class="tg-031e">400 ml of a £2.25 500 ml bottle</td><br />
<td class="tg-031e">1.80</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Total</td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv"></td><br />
<td class="tg-e3zv">2.27</td><br />
</tr><br />
<tr><br />
<td class="tg-e3zv">Product</td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
<td class="tg-031e"></td><br />
</tr><br />
<tr><br />
<td class="tg-e3zv">Component</td><br />
<td class="tg-e3zv">Quantity</td><br />
<td class="tg-e3zv">Source</td><br />
<td class="tg-e3zv">Price breakdown (£)</td><br />
<td class="tg-e3zv">Price per g (£)</td><br />
</tr><br />
<tr><br />
<td class="tg-031e">Bacterial cellulose yield</td><br />
<td class="tg-031e">60 cm by 40 cm = 0.24 m2</td><br />
<td class="tg-031e">production from single tray</td><br />
<td class="tg-031e">110 g/m2 x 0.24 m2 = 26.4g</td><br />
<td class="tg-e3zv">0.09</td><br />
</tr><br />
</table><br />
<figcaption>Figure 1: Cost analysis of Aqualose </figcaption><br />
<br />
<p>Microbial cellulose membranes are considerably cheaper than competitors. Whilst Polyvinylidene fluoride (PVDF) ultrafiltration membranes typically sell to the wastewater industry at upwards of $100 /m<sup>2</sup> (Shenzhen Youber Technology Co., Ltd. no date). Current bacterial cellulose production for nata de coco costs around $1.6 /gram (Lee et al. 2014) and our cellulose with <em>G. xylinus</em> igem cost only £0.09 ($0.14) /g. Our thin cellulose membranes for filtration weighed around 20 g/m<sup>2</sup> so would cost just $2.80 /m<sup>2</sup>. Whilst <a href="https://2014.igem.org/Team:Imperial/Mechanical_Testing"> Our studies</a> and existing literature (Fifield 2012) suggest bacterial cellulose is a strong and durable material however we would expect a shorter membrane life than the highly durable PVDF. Being considerably cheaper and easier to dispose of however, cellulose membranes could be more regularly replaced and still provide a more cost effective solution. This would also save on chemical washes regularly required to sustain the extended life of PVDF membranes. <br />
</p><br />
</section><br />
<section id="Future Functionalisation"><br />
<h2>Future Functionalisation</h2><br />
<p>Our system for functionalising cellulose membranes is inherently modular. Proteins to bind or degrade a limitless range of contaminants can be bound to cellulose membranes. Filtration can then be adapted to the particular contaminants, altering seasonally, geographically and adapting over time to emerging threats. Metaldehyde contamination in the English home counties (Mathiesen 2013) could be addressed with specific degradation enzymes and microcystin could be removed from supplies in Ohio contaminated from algal blooms (Queally 2014). <br />
</p><br />
</section><br />
<section id="Process Engineering"><br />
<h2>Process Engineering</h2><br />
<figure class="content-image image-right"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/3f/IC-14_Membrane_backwash.gif"><br />
<figcaption>Figure 2: Schematic for dead end membrane filtration with backwash (from www.meco.com)</figcaption><br />
</figure><br />
<p><br />
We envisage functionalised bacterial cellulose membranes utilised in a plate and frame, dead end filtration setup for easy backflush cleaning and replacement when fouled.<br />
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This process is particularly applicable for water recycling. Ultrafiltration combines well with activated sludge for purification when organic solids content is high and the additional membrane functionalisation can target removal of recalcitrant small molecules that can accumulate in recycled water. Current wastewater treatment plants could be easily retrofitted with the technology with dedicated facilities constructed in the longer term. </p><br />
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<h3>Disposal of Contaminants</h3><br />
<p>Heavy metal contaminants pose a serious problem for all water processing solutions as they ultimately cannot be destroyed. To prevent escape into the environment they must be recovered and disposed of into controlled landfill (European Commission 2002). Trapping contaminants on filtration membranes concentrates them for easy recovery. To prolong filter life, contaminants could be regularly eluted and collected. When membranes are spent they could either be disposed of directly into controlled landfill or to reduce volume - incinerated and the flue scrubbed. Incineration is the most effective solution to destroy recalcitrant organic pollutants and unlike for other materials, incineration of cellulose membranes produces no harmful by products.</p><br />
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</p><br />
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</section><br />
<div class="pure-u-1-1"><br />
<section id="References"><br />
<h2>References</h2><br />
<p>European Commission (2002) “Heavy Metals in Waste - Final Report” Accessed 17th October 2014 http://ec.europa.eu/environment/waste/studies/pdf/heavy_metalsreport.pdf<br />
<br />
Fifield, Leonard, “Bacterial Cellulose Composites Opportunities and Challenges” PNNL Applied Materials Science Group (2012). Accessed 17th October 2014<br />
http://www1.eere.energy.gov/manufacturing/rd/pdfs/sustainable_nanomaterials_workshop_pnnl.pdf<br />
<br />
<br />
Gatenholm, P., & Klemm, D. (2010). Bacterial nanocellulose as a renewable material for biomedical applications. MRS bulletin, 35(03), 208-213.<br />
<br />
James Queally “Toledo's tap water undrinkable for a second day; test results delayed” The LA times 3 August 2014. Accessed 17th October 2014<br />
http://www.latimes.com/nation/nationnow/la-na-nn-toledo-ohio-toxins-water-20140802-story.html#page=1<br />
Karl Mathiesen “Slug poison found in one in eight of England's drinking water sources” the Guardian, Wednesday 10 July 2013. Accessed 17th October 2014<br />
http://www.theguardian.com/environment/2013/jul/10/slug-poison-drinking-water-metaldehyde<br />
<br />
<br />
Lee, Koon‐Yang, Gizem Buldum, Athanasios Mantalaris, and Alexander Bismarck. "More than meets the eye in bacterial cellulose: Biosynthesis, bioprocessing, and applications in advanced fiber composites." Macromolecular bioscience 14, no. 1 (2014): 10-32.<br />
<br />
Mautner, A., Lee, K. Y., Lahtinen, P., Hakalahti, M., Tammelin, T., Li, K., & Bismarck, A. (2014). Nanopapers for organic solvent nanofiltration. Chemical Communications, 50(43), 5778-5781.<br />
<br />
Shenzhen Youber Technology Co., Ltd. Accessed 17th October 2014 http://water-treatment.en.alibaba.com/<br />
<br />
Figure references:<br />
http://www.edstrom.com/assets/1/7/Water_Purification_Spectrum1.JPG<br />
http://upload.wikimedia.org/wikipedia/en/c/c0/MBRvsASP_Schematic.jpg<br />
http://www.meco.com/meco-biopharmaceutical/products/membrane-filtration</p><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2014-10-18T02:36:45Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
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<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
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<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
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<li><a data-scroll href="#engineering">Engineering</a><br />
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<li><a data-scroll href="#results">Results</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus KI</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and KI, creating a genetic toolbox of consisting of five new plasmid backbones and around 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
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<div class="pure-u-1-2"><br />
<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em></li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem</li> <br />
<br />
<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em></li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a powerful platform for the synthesis of new cellulose-based biomaterials and water filters</li><br />
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<section id="introduction"><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
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<h2>Introduction</h2><br />
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<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
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</section><br />
<section id="aim"><br />
<h2>Aims</h2><br />
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<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and Kombucha-isolated strain and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
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</section><br />
<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
</figure><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.<br />
<br />
In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. We are currently in the process of completing the bioinformatics analyses of the genomes, and will publish the full genome sequences soon.<br />
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</section><br />
<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and KI strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and KI strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus KI strains to increase cellulose productivity and decrease its cost. In total, by cloning widely used parts into our <em>G. xylinus</em> shuttle vectors we created a toolkit consisting of:<br />
<ul><br />
<li> 5 new shuttle vectors </li> <br />
<li> 15 Anderson promoters </li><br />
<li> Fluorescent proteins RFP and sfGFP </li><br />
<li> 5 chromoproteins </li><br />
<li> 11 CBD fusion proteins </li> <br />
<li> Vitreoscilla hemoglobin gene </li> <br />
<li> TetR and Arabinose inducible promoters </li><br />
</ul><br />
</p><br />
<p> Because the toolkit is in the non-standard pSEVA331-Bb backbone, it can not be housed in the Parts Registry, however in order to make it accessible for the synthetic biology community, we have made it freely available on request.<br />
</p><br />
<div class="accordion"><br />
<h3>Characterization of G.xylinus ATCC 53582 and KI strains</h3><br />
<div><br />
<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
</p><br />
<p> Cellulose productivity of G.xylinus ATCC 53582 and KI on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the KI strain is approximately one fifth of that of ATCC 53582 strain (see Figure X for comparison of ATCC53582 and KI strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for KI cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus KI). <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 3. Cellulose production of G.xylinus ATCC 53582 and KI strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
</figure><br />
</p> <br />
<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure Y.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, KI strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 2 for comparison of ATCC and KI strains and details of he experiment). This could be due to the niche KI strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 4. Cellulose productivity of G.xylinum ATCC 53582 and KI strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
</figure><br />
</p> <br />
<p> Natural antibiotics resistance of ATCC 53582 and KI strains </p><br />
<p></p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 5. Natural antibiotics resistance of G.xylinus ATCC 53582 and KI strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and KI seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
</figure><br />
<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 5 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and KI strain). However, when using high cell densities for plating (after transformations), antibiotic -resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus KI is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the KI strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
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<h3>Plasmid backbones</h3><br />
<div><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 1. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus KI after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
</figure><br />
<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and Ki strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus KI and ATCC 53583 (See figure X). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
</ul><br />
</div><br />
<h3>Anderson promoters</h3><br />
<div><br />
<p> We inserted 15 Anderson promoter-RFP coding devices into pSEVA331-Bb backbone<br />
</p><br />
</div><br />
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<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p><i>G. xylinus</i> is obligatively aerobic because of which cellulose production is reduced below the medium surface in static culture[1] due to reduced oxygen concentrations, reducing the overall volume of the pellicle that is biologically active. Transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i> (another obligate aerobe that expresses this protein in oxygen-poor environments) has been shown to result in increased the metabolic activity and cellulose production of G.xylinus [2].<br />
Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
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<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure Z. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem wild type cells and cells transformed with pSEVA331-BBa_K1321200 plasmid were cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days, after which OD600 was measured. Samples were diluted 1:1 in HS-cellulase medium before measurement. Negative controls (HS-cellulase without inoculations) showed no growth. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
</figure><br />
<br />
<p>We cloned the Vhb gene behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS (part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a>) in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure Z) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
<br />
<p>We also made a hypothesis of the metabolic pathway involving the interaction of vHb, which could potentially explain its effect on enhancing bacterial cellulose yield. As described in the diagram above, vHb plays a role as oxygen storage: it will actively capture oxygen molecules when when the oxygen concentration is high in the cell whereas release oxygen molecules when it is low. As such, the presence of vHb will smooth out the oxygen level within the cell and therefore provides it with a relatively constant oxygen supply. Considering the fact that oxygen also bind to protein complex cG PDE (cyclic GMP phosphodiesterase), where the binding will impose an upregulating effect on bacterial cellulose production, a constant oxygen level maintained by vHb will inevitably contribute to enhance bacterial cellulose yield.</p><br />
<br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://lh5.googleusercontent.com/OkLBTdjMaO6PjVewhOFchlpEd3x4REDL53KYLp_sSxkD35RU4U8Gh21PJKUSfTA1GZ7ShEayn0Y=w751-h608"><br />
<figcaption>Figure Z. Effects of vHb interaction on cellular oxygen level.</figcaption><br />
</figure> <br />
<br />
</div><br />
<br />
<br />
</div><br />
</section><br />
<section id="engineering"><br />
<h2>Engineering</h2><br />
<br />
<div class="accordion"><br />
<h3>Sequencing the genomes of <em>G. xylinus</em> ATCC 53582 and Kombucha-isolated <em>G. xylinus</em> strains</h3><br />
<div><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of <em>G. xylinus</em> is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.</p><br />
<p>In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. <br />
We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction).<br />
We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq present at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9063619 reads with 98.71% reads identified). We completed the genome assembly with the support of Imperial Bioinformatics Support service using their in-house assembly pipeline. </p><br />
</div><br />
<h3>Creating a toolbox for Gluconacetobacter genetic engineering</h3><br />
<div><br />
<p>In parallel to sequencing the genome, we began building a large toolbox for <em>G. xylinus genetic engineering</em>. Only a few vectors have been developed for <em>G. xylinus</em>, and most of them were not available for us for various reasons (including patents, materials-transfer agreements, and retired or deceased researchers). For this reason, we first tested out a set of new plasmids, and engineered them to be Biobrick compatible. As some <em>E. coli</em> promoters and proteins have been shown to work in <em>Gluconacetobacter</em> or <em>Acetobacter</em> species (Setyawati, 2007; Chien et al., 2006), and similarly to <em>E.coli</em>, <em>G. xylinus</em> is a Gram- species, we decided to test out and modify already existing Biobricks in the parts registry in <em>G. xylinus</em>. Therefore, the toolkit consists of:</p><br />
<br />
<br />
<br />
<ul><br />
<li>New, Biobrick compatible shuttle vectors for <em>Gluconacetobacter</em> and <em>E. coli</em><br />
<ul><br />
<br />
<li><em>pSEVA321</em><br />
</li><br />
<li><em>pSEVA331</em><br />
</li><br />
<li><em>pSEVA351</em><br />
</li><br />
<li><em>pBAV1K</em><br />
</li><br />
<br />
</ul><br />
</li>f<br />
<br />
<li>Characterization and optimization of parts from the Registry, including:<br />
<ul><br />
<li>Fluorescent proteins, GFP, RFP, YFP, CFP<br />
</li><br />
<li>Chromoproteins</li><br />
<li>Vitreoscilla haemoglobin<br />
</li><br />
<li>The entire Anderson promoter library<br />
</li><br />
<li>Widely used gene expression control proteins AraC and TetR.<br />
</li><br />
<br />
<br />
</ul><br />
<br />
</li><br />
</ul><br />
</div><br />
<h3>Engineering a high cellulose-producing ATCC 53582 strain</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
<br />
<br />
</div><br />
<br />
</div><br />
</section><br />
<br />
<br />
<section id="results"><br />
<h2>Results</h2><br />
<p>We have isolated a new strain of Acetobacter from kombucha, and determined its growth rate, cellulose productivity, and natural antibiotic resistance. We have also developed a set of methods for genetic engineering of this strain and have determined five new plasmids capable of replication in this strain. Thus we are the first to genetically engineer it.</p><br />
<p>We have discovered four new plasmids capable of replication in Acetobacter species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K - both in G.xylinus ATCC 53582 strain, and in the strain we isolated from Kombucha. We have biobricked these plasmids, with the exception of pBAV1K, which had been originally engineered to be biobrick compatible (however, pBAV1K can not be uploaded to parts registry due to binding materials-transfer agreements).<br />
</p><br />
</section><br />
<br />
<section id="references"><br />
<h2>References</h2><br />
<ul><br />
<br />
<li>[1] https://www.researchgate.net/ publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2014-10-18T02:33:41Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
</li><br />
<li><a data-scroll href="#aim">Aims</a><br />
</li><br />
<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
</li><br />
<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
</li><br />
<li><a data-scroll href="#engineering">Engineering</a><br />
</li><br />
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<li><a data-scroll href="#results">Results</a><br />
</li><br />
<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
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<div class="pure-u-1-2"><br />
<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus KI</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and KI, creating a genetic toolbox of consisting of five new plasmid backbones and around 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
</div><br />
<div class="pure-u-1-2"><br />
<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em></li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem</li> <br />
<br />
<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em></li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a powerful platform for the synthesis of new cellulose-based biomaterials and water filters</li><br />
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<br />
<br />
<br />
</ul><br />
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</section><br />
<section id="introduction"><br />
<br/><br />
<figure class="content-image image-full"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
</figure><br />
<h2>Introduction</h2><br />
<br />
<br />
<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
</p><br />
<br />
</section><br />
<section id="aim"><br />
<h2>Aims</h2><br />
<br />
<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and Kombucha-isolated strain and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
<br />
<br />
</section><br />
<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
</figure><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.<br />
<br />
In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. We are currently in the process of completing the bioinformatics analyses of the genomes, and will publish the full genome sequences soon.<br />
<br />
</p><br />
</section><br />
<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and KI strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and KI strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus KI strains to increase cellulose productivity and decrease its cost. In total, by cloning widely used parts into our <em>G. xylinus</em> shuttle vectors and expressing them in<em>G. xylinus</em> we created a toolkit consisting of:<br />
<ul><br />
<li> 5 new shuttle vectors </li> <br />
<li> 15 Anderson promoters </li><br />
<li> Fluorescent proteins RFP and sfGFP </li><br />
<li> 5 chromoproteins </li><br />
<li> 11 CBD fusion proteins </li> <br />
<li> Vitreoscilla hemoglobin gene </li> <br />
<li> TetR and Arabinose inducible promoters </li><br />
</ul><br />
</p><br />
<p> Because the toolkit is in the non-standard pSEVA331-Bb backbone, it can not be housed in the Parts Registry, however in order to make it accessible for the synthetic biology community, we have made it freely available on request.<br />
</p><br />
<div class="accordion"><br />
<h3>Characterization of G.xylinus ATCC 53582 and KI strains</h3><br />
<div><br />
<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
</p><br />
<p> Cellulose productivity of G.xylinus ATCC 53582 and KI on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the KI strain is approximately one fifth of that of ATCC 53582 strain (see Figure X for comparison of ATCC53582 and KI strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for KI cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus KI). <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 3. Cellulose production of G.xylinus ATCC 53582 and KI strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
</figure><br />
</p> <br />
<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure Y.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, KI strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 2 for comparison of ATCC and KI strains and details of he experiment). This could be due to the niche KI strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 4. Cellulose productivity of G.xylinum ATCC 53582 and KI strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
</figure><br />
</p> <br />
<p> Natural antibiotics resistance of ATCC 53582 and KI strains </p><br />
<p></p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 5. Natural antibiotics resistance of G.xylinus ATCC 53582 and KI strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and KI seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
</figure><br />
<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 5 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and KI strain). However, when using high cell densities for plating (after transformations), antibiotic -resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus KI is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the KI strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
<br />
<br />
</div><br />
<h3>Plasmid backbones</h3><br />
<div><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 1. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus KI after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
</figure><br />
<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and Ki strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus KI and ATCC 53583 (See figure X). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
</ul><br />
</div><br />
<h3>Anderson promoters</h3><br />
<div><br />
<p> We inserted 15 Anderson promoter-RFP coding devices into pSEVA331-Bb backbone<br />
</p><br />
</div><br />
<br />
<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p><i>G. xylinus</i> is obligatively aerobic because of which cellulose production is reduced below the medium surface in static culture[1] due to reduced oxygen concentrations, reducing the overall volume of the pellicle that is biologically active. Transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i> (another obligate aerobe that expresses this protein in oxygen-poor environments) has been shown to result in increased the metabolic activity and cellulose production of G.xylinus [2].<br />
Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
<br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure Z. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem wild type cells and cells transformed with pSEVA331-BBa_K1321200 plasmid were cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days, after which OD600 was measured. Samples were diluted 1:1 in HS-cellulase medium before measurement. Negative controls (HS-cellulase without inoculations) showed no growth. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
</figure><br />
<br />
<p>We cloned the Vhb gene behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS (part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a>) in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure Z) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
<br />
<p>We also made a hypothesis of the metabolic pathway involving the interaction of vHb, which could potentially explain its effect on enhancing bacterial cellulose yield. As described in the diagram above, vHb plays a role as oxygen storage: it will actively capture oxygen molecules when when the oxygen concentration is high in the cell whereas release oxygen molecules when it is low. As such, the presence of vHb will smooth out the oxygen level within the cell and therefore provides it with a relatively constant oxygen supply. Considering the fact that oxygen also bind to protein complex cG PDE (cyclic GMP phosphodiesterase), where the binding will impose an upregulating effect on bacterial cellulose production, a constant oxygen level maintained by vHb will inevitably contribute to enhance bacterial cellulose yield.</p><br />
<br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://lh5.googleusercontent.com/OkLBTdjMaO6PjVewhOFchlpEd3x4REDL53KYLp_sSxkD35RU4U8Gh21PJKUSfTA1GZ7ShEayn0Y=w751-h608"><br />
<figcaption>Figure Z. Effects of vHb interaction on cellular oxygen level.</figcaption><br />
</figure> <br />
<br />
</div><br />
<br />
<br />
</div><br />
</section><br />
<section id="engineering"><br />
<h2>Engineering</h2><br />
<br />
<div class="accordion"><br />
<h3>Sequencing the genomes of <em>G. xylinus</em> ATCC 53582 and Kombucha-isolated <em>G. xylinus</em> strains</h3><br />
<div><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of <em>G. xylinus</em> is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.</p><br />
<p>In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. <br />
We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction).<br />
We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq present at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9063619 reads with 98.71% reads identified). We completed the genome assembly with the support of Imperial Bioinformatics Support service using their in-house assembly pipeline. </p><br />
</div><br />
<h3>Creating a toolbox for Gluconacetobacter genetic engineering</h3><br />
<div><br />
<p>In parallel to sequencing the genome, we began building a large toolbox for <em>G. xylinus genetic engineering</em>. Only a few vectors have been developed for <em>G. xylinus</em>, and most of them were not available for us for various reasons (including patents, materials-transfer agreements, and retired or deceased researchers). For this reason, we first tested out a set of new plasmids, and engineered them to be Biobrick compatible. As some <em>E. coli</em> promoters and proteins have been shown to work in <em>Gluconacetobacter</em> or <em>Acetobacter</em> species (Setyawati, 2007; Chien et al., 2006), and similarly to <em>E.coli</em>, <em>G. xylinus</em> is a Gram- species, we decided to test out and modify already existing Biobricks in the parts registry in <em>G. xylinus</em>. Therefore, the toolkit consists of:</p><br />
<br />
<br />
<br />
<ul><br />
<li>New, Biobrick compatible shuttle vectors for <em>Gluconacetobacter</em> and <em>E. coli</em><br />
<ul><br />
<br />
<li><em>pSEVA321</em><br />
</li><br />
<li><em>pSEVA331</em><br />
</li><br />
<li><em>pSEVA351</em><br />
</li><br />
<li><em>pBAV1K</em><br />
</li><br />
<br />
</ul><br />
</li>f<br />
<br />
<li>Characterization and optimization of parts from the Registry, including:<br />
<ul><br />
<li>Fluorescent proteins, GFP, RFP, YFP, CFP<br />
</li><br />
<li>Chromoproteins</li><br />
<li>Vitreoscilla haemoglobin<br />
</li><br />
<li>The entire Anderson promoter library<br />
</li><br />
<li>Widely used gene expression control proteins AraC and TetR.<br />
</li><br />
<br />
<br />
</ul><br />
<br />
</li><br />
</ul><br />
</div><br />
<h3>Engineering a high cellulose-producing ATCC 53582 strain</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
<br />
<br />
</div><br />
<br />
</div><br />
</section><br />
<br />
<br />
<section id="results"><br />
<h2>Results</h2><br />
<p>We have isolated a new strain of Acetobacter from kombucha, and determined its growth rate, cellulose productivity, and natural antibiotic resistance. We have also developed a set of methods for genetic engineering of this strain and have determined five new plasmids capable of replication in this strain. Thus we are the first to genetically engineer it.</p><br />
<p>We have discovered four new plasmids capable of replication in Acetobacter species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K - both in G.xylinus ATCC 53582 strain, and in the strain we isolated from Kombucha. We have biobricked these plasmids, with the exception of pBAV1K, which had been originally engineered to be biobrick compatible (however, pBAV1K can not be uploaded to parts registry due to binding materials-transfer agreements).<br />
</p><br />
</section><br />
<br />
<section id="references"><br />
<h2>References</h2><br />
<ul><br />
<br />
<li>[1] https://www.researchgate.net/ publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2014-10-18T02:26:49Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
</li><br />
<li><a data-scroll href="#aim">Aims</a><br />
</li><br />
<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
</li><br />
<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
</li><br />
<li><a data-scroll href="#engineering">Engineering</a><br />
</li><br />
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<li><a data-scroll href="#results">Results</a><br />
</li><br />
<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
<div class="pure-g"><br />
<div class="pure-u-1-2"><br />
<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus KI</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and KI, creating a genetic toolbox of consisting of five new plasmid backbones and 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
</div><br />
<div class="pure-u-1-2"><br />
<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em></li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem</li> <br />
<br />
<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em></li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a powerful platform for the synthesis of new cellulose-based biomaterials and water filters</li><br />
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<br />
<br />
<br />
<br />
</ul><br />
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</section><br />
<section id="introduction"><br />
<br/><br />
<figure class="content-image image-full"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
</figure><br />
<h2>Introduction</h2><br />
<br />
<br />
<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
</p><br />
<br />
</section><br />
<section id="aim"><br />
<h2>Aims</h2><br />
<br />
<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and Kombucha-isolated strain and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
<br />
<br />
</section><br />
<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
</figure><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.<br />
<br />
In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. We are currently in the process of completing the bioinformatics analyses of the genomes, and will publish the full genome sequences soon.<br />
<br />
</p><br />
</section><br />
<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and KI strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and KI strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus KI strains to increase cellulose productivity and decrease its cost. In total, by cloning widely used parts into our <em>G. xylinus</em> shuttle vectors and expressing them in<em>G. xylinus</em> we created a toolkit consisting of:<br />
<ul><br />
<li> 5 new shuttle vectors </li> <br />
<li> 15 Anderson promoters </li><br />
<li> Fluorescent proteins RFP and sfGFP </li><br />
<li> 5 chromoproteins </li><br />
<li> 11 CBD fusion proteins </li> <br />
<li> Vitreoscilla hemoglobin gene </li> <br />
<li> TetR and Arabinose inducible promoters </li><br />
</ul><br />
</p><br />
<p> Because the toolkit is in the non-standard pSEVA331-Bb backbone, it can not be housed in the Parts Registry, however in order to make it accessible for the synthetic biology community, we have made it freely available on request.<br />
</p><br />
<div class="accordion"><br />
<h3>Characterization of G.xylinus ATCC 53582 and KI strains</h3><br />
<div><br />
<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
</p><br />
<p> Cellulose productivity of G.xylinus ATCC 53582 and KI on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the KI strain is approximately one fifth of that of ATCC 53582 strain (see Figure X for comparison of ATCC53582 and KI strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for KI cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus KI). <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 3. Cellulose production of G.xylinus ATCC 53582 and KI strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
</figure><br />
</p> <br />
<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure Y.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, KI strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 2 for comparison of ATCC and KI strains and details of he experiment). This could be due to the niche KI strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 4. Cellulose productivity of G.xylinum ATCC 53582 and KI strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
</figure><br />
</p> <br />
<p> Natural antibiotics resistance of ATCC 53582 and KI strains </p><br />
<p></p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 5. Natural antibiotics resistance of G.xylinus ATCC 53582 and KI strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and KI seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
</figure><br />
<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 5 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and KI strain). However, when using high cell densities for plating (after transformations), antibiotic -resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus KI is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the KI strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
<br />
<br />
</div><br />
<h3>Plasmid backbones</h3><br />
<div><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 1. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus KI after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
</figure><br />
<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and Ki strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus KI and ATCC 53583 (See figure X). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
</ul><br />
</div><br />
<h3>Anderson promoters</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
</div><br />
<h3>Inducible promoters</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p> <br />
</div><br />
<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p><i>G. xylinus</i> is obligatively aerobic because of which cellulose production is reduced below the medium surface in static culture[1] due to reduced oxygen concentrations, reducing the overall volume of the pellicle that is biologically active. Transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i> (another obligate aerobe that expresses this protein in oxygen-poor environments) has been shown to result in increased the metabolic activity and cellulose production of G.xylinus [2].<br />
Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
<br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure Z. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem wild type cells and cells transformed with pSEVA331-BBa_K1321200 plasmid were cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days, after which OD600 was measured. Samples were diluted 1:1 in HS-cellulase medium before measurement. Negative controls (HS-cellulase without inoculations) showed no growth. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
</figure><br />
<br />
<p>We cloned the Vhb gene behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS (part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a>) in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure Z) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
<br />
<p>We also made a hypothesis of the metabolic pathway involving the interaction of vHb, which could potentially explain its effect on enhancing bacterial cellulose yield. As described in the diagram above, vHb plays a role as oxygen storage: it will actively capture oxygen molecules when when the oxygen concentration is high in the cell whereas release oxygen molecules when it is low. As such, the presence of vHb will smooth out the oxygen level within the cell and therefore provides it with a relatively constant oxygen supply. Considering the fact that oxygen also bind to protein complex cG PDE (cyclic GMP phosphodiesterase), where the binding will impose an upregulating effect on bacterial cellulose production, a constant oxygen level maintained by vHb will inevitably contribute to enhance bacterial cellulose yield.</p><br />
<br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://lh5.googleusercontent.com/OkLBTdjMaO6PjVewhOFchlpEd3x4REDL53KYLp_sSxkD35RU4U8Gh21PJKUSfTA1GZ7ShEayn0Y=w751-h608"><br />
<figcaption>Figure Z. Effects of vHb interaction on cellular oxygen level.</figcaption><br />
</figure> <br />
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</div><br />
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</div><br />
</section><br />
<section id="engineering"><br />
<h2>Engineering</h2><br />
<br />
<div class="accordion"><br />
<h3>Sequencing the genomes of <em>G. xylinus</em> ATCC 53582 and Kombucha-isolated <em>G. xylinus</em> strains</h3><br />
<div><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of <em>G. xylinus</em> is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.</p><br />
<p>In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. <br />
We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction).<br />
We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq present at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9063619 reads with 98.71% reads identified). We completed the genome assembly with the support of Imperial Bioinformatics Support service using their in-house assembly pipeline. </p><br />
</div><br />
<h3>Creating a toolbox for Gluconacetobacter genetic engineering</h3><br />
<div><br />
<p>In parallel to sequencing the genome, we began building a large toolbox for <em>G. xylinus genetic engineering</em>. Only a few vectors have been developed for <em>G. xylinus</em>, and most of them were not available for us for various reasons (including patents, materials-transfer agreements, and retired or deceased researchers). For this reason, we first tested out a set of new plasmids, and engineered them to be Biobrick compatible. As some <em>E. coli</em> promoters and proteins have been shown to work in <em>Gluconacetobacter</em> or <em>Acetobacter</em> species (Setyawati, 2007; Chien et al., 2006), and similarly to <em>E.coli</em>, <em>G. xylinus</em> is a Gram- species, we decided to test out and modify already existing Biobricks in the parts registry in <em>G. xylinus</em>. Therefore, the toolkit consists of:</p><br />
<br />
<br />
<br />
<ul><br />
<li>New, Biobrick compatible shuttle vectors for <em>Gluconacetobacter</em> and <em>E. coli</em><br />
<ul><br />
<br />
<li><em>pSEVA321</em><br />
</li><br />
<li><em>pSEVA331</em><br />
</li><br />
<li><em>pSEVA351</em><br />
</li><br />
<li><em>pBAV1K</em><br />
</li><br />
<br />
</ul><br />
</li>f<br />
<br />
<li>Characterization and optimization of parts from the Registry, including:<br />
<ul><br />
<li>Fluorescent proteins, GFP, RFP, YFP, CFP<br />
</li><br />
<li>Chromoproteins</li><br />
<li>Vitreoscilla haemoglobin<br />
</li><br />
<li>The entire Anderson promoter library<br />
</li><br />
<li>Widely used gene expression control proteins AraC and TetR.<br />
</li><br />
<br />
<br />
</ul><br />
<br />
</li><br />
</ul><br />
</div><br />
<h3>Engineering a high cellulose-producing ATCC 53582 strain</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
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</div><br />
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</div><br />
</section><br />
<br />
<br />
<section id="results"><br />
<h2>Results</h2><br />
<p>We have isolated a new strain of Acetobacter from kombucha, and determined its growth rate, cellulose productivity, and natural antibiotic resistance. We have also developed a set of methods for genetic engineering of this strain and have determined five new plasmids capable of replication in this strain. Thus we are the first to genetically engineer it.</p><br />
<p>We have discovered four new plasmids capable of replication in Acetobacter species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K - both in G.xylinus ATCC 53582 strain, and in the strain we isolated from Kombucha. We have biobricked these plasmids, with the exception of pBAV1K, which had been originally engineered to be biobrick compatible (however, pBAV1K can not be uploaded to parts registry due to binding materials-transfer agreements).<br />
</p><br />
</section><br />
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<section id="references"><br />
<h2>References</h2><br />
<ul><br />
<br />
<li>[1] https://www.researchgate.net/ publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2014-10-18T02:18:06Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
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<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
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<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
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<li><a data-scroll href="#engineering">Engineering</a><br />
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<li><a data-scroll href="#results">Results</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
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<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus KI</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and KI, creating a genetic toolbox of consisting of five new plasmid backbones and 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
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<div class="pure-u-1-2"><br />
<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em></li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem</li> <br />
<br />
<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em></li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a powerful platform for the synthesis of new cellulose-based biomaterials and water filters</li><br />
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<section id="introduction"><br />
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<figure class="content-image image-full"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
</figure><br />
<h2>Introduction</h2><br />
<br />
<br />
<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
</p><br />
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</section><br />
<section id="aim"><br />
<h2>Aims</h2><br />
<br />
<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and Kombucha-isolated strain and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
<br />
<br />
</section><br />
<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
</figure><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.<br />
<br />
In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. We are currently in the process of completing the bioinformatics analyses of the genomes, and will publish the full genome sequences soon.<br />
<br />
</p><br />
</section><br />
<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and KI strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and KI strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus KI strains to increase cellulose productivity and decrease its cost. In total, by cloning widely used parts into our <em>G. xylinus</em> shuttle vectors and expressing them in<em>G. xylinus</em> we created a toolkit consisting of:<br />
<ul><br />
<li> 5 new shuttle vectors </li> <br />
<li> 15 Anderson promoters </li><br />
<li> Fluorescent proteins RFP and sfGFP </li><br />
<li> 5 chromoproteins </li><br />
<li> 11 CBD fusion proteins </li> <br />
<li> Vitreoscilla hemoglobin gene </li> <br />
<li> TetR and Arabinose inducible promoters <li><br />
</ul><br />
</p><br />
<div class="accordion"><br />
<h3>Characterization of G.xylinus ATCC 53582 and KI strains</h3><br />
<div><br />
<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
</p><br />
<p> Cellulose productivity of G.xylinus ATCC 53582 and KI on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the KI strain is approximately one fifth of that of ATCC 53582 strain (see Figure X for comparison of ATCC53582 and KI strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for KI cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus KI). <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 3. Cellulose production of G.xylinus ATCC 53582 and KI strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
</figure><br />
</p> <br />
<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure Y.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, KI strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 2 for comparison of ATCC and KI strains and details of he experiment). This could be due to the niche KI strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 4. Cellulose productivity of G.xylinum ATCC 53582 and KI strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
</figure><br />
</p> <br />
<p> Natural antibiotics resistance of ATCC 53582 and KI strains </p><br />
<p></p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 5. Natural antibiotics resistance of G.xylinus ATCC 53582 and KI strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and KI seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
</figure><br />
<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 5 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and KI strain). However, when using high cell densities for plating (after transformations), antibiotic -resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus KI is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the KI strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
<br />
<br />
</div><br />
<h3>Plasmid backbones</h3><br />
<div><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 1. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus KI after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
</figure><br />
<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and Ki strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus KI and ATCC 53583 (See figure X). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
</ul><br />
</div><br />
<h3>Anderson promoters</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
</div><br />
<h3>Inducible promoters</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p> <br />
</div><br />
<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p><i>G. xylinus</i> is obligatively aerobic because of which cellulose production is reduced below the medium surface in static culture[1] due to reduced oxygen concentrations, reducing the overall volume of the pellicle that is biologically active. Transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i> (another obligate aerobe that expresses this protein in oxygen-poor environments) has been shown to result in increased the metabolic activity and cellulose production of G.xylinus [2].<br />
Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
<br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure Z. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem wild type cells and cells transformed with pSEVA331-BBa_K1321200 plasmid were cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days, after which OD600 was measured. Samples were diluted 1:1 in HS-cellulase medium before measurement. Negative controls (HS-cellulase without inoculations) showed no growth. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
</figure><br />
<br />
<p>We cloned the Vhb gene behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS (part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a>) in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure Z) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
<br />
<p>We also made a hypothesis of the metabolic pathway involving the interaction of vHb, which could potentially explain its effect on enhancing bacterial cellulose yield. As described in the diagram above, vHb plays a role as oxygen storage: it will actively capture oxygen molecules when when the oxygen concentration is high in the cell whereas release oxygen molecules when it is low. As such, the presence of vHb will smooth out the oxygen level within the cell and therefore provides it with a relatively constant oxygen supply. Considering the fact that oxygen also bind to protein complex cG PDE (cyclic GMP phosphodiesterase), where the binding will impose an upregulating effect on bacterial cellulose production, a constant oxygen level maintained by vHb will inevitably contribute to enhance bacterial cellulose yield.</p><br />
<br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://lh5.googleusercontent.com/OkLBTdjMaO6PjVewhOFchlpEd3x4REDL53KYLp_sSxkD35RU4U8Gh21PJKUSfTA1GZ7ShEayn0Y=w751-h608"><br />
<figcaption>Figure Z. Effects of vHb interaction on cellular oxygen level.</figcaption><br />
</figure> <br />
<br />
</div><br />
<br />
<br />
</div><br />
</section><br />
<section id="engineering"><br />
<h2>Engineering</h2><br />
<br />
<div class="accordion"><br />
<h3>Sequencing the genomes of <em>G. xylinus</em> ATCC 53582 and Kombucha-isolated <em>G. xylinus</em> strains</h3><br />
<div><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of <em>G. xylinus</em> is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.</p><br />
<p>In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. <br />
We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction).<br />
We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq present at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9063619 reads with 98.71% reads identified). We completed the genome assembly with the support of Imperial Bioinformatics Support service using their in-house assembly pipeline. </p><br />
</div><br />
<h3>Creating a toolbox for Gluconacetobacter genetic engineering</h3><br />
<div><br />
<p>In parallel to sequencing the genome, we began building a large toolbox for <em>G. xylinus genetic engineering</em>. Only a few vectors have been developed for <em>G. xylinus</em>, and most of them were not available for us for various reasons (including patents, materials-transfer agreements, and retired or deceased researchers). For this reason, we first tested out a set of new plasmids, and engineered them to be Biobrick compatible. As some <em>E. coli</em> promoters and proteins have been shown to work in <em>Gluconacetobacter</em> or <em>Acetobacter</em> species (Setyawati, 2007; Chien et al., 2006), and similarly to <em>E.coli</em>, <em>G. xylinus</em> is a Gram- species, we decided to test out and modify already existing Biobricks in the parts registry in <em>G. xylinus</em>. Therefore, the toolkit consists of:</p><br />
<br />
<br />
<br />
<ul><br />
<li>New, Biobrick compatible shuttle vectors for <em>Gluconacetobacter</em> and <em>E. coli</em><br />
<ul><br />
<br />
<li><em>pSEVA321</em><br />
</li><br />
<li><em>pSEVA331</em><br />
</li><br />
<li><em>pSEVA351</em><br />
</li><br />
<li><em>pBAV1K</em><br />
</li><br />
<br />
</ul><br />
</li>f<br />
<br />
<li>Characterization and optimization of parts from the Registry, including:<br />
<ul><br />
<li>Fluorescent proteins, GFP, RFP, YFP, CFP<br />
</li><br />
<li>Chromoproteins</li><br />
<li>Vitreoscilla haemoglobin<br />
</li><br />
<li>The entire Anderson promoter library<br />
</li><br />
<li>Widely used gene expression control proteins AraC and TetR.<br />
</li><br />
<br />
<br />
</ul><br />
<br />
</li><br />
</ul><br />
</div><br />
<h3>Engineering a high cellulose-producing ATCC 53582 strain</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
<br />
<br />
</div><br />
<br />
</div><br />
</section><br />
<br />
<br />
<section id="results"><br />
<h2>Results</h2><br />
<p>We have isolated a new strain of Acetobacter from kombucha, and determined its growth rate, cellulose productivity, and natural antibiotic resistance. We have also developed a set of methods for genetic engineering of this strain and have determined five new plasmids capable of replication in this strain. Thus we are the first to genetically engineer it.</p><br />
<p>We have discovered four new plasmids capable of replication in Acetobacter species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K - both in G.xylinus ATCC 53582 strain, and in the strain we isolated from Kombucha. We have biobricked these plasmids, with the exception of pBAV1K, which had been originally engineered to be biobrick compatible (however, pBAV1K can not be uploaded to parts registry due to binding materials-transfer agreements).<br />
</p><br />
</section><br />
<br />
<section id="references"><br />
<h2>References</h2><br />
<ul><br />
<br />
<li>[1] https://www.researchgate.net/ publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2014-10-18T02:13:25Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
</li><br />
<li><a data-scroll href="#aim">Aims</a><br />
</li><br />
<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
</li><br />
<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
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<li><a data-scroll href="#engineering">Engineering</a><br />
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<li><a data-scroll href="#results">Results</a><br />
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<li><a data-scroll href="#references">References</a><br />
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</div><br />
<section id="overview"><br />
<div class="pure-g"><br />
<div class="pure-u-1-2"><br />
<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus KI</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and KI, creating a genetic toolbox of consisting of five new plasmid backbones and 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
</div><br />
<div class="pure-u-1-2"><br />
<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em></li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem</li> <br />
<br />
<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em></li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a powerful platform for the synthesis of new cellulose-based biomaterials and water filters</li><br />
<br />
<br />
<br />
<br />
<br />
</ul><br />
</div><br />
<br />
<br />
</div><br />
<br />
</section><br />
<section id="introduction"><br />
<br/><br />
<figure class="content-image image-full"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
</figure><br />
<h2>Introduction</h2><br />
<br />
<br />
<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
</p><br />
<br />
</section><br />
<section id="aim"><br />
<h2>Aims</h2><br />
<br />
<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and Kombucha-isolated strain and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
<br />
<br />
</section><br />
<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
</figure><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.<br />
<br />
In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. We are currently in the process of completing the bioinformatics analyses of the genomes, and will publish the full genome sequences soon.<br />
<br />
</p><br />
</section><br />
<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and KI strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and KI strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus KI strains to increase cellulose productivity and decrease its cost. In total, we created a toolkit for <em>G. xylinus</em> which consists of:<br />
<ul><br />
<li> 5 new shuttle vectors </li> <br />
<li> 15 Anderson promoters </li><br />
<li> Fluorescent proteins RFP and sfGFP </li><br />
<li> 5 chromoproteins </li><br />
<li> 11 CBD fusion proteins </li> <br />
<li> Vitreoscilla hemoglobin gene </li> <br />
<li> TetR and Arabinose inducible promoters <li><br />
</p><br />
<div class="accordion"><br />
<h3>Characterization of G.xylinus ATCC 53582 and KI strains</h3><br />
<div><br />
<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
</p><br />
<p> Cellulose productivity of G.xylinus ATCC 53582 and KI on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the KI strain is approximately one fifth of that of ATCC 53582 strain (see Figure X for comparison of ATCC53582 and KI strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for KI cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus KI). <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 3. Cellulose production of G.xylinus ATCC 53582 and KI strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
</figure><br />
</p> <br />
<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure Y.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, KI strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 2 for comparison of ATCC and KI strains and details of he experiment). This could be due to the niche KI strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 4. Cellulose productivity of G.xylinum ATCC 53582 and KI strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
</figure><br />
</p> <br />
<p> Natural antibiotics resistance of ATCC 53582 and KI strains </p><br />
<p></p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 5. Natural antibiotics resistance of G.xylinus ATCC 53582 and KI strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and KI seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
</figure><br />
<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 5 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and KI strain). However, when using high cell densities for plating (after transformations), antibiotic -resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus KI is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the KI strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
<br />
<br />
</div><br />
<h3>Plasmid backbones</h3><br />
<div><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 1. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus KI after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
</figure><br />
<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and Ki strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus KI and ATCC 53583 (See figure X). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
</ul><br />
</div><br />
<h3>Anderson promoters</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
</div><br />
<h3>Inducible promoters</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p> <br />
</div><br />
<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p><i>G. xylinus</i> is obligatively aerobic because of which cellulose production is reduced below the medium surface in static culture[1] due to reduced oxygen concentrations, reducing the overall volume of the pellicle that is biologically active. Transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i> (another obligate aerobe that expresses this protein in oxygen-poor environments) has been shown to result in increased the metabolic activity and cellulose production of G.xylinus [2].<br />
Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
<br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure Z. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem wild type cells and cells transformed with pSEVA331-BBa_K1321200 plasmid were cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days, after which OD600 was measured. Samples were diluted 1:1 in HS-cellulase medium before measurement. Negative controls (HS-cellulase without inoculations) showed no growth. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
</figure><br />
<br />
<p>We cloned the Vhb gene behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS (part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a>) in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure Z) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
<br />
<p>We also made a hypothesis of the metabolic pathway involving the interaction of vHb, which could potentially explain its effect on enhancing bacterial cellulose yield. As described in the diagram above, vHb plays a role as oxygen storage: it will actively capture oxygen molecules when when the oxygen concentration is high in the cell whereas release oxygen molecules when it is low. As such, the presence of vHb will smooth out the oxygen level within the cell and therefore provides it with a relatively constant oxygen supply. Considering the fact that oxygen also bind to protein complex cG PDE (cyclic GMP phosphodiesterase), where the binding will impose an upregulating effect on bacterial cellulose production, a constant oxygen level maintained by vHb will inevitably contribute to enhance bacterial cellulose yield.</p><br />
<br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://lh5.googleusercontent.com/OkLBTdjMaO6PjVewhOFchlpEd3x4REDL53KYLp_sSxkD35RU4U8Gh21PJKUSfTA1GZ7ShEayn0Y=w751-h608"><br />
<figcaption>Figure Z. Effects of vHb interaction on cellular oxygen level.</figcaption><br />
</figure> <br />
<br />
</div><br />
<br />
<br />
</div><br />
</section><br />
<section id="engineering"><br />
<h2>Engineering</h2><br />
<br />
<div class="accordion"><br />
<h3>Sequencing the genomes of <em>G. xylinus</em> ATCC 53582 and Kombucha-isolated <em>G. xylinus</em> strains</h3><br />
<div><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of <em>G. xylinus</em> is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.</p><br />
<p>In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. <br />
We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction).<br />
We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq present at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9063619 reads with 98.71% reads identified). We completed the genome assembly with the support of Imperial Bioinformatics Support service using their in-house assembly pipeline. </p><br />
</div><br />
<h3>Creating a toolbox for Gluconacetobacter genetic engineering</h3><br />
<div><br />
<p>In parallel to sequencing the genome, we began building a large toolbox for <em>G. xylinus genetic engineering</em>. Only a few vectors have been developed for <em>G. xylinus</em>, and most of them were not available for us for various reasons (including patents, materials-transfer agreements, and retired or deceased researchers). For this reason, we first tested out a set of new plasmids, and engineered them to be Biobrick compatible. As some <em>E. coli</em> promoters and proteins have been shown to work in <em>Gluconacetobacter</em> or <em>Acetobacter</em> species (Setyawati, 2007; Chien et al., 2006), and similarly to <em>E.coli</em>, <em>G. xylinus</em> is a Gram- species, we decided to test out and modify already existing Biobricks in the parts registry in <em>G. xylinus</em>. Therefore, the toolkit consists of:</p><br />
<br />
<br />
<br />
<ul><br />
<li>New, Biobrick compatible shuttle vectors for <em>Gluconacetobacter</em> and <em>E. coli</em><br />
<ul><br />
<br />
<li><em>pSEVA321</em><br />
</li><br />
<li><em>pSEVA331</em><br />
</li><br />
<li><em>pSEVA351</em><br />
</li><br />
<li><em>pBAV1K</em><br />
</li><br />
<br />
</ul><br />
</li>f<br />
<br />
<li>Characterization and optimization of parts from the Registry, including:<br />
<ul><br />
<li>Fluorescent proteins, GFP, RFP, YFP, CFP<br />
</li><br />
<li>Chromoproteins</li><br />
<li>Vitreoscilla haemoglobin<br />
</li><br />
<li>The entire Anderson promoter library<br />
</li><br />
<li>Widely used gene expression control proteins AraC and TetR.<br />
</li><br />
<br />
<br />
</ul><br />
<br />
</li><br />
</ul><br />
</div><br />
<h3>Engineering a high cellulose-producing ATCC 53582 strain</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
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<h2>Results</h2><br />
<p>We have isolated a new strain of Acetobacter from kombucha, and determined its growth rate, cellulose productivity, and natural antibiotic resistance. We have also developed a set of methods for genetic engineering of this strain and have determined five new plasmids capable of replication in this strain. Thus we are the first to genetically engineer it.</p><br />
<p>We have discovered four new plasmids capable of replication in Acetobacter species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K - both in G.xylinus ATCC 53582 strain, and in the strain we isolated from Kombucha. We have biobricked these plasmids, with the exception of pBAV1K, which had been originally engineered to be biobrick compatible (however, pBAV1K can not be uploaded to parts registry due to binding materials-transfer agreements).<br />
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<h2>References</h2><br />
<ul><br />
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<li>[1] https://www.researchgate.net/ publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/File:IC14-mass-production3.jpgFile:IC14-mass-production3.jpg2014-10-18T01:40:01Z<p>Mf1512: Granular sheet of bacterial cellulose</p>
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<div>Granular sheet of bacterial cellulose</div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2014-10-18T01:14:49Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
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<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
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<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
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<li><a data-scroll href="#engineering">Engineering</a><br />
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<li><a data-scroll href="#results">Results</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
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<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus KI</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and KI, creating a genetic toolbox of consisting of five new plasmid backbones and 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
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<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em></li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem</li> <br />
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<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em></li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a powerful platform for the synthesis of new cellulose-based biomaterials and water filters</li><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
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<h2>Introduction</h2><br />
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<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
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<section id="aim"><br />
<h2>Aims</h2><br />
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<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and Kombucha-isolated strain and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
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<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
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<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.<br />
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In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. We are currently in the process of completing the bioinformatics analyses of the genomes, and will publish the full genome sequences soon.<br />
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<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and KI strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and KI strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus KI strains to increase cellulose productivity and decrease its cost.</p><br />
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<h3>Characterization of G.xylinus ATCC 53582 and KI strains</h3><br />
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<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
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<p> Cellulose productivity of G.xylinus ATCC 53582 and KI on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the KI strain is approximately one fifth of that of ATCC 53582 strain (see Figure X for comparison of ATCC53582 and KI strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for KI cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus KI). <br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 3. Cellulose production of G.xylinus ATCC 53582 and KI strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
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<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure Y.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, KI strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 2 for comparison of ATCC and KI strains and details of he experiment). This could be due to the niche KI strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 4. Cellulose productivity of G.xylinum ATCC 53582 and KI strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
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<p> Natural antibiotics resistance of ATCC 53582 and KI strains </p><br />
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<figcaption> Figure 5. Natural antibiotics resistance of G.xylinus ATCC 53582 and KI strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and KI seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
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<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 5 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and KI strain). However, when using high cell densities for plating (after transformations), antibiotic -resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus KI is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the KI strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
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<h3>Plasmid backbones</h3><br />
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<figcaption>Figure 1. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus KI after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
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<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and Ki strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus KI and ATCC 53583 (See figure X). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
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<h3>Anderson promoters</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
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<h3>Inducible promoters</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p> <br />
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<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p><i>G. xylinus</i> is obligatively aerobic because of which cellulose production is reduced below the medium surface in static culture[1] due to reduced oxygen concentrations, reducing the overall volume of the pellicle that is biologically active. Transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i> (another obligate aerobe that expresses this protein in oxygen-poor environments) has been shown to result in increased the metabolic activity and cellulose production of G.xylinus [2].<br />
Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure Z. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem wild type cells and cells transformed with pSEVA331-BBa_K1321200 plasmid were cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days, after which OD600 was measured. Samples were diluted 1:1 in HS-cellulase medium before measurement. Negative controls (HS-cellulase without inoculations) showed no growth. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
</figure><br />
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<p>We cloned the Vhb gene behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS (part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a>) in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure Z) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
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<section id="engineering"><br />
<h2>Engineering</h2><br />
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<div class="accordion"><br />
<h3>Sequencing the genomes of <em>G. xylinus</em> ATCC 53582 and Kombucha-isolated <em>G. xylinus</em> strains</h3><br />
<div><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of <em>G. xylinus</em> is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.</p><br />
<p>In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. <br />
We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction).<br />
We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq present at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9063619 reads with 98.71% reads identified). We completed the genome assembly with the support of Imperial Bioinformatics Support service using their in-house assembly pipeline. </p><br />
</div><br />
<h3>Creating a toolbox for Gluconacetobacter genetic engineering</h3><br />
<div><br />
<p>In parallel to sequencing the genome, we began building a large toolbox for <em>G. xylinus genetic engineering</em>. Only a few vectors have been developed for <em>G. xylinus</em>, and most of them were not available for us for various reasons (including patents, materials-transfer agreements, and retired or deceased researchers). For this reason, we first tested out a set of new plasmids, and engineered them to be Biobrick compatible. As some <em>E. coli</em> promoters and proteins have been shown to work in <em>Gluconacetobacter</em> or <em>Acetobacter</em> species (Setyawati, 2007; Chien et al., 2006), and similarly to <em>E.coli</em>, <em>G. xylinus</em> is a Gram- species, we decided to test out and modify already existing Biobricks in the parts registry in <em>G. xylinus</em>. Therefore, the toolkit consists of:</p><br />
<ul><br />
<li>New, Biobrick compatible shuttle vectors for <em>Gluconacetobacter</em> and <em>E. coli</em><br />
<ul><br />
<br />
<li><em>pSEVA321</em><br />
</li><br />
<li><em>pSEVA331</em><br />
</li><br />
<li><em>pSEVA351</em><br />
</li><br />
<li><em>pBAV1K</em><br />
</li><br />
<br />
</ul><br />
</li>f<br />
<br />
<li>Characterization and optimization of parts from the Registry, including:<br />
<ul><br />
<li>Fluorescent proteins, GFP, RFP, YFP, CFP<br />
</li><br />
<li>Chromoproteins</li><br />
<li>Vitreoscilla haemoglobin<br />
</li><br />
<li>The entire Anderson promoter library<br />
</li><br />
<li>Widely used gene expression control proteins AraC and TetR.<br />
</li><br />
<br />
<br />
</ul><br />
<br />
</li><br />
</ul><br />
</div><br />
<h3>Engineering a high cellulose-producing ATCC 53582 strain</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
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<section id="results"><br />
<h2>Results</h2><br />
<p>We have isolated a new strain of Acetobacter from kombucha, and determined its growth rate, cellulose productivity, and natural antibiotic resistance. We have also developed a set of methods for genetic engineering of this strain and have determined five new plasmids capable of replication in this strain. Thus we are the first to genetically engineer it.</p><br />
<p>We have discovered four new plasmids capable of replication in Acetobacter species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K - both in G.xylinus ATCC 53582 strain, and in the strain we isolated from Kombucha. We have biobricked these plasmids, with the exception of pBAV1K, which had been originally engineered to be biobrick compatible (however, pBAV1K can not be uploaded to parts registry due to binding materials-transfer agreements).<br />
</p><br />
</section><br />
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<h3>References</h3><br />
<ul><br />
<br />
<li>[1]https://www.researchgate.net/publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/File:IC14_Cellulose_processing1.mp4File:IC14 Cellulose processing1.mp42014-10-18T01:05:32Z<p>Mf1512: Short video from the Vine Channel of Imperial College showing bacterial cellulose being processed to become blue, flexible and durable.</p>
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<div>Short video from the Vine Channel of Imperial College showing bacterial cellulose being processed to become blue, flexible and durable.</div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2014-10-18T01:05:22Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
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<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
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<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
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<li><a data-scroll href="#engineering">Engineering</a><br />
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<li><a data-scroll href="#results">Results</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
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<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus KI</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and KI, creating a genetic toolbox of consisting of five new plasmid backbones and 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
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<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em></li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem</li> <br />
<br />
<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em></li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a powerful platform for the synthesis of new cellulose-based biomaterials and water filters</li><br />
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<section id="introduction"><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
</figure><br />
<h2>Introduction</h2><br />
<br />
<br />
<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
</p><br />
<br />
</section><br />
<section id="aim"><br />
<h2>Aims</h2><br />
<br />
<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and Kombucha-isolated strain and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
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</section><br />
<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
</figure><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.<br />
<br />
In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. We are currently in the process of completing the bioinformatics analyses of the genomes, and will publish the full genome sequences soon.<br />
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</p><br />
</section><br />
<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and KI strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and KI strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus KI strains to increase cellulose productivity and decrease its cost.</p><br />
<div class="accordion"><br />
<h3>Characterization of G.xylinus ATCC 53582 and KI strains</h3><br />
<div><br />
<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
</p><br />
<p> Cellulose productivity of G.xylinus ATCC 53582 and KI on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the KI strain is approximately one fifth of that of ATCC 53582 strain (see Figure X for comparison of ATCC53582 and KI strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for KI cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus KI). <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 3. Cellulose production of G.xylinus ATCC 53582 and KI strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
</figure><br />
</p> <br />
<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure Y.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, KI strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 2 for comparison of ATCC and KI strains and details of he experiment). This could be due to the niche KI strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 4. Cellulose productivity of G.xylinum ATCC 53582 and KI strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
</figure><br />
</p> <br />
<p> Natural antibiotics resistance of ATCC 53582 and KI strains </p><br />
<p></p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 5. Natural antibiotics resistance of G.xylinus ATCC 53582 and KI strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and KI seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
</figure><br />
<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 5 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and KI strain). However, when using high cell densities for plating (after transformations), antibiotic -resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus KI is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the KI strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
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<h3>Plasmid backbones</h3><br />
<div><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 1. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus KI after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
</figure><br />
<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and Ki strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus KI and ATCC 53583 (See figure X). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
</ul><br />
</div><br />
<h3>Anderson promoters</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
</div><br />
<h3>Inducible promoters</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p> <br />
</div><br />
<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p>One key drawback of producing cellulose using the obligate aerobe <i>G. xylinus</i> is that it cannot grow below the medium surface in static culture[1], reducing the overall volume of the pellicle that is biologically active. This drawback has been overcome to some extent by transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i>[2], another obligate aerobe that expresses this protein in oxygen-poor environments.</p><br />
<br />
<p><i>Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
<br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure Z. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem wild type cells and cells transformed with pSEVA331-BBa_K1321200 plasmid were cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days, after which OD600 was measured. Samples were diluted 1:1 in HS-cellulase medium before measurement. Negative controls (HS-cellulase without inoculations) showed no growth. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
</figure><br />
<br />
<p>We cloned the Vitreoscilla hemoglobin gene (<em> Vhb </em>) behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS - part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a> in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure Z) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
<br />
</div><br />
<br />
<br />
</div><br />
</section><br />
<section id="engineering"><br />
<h2>Engineering</h2><br />
<br />
<div class="accordion"><br />
<h3>Sequencing the genomes of <em>G. xylinus</em> ATCC 53582 and Kombucha-isolated <em>G. xylinus</em> strains</h3><br />
<div><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of <em>G. xylinus</em> is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.</p><br />
<p>In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. <br />
We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction).<br />
We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq present at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9063619 reads with 98.71% reads identified). We completed the genome assembly with the support of Imperial Bioinformatics Support service using their in-house assembly pipeline. </p><br />
</div><br />
<h3>Creating a toolbox for Gluconacetobacter genetic engineering</h3><br />
<div><br />
<p>In parallel to sequencing the genome, we began building a large toolbox for <em>G. xylinus genetic engineering</em>. Only a few vectors have been developed for <em>G. xylinus</em>, and most of them were not available for us for various reasons (including patents, materials-transfer agreements, and retired or deceased researchers). For this reason, we first tested out a set of new plasmids, and engineered them to be Biobrick compatible. As some <em>E. coli</em> promoters and proteins have been shown to work in <em>Gluconacetobacter</em> or <em>Acetobacter</em> species (Setyawati, 2007; Chien et al., 2006), and similarly to <em>E.coli</em>, <em>G. xylinus</em> is a Gram- species, we decided to test out and modify already existing Biobricks in the parts registry in <em>G. xylinus</em>. Therefore, the toolkit consists of:</p><br />
<ul><br />
<li>New, Biobrick compatible shuttle vectors for <em>Gluconacetobacter</em> and <em>E. coli</em><br />
<ul><br />
<br />
<li><em>pSEVA321</em><br />
</li><br />
<li><em>pSEVA331</em><br />
</li><br />
<li><em>pSEVA351</em><br />
</li><br />
<li><em>pBAV1K</em><br />
</li><br />
<br />
</ul><br />
</li>f<br />
<br />
<li>Characterization and optimization of parts from the Registry, including:<br />
<ul><br />
<li>Fluorescent proteins, GFP, RFP, YFP, CFP<br />
</li><br />
<li>Chromoproteins</li><br />
<li>Vitreoscilla haemoglobin<br />
</li><br />
<li>The entire Anderson promoter library<br />
</li><br />
<li>Widely used gene expression control proteins AraC and TetR.<br />
</li><br />
<br />
<br />
</ul><br />
<br />
</li><br />
</ul><br />
</div><br />
<h3>Engineering a high cellulose-producing ATCC 53582 strain</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
<br />
<br />
</div><br />
<br />
</div><br />
</section><br />
<br />
<br />
<section id="results"><br />
<h2>Results</h2><br />
<p>We have isolated a new strain of Acetobacter from kombucha, and determined its growth rate, cellulose productivity, and natural antibiotic resistance. We have also developed a set of methods for genetic engineering of this strain and have determined five new plasmids capable of replication in this strain. Thus we are the first to genetically engineer it.</p><br />
<p>We have discovered four new plasmids capable of replication in Acetobacter species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K - both in G.xylinus ATCC 53582 strain, and in the strain we isolated from Kombucha. We have biobricked these plasmids, with the exception of pBAV1K, which had been originally engineered to be biobrick compatible (however, pBAV1K can not be uploaded to parts registry due to binding materials-transfer agreements).<br />
</p><br />
</section><br />
<br />
<section id="references"><br />
<h3>References</h3><br />
<ul><br />
<br />
<li>[1] https://www.researchgate.net/publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2014-10-18T00:58:54Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
</li><br />
<li><a data-scroll href="#aim">Aims</a><br />
</li><br />
<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
</li><br />
<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
</li><br />
<li><a data-scroll href="#engineering">Engineering</a><br />
</li><br />
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<li><a data-scroll href="#results">Results</a><br />
</li><br />
<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
<div class="pure-g"><br />
<div class="pure-u-1-2"><br />
<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus KI</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and KI, creating a genetic toolbox of consisting of five new plasmid backbones and 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
</div><br />
<div class="pure-u-1-2"><br />
<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em></li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem</li> <br />
<br />
<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em></li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a powerful platform for the synthesis of new cellulose-based biomaterials and water filters</li><br />
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<br />
<br />
<br />
<br />
</ul><br />
</div><br />
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</section><br />
<section id="introduction"><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
</figure><br />
<h2>Introduction</h2><br />
<br />
<br />
<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
</p><br />
<br />
</section><br />
<section id="aim"><br />
<h2>Aims</h2><br />
<br />
<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and Kombucha-isolated strain and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
<br />
<br />
</section><br />
<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
</figure><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.<br />
<br />
In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. We are currently in the process of completing the bioinformatics analyses of the genomes, and will publish the full genome sequences soon.<br />
<br />
</p><br />
</section><br />
<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and KI strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and KI strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus KI strains to increase cellulose productivity and decrease its cost.</p><br />
<div class="accordion"><br />
<h3>Characterization of G.xylinus ATCC 53582 and KI strains</h3><br />
<div><br />
<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
</p><br />
<p> Cellulose productivity of G.xylinus ATCC 53582 and KI on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the KI strain is approximately one fifth of that of ATCC 53582 strain (see Figure X for comparison of ATCC53582 and KI strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for KI cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus KI). <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 3. Cellulose production of G.xylinus ATCC 53582 and KI strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
</figure><br />
</p> <br />
<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure Y.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, KI strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 2 for comparison of ATCC and KI strains and details of he experiment). This could be due to the niche KI strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 4. Cellulose productivity of G.xylinum ATCC 53582 and KI strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
</figure><br />
</p> <br />
<p> Natural antibiotics resistance of ATCC 53582 and KI strains </p><br />
<p></p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 5. Natural antibiotics resistance of G.xylinus ATCC 53582 and KI strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and KI seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
</figure><br />
<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 5 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and KI strain). However, when using high cell densities for plating (after transformations), antibiotic -resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus KI is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the KI strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
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</div><br />
<h3>Plasmid backbones</h3><br />
<div><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 1. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus KI after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
</figure><br />
<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and Ki strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus KI and ATCC 53583 (See figure X). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
</ul><br />
</div><br />
<h3>Anderson promoters</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
</div><br />
<h3>Inducible promoters</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p> <br />
</div><br />
<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p>One key drawback of producing cellulose using the obligate aerobe <i>G. xylinus</i> is that it cannot grow below the medium surface in static culture[1], reducing the overall volume of the pellicle that is biologically active. This drawback has been overcome to some extent by transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i>[2], another obligate aerobe that expresses this protein in oxygen-poor environments.</p><br />
<br />
<p><i>Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
<br />
<figure class="content-image"><br />
<img class="image-full image-left" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure Z. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem was cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days. OD600 was measured using a spectrophotometer. Samples were diluted 1:1 in HS-cellulase medium before measurement. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
</figure><br />
<br />
<p>We cloned the Vitreoscilla hemoglobin gene (<em> Vhb </em>) behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS - part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a> in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure Z) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
<br />
</div><br />
<br />
<br />
</div><br />
</section><br />
<section id="engineering"><br />
<h2>Engineering</h2><br />
<br />
<div class="accordion"><br />
<h3>Sequencing the genomes of <em>G. xylinus</em> ATCC 53582 and Kombucha-isolated <em>G. xylinus</em> strains</h3><br />
<div><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of <em>G. xylinus</em> is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.</p><br />
<p>In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. <br />
We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction).<br />
We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq present at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9063619 reads with 98.71% reads identified). We completed the genome assembly with the support of Imperial Bioinformatics Support service using their in-house assembly pipeline. </p><br />
</div><br />
<h3>Creating a toolbox for Gluconacetobacter genetic engineering</h3><br />
<div><br />
<p>In parallel to sequencing the genome, we began building a large toolbox for <em>G. xylinus genetic engineering</em>. Only a few vectors have been developed for <em>G. xylinus</em>, and most of them were not available for us for various reasons (including patents, materials-transfer agreements, and retired or deceased researchers). For this reason, we first tested out a set of new plasmids, and engineered them to be Biobrick compatible. As some <em>E. coli</em> promoters and proteins have been shown to work in <em>Gluconacetobacter</em> or <em>Acetobacter</em> species (Setyawati, 2007; Chien et al., 2006), and similarly to <em>E.coli</em>, <em>G. xylinus</em> is a Gram- species, we decided to test out and modify already existing Biobricks in the parts registry in <em>G. xylinus</em>. Therefore, the toolkit consists of:</p><br />
<ul><br />
<li>New, Biobrick compatible shuttle vectors for <em>Gluconacetobacter</em> and <em>E. coli</em><br />
<ul><br />
<br />
<li><em>pSEVA321</em><br />
</li><br />
<li><em>pSEVA331</em><br />
</li><br />
<li><em>pSEVA351</em><br />
</li><br />
<li><em>pBAV1K</em><br />
</li><br />
<br />
</ul><br />
</li>f<br />
<br />
<li>Characterization and optimization of parts from the Registry, including:<br />
<ul><br />
<li>Fluorescent proteins, GFP, RFP, YFP, CFP<br />
</li><br />
<li>Chromoproteins</li><br />
<li>Vitreoscilla haemoglobin<br />
</li><br />
<li>The entire Anderson promoter library<br />
</li><br />
<li>Widely used gene expression control proteins AraC and TetR.<br />
</li><br />
<br />
<br />
</ul><br />
<br />
</li><br />
</ul><br />
</div><br />
<h3>Engineering a high cellulose-producing ATCC 53582 strain</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
<br />
<br />
</div><br />
<br />
</div><br />
</section><br />
<br />
<br />
<section id="results"><br />
<h2>Results</h2><br />
<p>We have isolated a new strain of Acetobacter from kombucha, and determined its growth rate, cellulose productivity, and natural antibiotic resistance. We have also developed a set of methods for genetic engineering of this strain and have determined five new plasmids capable of replication in this strain. Thus we are the first to genetically engineer it.</p><br />
<p>We have discovered four new plasmids capable of replication in Acetobacter species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K - both in G.xylinus ATCC 53582 strain, and in the strain we isolated from Kombucha. We have biobricked these plasmids, with the exception of pBAV1K, which had been originally engineered to be biobrick compatible (however, pBAV1K can not be uploaded to parts registry due to binding materials-transfer agreements).<br />
</p><br />
</section><br />
<br />
<section id="references"><br />
<h3>References</h3><br />
<ul><br />
<br />
<li>[1] https://www.researchgate.net/publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2014-10-18T00:55:31Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
</li><br />
<li><a data-scroll href="#aim">Aims</a><br />
</li><br />
<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
</li><br />
<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
</li><br />
<li><a data-scroll href="#engineering">Engineering</a><br />
</li><br />
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<li><a data-scroll href="#results">Results</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
<div class="pure-g"><br />
<div class="pure-u-1-2"><br />
<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus KI</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and KI, creating a genetic toolbox of consisting of five new plasmid backbones and 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
</div><br />
<div class="pure-u-1-2"><br />
<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em></li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem</li> <br />
<br />
<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em></li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a powerful platform for the synthesis of new cellulose-based biomaterials and water filters</li><br />
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<br />
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</ul><br />
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</section><br />
<section id="introduction"><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
</figure><br />
<h2>Introduction</h2><br />
<br />
<br />
<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
</p><br />
<br />
</section><br />
<section id="aim"><br />
<h2>Aims</h2><br />
<br />
<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and Kombucha-isolated strain and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
<br />
<br />
</section><br />
<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
</figure><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.<br />
<br />
In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. We are currently in the process of completing the bioinformatics analyses of the genomes, and will publish the full genome sequences soon.<br />
<br />
</p><br />
</section><br />
<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and KI strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and KI strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus KI strains to increase cellulose productivity and decrease its cost.</p><br />
<div class="accordion"><br />
<h3>Characterization of G.xylinus ATCC 53582 and KI strains</h3><br />
<div><br />
<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
</p><br />
<p> Cellulose productivity of G.xylinus ATCC 53582 and KI on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the KI strain is approximately one fifth of that of ATCC 53582 strain (see Figure X for comparison of ATCC53582 and KI strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for KI cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus KI). <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 3. Cellulose production of G.xylinus ATCC 53582 and KI strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
</figure><br />
</p> <br />
<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure Y.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, KI strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 2 for comparison of ATCC and KI strains and details of he experiment). This could be due to the niche KI strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 4. Cellulose productivity of G.xylinum ATCC 53582 and KI strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
</figure><br />
</p> <br />
<p> Natural antibiotics resistance of ATCC 53582 and KI strains </p><br />
<p></p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 5. Natural antibiotics resistance of G.xylinus ATCC 53582 and KI strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and KI seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
</figure><br />
<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 5 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and KI strain). However, when using high cell densities for plating (after transformations), antibiotic -resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus KI is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the KI strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
<br />
<br />
</div><br />
<h3>Plasmid backbones</h3><br />
<div><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 1. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus KI after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
</figure><br />
<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and Ki strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus KI and ATCC 53583 (See figure X). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
</ul><br />
</div><br />
<h3>Anderson promoters</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
</div><br />
<h3>Inducible promoters</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p> <br />
</div><br />
<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p>One key drawback of producing cellulose using the obligate aerobe <i>G. xylinus</i> is that it cannot grow below the medium surface in static culture[1], reducing the overall volume of the pellicle that is biologically active. This drawback has been overcome to some extent by transforming <i>G. xylinus</i> with the gene for bacterial hemoglobin from <i>Vitreoscilla</i>[2], another obligate aerobe that expresses this protein in oxygen-poor environments.</p><br />
<br />
<p><i>Vitreoscilla</i> hemoglobin (VHb) is a monomeric heme-containing protein that appears to improve the metabolic function of obligate aerobes and facultative anaerobes in low-oxygen conditions[3][4][5][6]. Evidence suggests that the protein binds oxygen, then shuttles it to at least one cytochrome in the electron transport chain[7], improving the rate of oxidative phosphorylation and therefore ATP production even when dissolved oxygen is scarce.</p><br />
<br />
<p>We cloned the Vitreoscilla hemoglobin gene (<em> Vhb </em>) behind a medium-to-strong Anderson promoter J23101 and the Elowitz RBS - part <a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a> in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure Z) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass. </p><br />
<figure class="content-image"><br />
<img class="image-full image-right" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure Z. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. <em>G.xylinus </em> igem was cultured in 5ml of HS-cellulase medium (in 50ml Falcon tubes with loose caps) at 30degC, 180rpm shaking for 4 days. OD600 was measured using a spectrophotometer. Samples were diluted 1:1 in HS-cellulase medium before measurement. N=3 (Vhb) and 4(wild-type), error bars denote SD. </figcaption><br />
</figure><br />
</div><br />
<br />
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</div><br />
</section><br />
<section id="engineering"><br />
<h2>Engineering</h2><br />
<br />
<div class="accordion"><br />
<h3>Sequencing the genomes of <em>G. xylinus</em> ATCC 53582 and Kombucha-isolated <em>G. xylinus</em> strains</h3><br />
<div><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of <em>G. xylinus</em> is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.</p><br />
<p>In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. <br />
We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction).<br />
We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq present at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9063619 reads with 98.71% reads identified). We completed the genome assembly with the support of Imperial Bioinformatics Support service using their in-house assembly pipeline. </p><br />
</div><br />
<h3>Creating a toolbox for Gluconacetobacter genetic engineering</h3><br />
<div><br />
<p>In parallel to sequencing the genome, we began building a large toolbox for <em>G. xylinus genetic engineering</em>. Only a few vectors have been developed for <em>G. xylinus</em>, and most of them were not available for us for various reasons (including patents, materials-transfer agreements, and retired or deceased researchers). For this reason, we first tested out a set of new plasmids, and engineered them to be Biobrick compatible. As some <em>E. coli</em> promoters and proteins have been shown to work in <em>Gluconacetobacter</em> or <em>Acetobacter</em> species (Setyawati, 2007; Chien et al., 2006), and similarly to <em>E.coli</em>, <em>G. xylinus</em> is a Gram- species, we decided to test out and modify already existing Biobricks in the parts registry in <em>G. xylinus</em>. Therefore, the toolkit consists of:</p><br />
<ul><br />
<li>New, Biobrick compatible shuttle vectors for <em>Gluconacetobacter</em> and <em>E. coli</em><br />
<ul><br />
<br />
<li><em>pSEVA321</em><br />
</li><br />
<li><em>pSEVA331</em><br />
</li><br />
<li><em>pSEVA351</em><br />
</li><br />
<li><em>pBAV1K</em><br />
</li><br />
<br />
</ul><br />
</li>f<br />
<br />
<li>Characterization and optimization of parts from the Registry, including:<br />
<ul><br />
<li>Fluorescent proteins, GFP, RFP, YFP, CFP<br />
</li><br />
<li>Chromoproteins</li><br />
<li>Vitreoscilla haemoglobin<br />
</li><br />
<li>The entire Anderson promoter library<br />
</li><br />
<li>Widely used gene expression control proteins AraC and TetR.<br />
</li><br />
<br />
<br />
</ul><br />
<br />
</li><br />
</ul><br />
</div><br />
<h3>Engineering a high cellulose-producing ATCC 53582 strain</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
<br />
<br />
</div><br />
<br />
</div><br />
</section><br />
<br />
<br />
<section id="results"><br />
<h2>Results</h2><br />
<p>We have isolated a new strain of Acetobacter from kombucha, and determined its growth rate, cellulose productivity, and natural antibiotic resistance. We have also developed a set of methods for genetic engineering of this strain and have determined five new plasmids capable of replication in this strain. Thus we are the first to genetically engineer it.</p><br />
<p>We have discovered four new plasmids capable of replication in Acetobacter species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K - both in G.xylinus ATCC 53582 strain, and in the strain we isolated from Kombucha. We have biobricked these plasmids, with the exception of pBAV1K, which had been originally engineered to be biobrick compatible (however, pBAV1K can not be uploaded to parts registry due to binding materials-transfer agreements).<br />
</p><br />
</section><br />
<br />
<section id="references"><br />
<h3>References</h3><br />
<ul><br />
<br />
<li>[1] https://www.researchgate.net/publication/227802179_Optimizing_the_Production_of_Bacterial_Cellulose_in_Surface_Culture_Evaluation_of_Substrate_Mass_Transfer_Influences_on_the_Bioreaction_%28Part_1%29 - Optimizing the production of bacterial cellulose in surface culture: Evaluation of substrate mass transfer influences on the bioreaction</li><br />
<li>[2] http://www.ncbi.nlm.nih.gov/pubmed/17868946 - Expressing Vitreoscilla hemoglobin in statically cultured Acetobacter xylinum with reduced O2 tension maximizes bacterial cellulose pellicle production</li><br />
<li>[3] http://www.ncbi.nlm.nih.gov/pubmed/2850971 - Cloning, characterisation and expression of the hemoglobin gene from Vitreoscilla in Escherichia coli</li><br />
<li>[4] http://www.ncbi.nlm.nih.gov/pubmed/11478898 - Monomer-dimer equilibrium and oxygen-binding properties of ferrous Vitreoscilla hemoglobin</li><br />
<li>[5] http://onlinelibrary.wiley.com/doi/10.1021/bp960071v/full - Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin for enhancing Escherichia coli growth in a microaerobic bioreactor</li><br />
<li>[6] http://www.nature.com/nbt/journal/v11/n8/full/nbt0893-926.html - The production of cephalosporin C by Aecremonium chrysogenum is improved by the intracellular expression of bacterial hemoglobin</li><br />
<li>[7] http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1994.tb19931.x/full - Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions</li><br />
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<li><a data-scroll href="#featured">Featured Parts</a><br />
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<h3><a href="http://parts.igem.org/Part:BBa_K1321340">BBa_K1321340</a></h3><br />
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<div class="pure-u-1-3"><br />
<p>N-terminal linker + double CBD</p><br />
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<p>Double cellulose binding domain (dCBD) using two cellulose binding domains from <i>Trichoderma reesei</i> cellobiohydrolases, with an N-terminal linker and internal linker sequence between the two domains which are derived from the endogenous cellobiohydrolase linker sequence. This part is in RFC(25) Freiberg fusion format to allow for easy use in protein fusions. dCBD binding affinity for cellulose was <a href="https://2014.igem.org/Team:Imperial/Functionalisation"> characterised </a> using sfGFP fusions.<br />
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<h3><a href="http://parts.igem.org/Part:BBa_K1321339">BBa_K1321339</a></h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p>CBDcenA + Linker</p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>Cellulose binding domain (CBD) of Endoglucanase A (cenA) from <i>Cellulomonas fimi</i> with an endogenous C-terminal linker. The part is in Freiburg format (RFC 25) for ease of use in protein fusions.</p><br />
</div><br />
</div><br />
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<br />
</div><br />
<h3><a href="http://parts.igem.org/Part:BBa_K1321014"> BBa_K1321014</a></h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p>CBDcipA with N and C-terminal linker </p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>This CBD is from the Cellulosomal -scaffolding protein A (cipA) of <i>Clostridium thermocellum </i> including the endogenous linker sequences at the N and C-terminus. It is in RFC25 format to allow for easy use in protein fusions. At present the cloning for these constructs is still in progress to correct an illegal EcoRI site which was identified in the parts with this CBD. This can be achieved with a silent mutation via site-directed mutagenesis and we aim to send these parts to the registry once this is complete.<br />
</p><br />
</div><br />
</div><br />
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<h3><a href="http://parts.igem.org/Part:BBa_K1321005">BBa_K1321005</a></h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p>Synthetic Phytochelatin (PC) EC20</p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>A general heavy metal-binding peptide consisting of 20 Glu-Cys (EC) repeats in Freiburg format (RFC[25]) to allow for easy use in fusion proteins. We created a library of CBD fusions with this part and demonstrated [INSERT FINAL RESULTS SENTENCE HERE]<br />
</p><br />
</div><br />
</div><br />
</div><br />
<h3><a href="http://parts.igem.org/Part:BBa_K1321200">BBa_K1321200</a></h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p><i>Vitreoscilla</i> haemoglobin (VHb)</p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>This part is a haemoglobin isolated from Vitreoscilla (VHb) which increases cell metabolism. We used it to increase cellulose production and growth rate in <i> G. xylinus </i>.<br />
</p><br />
</div><br />
</div><br />
<br />
</div><br />
<h3><a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a>, <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p>pSEVA331 and pSEVA321 backbones</p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>These are two broad host range vector backbones for which we have demonstrated use in <i>E. coli</i> and <i>Gluconacetobacter xylinus </i>.<br />
</p><br />
</div><br />
</div> <br />
</div><br />
<h3><a href="http://parts.igem.org/Part:BBa_K1321334"> BBa_K1321334</a>, <a href="http://parts.igem.org/Part:BBa_K1321335">BBa_K1321335</a></h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p>Cellulose synthase operon AcsAB and AcsCD</p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>Designed and refactored from <i>G. xylinus</i>, this operon was inserted into <i>E.coli </i> for cellulose synthesis. <br />
</p><br />
</div><br />
</div> <br />
<br />
</div><br />
<h3><a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a>,<a href="http://parts.igem.org/Part:BBa_K1321306"> BBa_K1321306</a></h3><br />
<div><br />
<div class="pure-g"><br />
<div class="pure-u-1-3"><br />
<p><i>Gluconacetobacter xylinus</i> strains ATCC53582 and Kombucha Isolate</p><br />
</div><br />
<div class="pure-u-2-3"><br />
<p>We created and characterised a library of parts for this organism, with the aim to increase the accessibility the chassis for future work in iGEM. By sharing these strains and their genomes on the registry we hope this will contribute to their ease of use and characterisation.<br />
</p><br />
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<h2>Table of Parts</h2><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2014-10-18T00:38:17Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
</li><br />
<li><a data-scroll href="#aim">Aims</a><br />
</li><br />
<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
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<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
</li><br />
<li><a data-scroll href="#engineering">Engineering</a><br />
</li><br />
<br />
<li><a data-scroll href="#results">Results</a><br />
</li><br />
<li><a data-scroll href="#references">References</a><br />
</li><br />
</ul><br />
<br class="clearLeft" /><br />
</div><br />
</div><br />
<section id="overview"><br />
<div class="pure-g"><br />
<div class="pure-u-1-2"><br />
<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus KI</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and KI, creating a genetic toolbox of consisting of five new plasmid backbones and 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
</div><br />
<div class="pure-u-1-2"><br />
<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em></li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem</li> <br />
<br />
<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em></li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a powerful platform for the synthesis of new cellulose-based biomaterials and water filters</li><br />
<br />
<br />
<br />
<br />
<br />
</ul><br />
</div><br />
<br />
<br />
</div><br />
<br />
</section><br />
<section id="introduction"><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
</figure><br />
<h2>Introduction</h2><br />
<br />
<br />
<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
</p><br />
<br />
</section><br />
<section id="aim"><br />
<h2>Aims</h2><br />
<br />
<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and Kombucha-isolated strain and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
<br />
<br />
</section><br />
<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
</figure><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.<br />
<br />
In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. We are currently in the process of completing the bioinformatics analyses of the genomes, and will publish the full genome sequences soon.<br />
<br />
</p><br />
</section><br />
<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and KI strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and KI strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus KI strains to increase cellulose productivity and decrease its cost.</p><br />
<div class="accordion"><br />
<h3>Characterization of G.xylinus ATCC 53582 and KI strains</h3><br />
<div><br />
<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
</p><br />
<p> Cellulose productivity of G.xylinus ATCC 53582 and KI on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the KI strain is approximately one fifth of that of ATCC 53582 strain (see Figure X for comparison of ATCC53582 and KI strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for KI cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus KI). <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 3. Cellulose production of G.xylinus ATCC 53582 and KI strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
</figure><br />
</p> <br />
<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure Y.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, KI strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 2 for comparison of ATCC and KI strains and details of he experiment). This could be due to the niche KI strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 4. Cellulose productivity of G.xylinum ATCC 53582 and KI strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
</figure><br />
</p> <br />
<p> Natural antibiotics resistance of ATCC 53582 and KI strains </p><br />
<p></p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 5. Natural antibiotics resistance of G.xylinus ATCC 53582 and KI strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and KI seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
</figure><br />
<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 5 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and KI strain). However, when using high cell densities for plating (after transformations), antibiotic -resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus KI is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the KI strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
<br />
<br />
</div><br />
<h3>Plasmid backbones</h3><br />
<div><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 1. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus KI after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
</figure><br />
<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and Ki strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus KI and ATCC 53583 (See figure X). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
</ul><br />
</div><br />
<h3>Anderson promoters</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
</div><br />
<h3>Inducible promoters</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p> <br />
</div><br />
<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p>We cloned the Vitreoscilla hemoglobin gene (<em> Vhb </em>) behind a medium-to-strong Anderson promoter <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure Z) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass, most likely by increasing oxygen availability to cells. </p><br />
<figure class="content-image"><br />
<img class="image-full image-right" src="https://static.igem.org/mediawiki/2014/f/f6/IC14_Vhb_effects_on_growth_correct.jpg"><br />
<figcaption>Figure Z. Effects of Vitreoscilla hemoglobin expression on G.xylinus maximum biomass production. </figcaption><br />
</figure><br />
</div><br />
<br />
<br />
</div><br />
</section><br />
<section id="engineering"><br />
<h2>Engineering</h2><br />
<br />
<div class="accordion"><br />
<h3>Sequencing the genomes of <em>G. xylinus</em> ATCC 53582 and Kombucha-isolated <em>G. xylinus</em> strains</h3><br />
<div><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of <em>G. xylinus</em> is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.</p><br />
<p>In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. <br />
We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction).<br />
We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq present at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9063619 reads with 98.71% reads identified). We completed the genome assembly with the support of Imperial Bioinformatics Support service using their in-house assembly pipeline. </p><br />
</div><br />
<h3>Creating a toolbox for Gluconacetobacter genetic engineering</h3><br />
<div><br />
<p>In parallel to sequencing the genome, we began building a large toolbox for <em>G. xylinus genetic engineering</em>. Only a few vectors have been developed for <em>G. xylinus</em>, and most of them were not available for us for various reasons (including patents, materials-transfer agreements, and retired or deceased researchers). For this reason, we first tested out a set of new plasmids, and engineered them to be Biobrick compatible. As some <em>E. coli</em> promoters and proteins have been shown to work in <em>Gluconacetobacter</em> or <em>Acetobacter</em> species (Setyawati, 2007; Chien et al., 2006), and similarly to <em>E.coli</em>, <em>G. xylinus</em> is a Gram- species, we decided to test out and modify already existing Biobricks in the parts registry in <em>G. xylinus</em>. Therefore, the toolkit consists of:</p><br />
<ul><br />
<li>New, Biobrick compatible shuttle vectors for <em>Gluconacetobacter</em> and <em>E. coli</em><br />
<ul><br />
<br />
<li><em>pSEVA321</em><br />
</li><br />
<li><em>pSEVA331</em><br />
</li><br />
<li><em>pSEVA351</em><br />
</li><br />
<li><em>pBAV1K</em><br />
</li><br />
<br />
</ul><br />
</li>f<br />
<br />
<li>Characterization and optimization of parts from the Registry, including:<br />
<ul><br />
<li>Fluorescent proteins, GFP, RFP, YFP, CFP<br />
</li><br />
<li>Chromoproteins</li><br />
<li>Vitreoscilla haemoglobin<br />
</li><br />
<li>The entire Anderson promoter library<br />
</li><br />
<li>Widely used gene expression control proteins AraC and TetR.<br />
</li><br />
<br />
<br />
</ul><br />
<br />
</li><br />
</ul><br />
</div><br />
<h3>Engineering a high cellulose-producing ATCC 53582 strain</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
<br />
<br />
</div><br />
<br />
</div><br />
</section><br />
<br />
<br />
<section id="results"><br />
<h2>Results</h2><br />
<p>We have isolated a new strain of Acetobacter from kombucha, and determined its growth rate, cellulose productivity, and natural antibiotic resistance. We have also developed a set of methods for genetic engineering of this strain and have determined five new plasmids capable of replication in this strain. Thus we are the first to genetically engineer it.</p><br />
<p>We have discovered four new plasmids capable of replication in Acetobacter species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K - both in G.xylinus ATCC 53582 strain, and in the strain we isolated from Kombucha. We have biobricked these plasmids, with the exception of pBAV1K, which had been originally engineered to be biobrick compatible (however, pBAV1K can not be uploaded to parts registry due to binding materials-transfer agreements).<br />
</p><br />
</section><br />
<br />
<section id="references"><br />
<h3>References</h3><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2014-10-18T00:31:04Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
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<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
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<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
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<li><a data-scroll href="#engineering">Engineering</a><br />
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<li><a data-scroll href="#results">Results</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
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<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus KI</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and KI, creating a genetic toolbox of consisting of five new plasmid backbones and 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
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<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em></li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem</li> <br />
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<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em></li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a powerful platform for the synthesis of new cellulose-based biomaterials and water filters</li><br />
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<section id="introduction"><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
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<h2>Introduction</h2><br />
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<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
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</section><br />
<section id="aim"><br />
<h2>Aims</h2><br />
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<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and Kombucha-isolated strain and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
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</section><br />
<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
</figure><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.<br />
<br />
In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. We are currently in the process of completing the bioinformatics analyses of the genomes, and will publish the full genome sequences soon.<br />
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<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and KI strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and KI strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus KI strains to increase cellulose productivity and decrease its cost.</p><br />
<div class="accordion"><br />
<h3>Characterization of G.xylinus ATCC 53582 and KI strains</h3><br />
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<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
</p><br />
<p> Cellulose productivity of G.xylinus ATCC 53582 and KI on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the KI strain is approximately one fifth of that of ATCC 53582 strain (see Figure X for comparison of ATCC53582 and KI strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for KI cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus KI). <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 3. Cellulose production of G.xylinus ATCC 53582 and KI strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
</figure><br />
</p> <br />
<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure Y.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, KI strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 2 for comparison of ATCC and KI strains and details of he experiment). This could be due to the niche KI strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 4. Cellulose productivity of G.xylinum ATCC 53582 and KI strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
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<p> Natural antibiotics resistance of ATCC 53582 and KI strains </p><br />
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<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 5. Natural antibiotics resistance of G.xylinus ATCC 53582 and KI strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and KI seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
</figure><br />
<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 5 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and KI strain). However, when using high cell densities for plating (after transformations), antibiotic -resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus KI is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the KI strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
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<h3>Plasmid backbones</h3><br />
<div><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 1. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus KI after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
</figure><br />
<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and Ki strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus KI and ATCC 53583 (See figure X). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
</ul><br />
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<h3>Anderson promoters</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
</div><br />
<h3>Inducible promoters</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p> <br />
</div><br />
<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p>We cloned the Vitreoscilla hemoglobin gene (<em> Vhb </em>) behind a medium-to-strong Anderson promoter <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> in pSEVA331-Bb backbone in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb. Expression of Vhb increased the final culture density of <em>G.xylinus</em> igem strain almost two-fold (with a high statistical significance of p<0.0001; see Figure Z) when grown to stationary phase in HS-cellulase medium, demonstrating that expression of Vhb increases the maximum G.xylinus biomass, most likely by increasing oxygen availability to cells. </p><br />
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</section><br />
<section id="engineering"><br />
<h2>Engineering</h2><br />
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<div class="accordion"><br />
<h3>Sequencing the genomes of <em>G. xylinus</em> ATCC 53582 and Kombucha-isolated <em>G. xylinus</em> strains</h3><br />
<div><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of <em>G. xylinus</em> is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.</p><br />
<p>In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. <br />
We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction).<br />
We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq present at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9063619 reads with 98.71% reads identified). We completed the genome assembly with the support of Imperial Bioinformatics Support service using their in-house assembly pipeline. </p><br />
</div><br />
<h3>Creating a toolbox for Gluconacetobacter genetic engineering</h3><br />
<div><br />
<p>In parallel to sequencing the genome, we began building a large toolbox for <em>G. xylinus genetic engineering</em>. Only a few vectors have been developed for <em>G. xylinus</em>, and most of them were not available for us for various reasons (including patents, materials-transfer agreements, and retired or deceased researchers). For this reason, we first tested out a set of new plasmids, and engineered them to be Biobrick compatible. As some <em>E. coli</em> promoters and proteins have been shown to work in <em>Gluconacetobacter</em> or <em>Acetobacter</em> species (Setyawati, 2007; Chien et al., 2006), and similarly to <em>E.coli</em>, <em>G. xylinus</em> is a Gram- species, we decided to test out and modify already existing Biobricks in the parts registry in <em>G. xylinus</em>. Therefore, the toolkit consists of:</p><br />
<ul><br />
<li>New, Biobrick compatible shuttle vectors for <em>Gluconacetobacter</em> and <em>E. coli</em><br />
<ul><br />
<br />
<li><em>pSEVA321</em><br />
</li><br />
<li><em>pSEVA331</em><br />
</li><br />
<li><em>pSEVA351</em><br />
</li><br />
<li><em>pBAV1K</em><br />
</li><br />
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</ul><br />
</li><br />
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<li>Characterization and optimization of parts from the Registry, including:<br />
<ul><br />
<li>Fluorescent proteins, GFP, RFP, YFP, CFP<br />
</li><br />
<li>Chromoproteins</li><br />
<li>Vitreoscilla haemoglobin<br />
</li><br />
<li>The entire Anderson promoter library<br />
</li><br />
<li>Widely used gene expression control proteins AraC and TetR.<br />
</li><br />
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</ul><br />
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</li><br />
</ul><br />
</div><br />
<h3>Engineering a high cellulose-producing ATCC 53582 strain</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
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</section><br />
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<section id="results"><br />
<h2>Results</h2><br />
<p>We have isolated a new strain of Acetobacter from kombucha, and determined its growth rate, cellulose productivity, and natural antibiotic resistance. We have also developed a set of methods for genetic engineering of this strain and have determined five new plasmids capable of replication in this strain. Thus we are the first to genetically engineer it.</p><br />
<p>We have discovered four new plasmids capable of replication in Acetobacter species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K - both in G.xylinus ATCC 53582 strain, and in the strain we isolated from Kombucha. We have biobricked these plasmids, with the exception of pBAV1K, which had been originally engineered to be biobrick compatible (however, pBAV1K can not be uploaded to parts registry due to binding materials-transfer agreements).<br />
</p><br />
</section><br />
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<h3>References</h3><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/File:IC14-mass_production2.pngFile:IC14-mass production2.png2014-10-18T00:25:36Z<p>Mf1512: uploaded a new version of &quot;File:IC14-mass production2.png&quot;: Removed charred line</p>
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<div>Workflow of the processing steps for making cellulose shapes and dyed flexible cellulose.</div>Mf1512http://2014.igem.org/Team:Imperial/GluconacetobacterTeam:Imperial/Gluconacetobacter2014-10-18T00:19:45Z<p>Mf1512: </p>
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<h1><em>G. xylinus</em></h1><br />
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<ul><br />
<li><a data-scroll href="#introduction">Introduction</a><br />
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<li><a data-scroll href="#aim">Aims</a><br />
</li><br />
<li><a data-scroll href="#genome_sequencing">Genome Sequencing</a><br />
</li><br />
<li><a data-scroll href="#toolbox"><em>G. xylinus</em> toolbox</a><br />
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<li><a data-scroll href="#engineering">Engineering</a><br />
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<li><a data-scroll href="#results">Results</a><br />
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<li><a data-scroll href="#references">References</a><br />
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<section id="overview"><br />
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<h2>Overview</h2><br />
<p>Bacterial cellulose has great potential in many areas, including water purification, tissue scaffolds, wound dressings, etc., however, until now, all bacterial cellulose-based materials have been created using chemical or physical post-production processing, not genetic engineering. This is due to the lack of well-developed tools and methods for <em>Gluconacetobacter</em> genetic engineering, as well as the lack of genome sequence of the highest cellulose-producing strain ATCC 53582. We have overcome the numerous difficulties associated with <em>G.xylinus</em> genetic engineering, and turned <em>G.xylinus KI</em> and ATCC 53852 strains into new platforms for the production of cellulose-based biomaterials by sequencing the genomes of ATCC 53582 and KI, creating a genetic toolbox of consisting of five new plasmid backbones and 40 widely used genes, and developing a set of new and improved protocols for <em>G.xylinus</em> genetic engineering. </p><br />
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<div class="pure-u-1-2"><br />
<h2>Key Achievements </h2><br />
<ul><br />
<li>Isolated a new strain of <em>Gluconacetobacter</em> (named <em>G. xylinus</em> igem) from Kombucha tea and characterized its properties fully.</li><br />
<li>Sequenced the previously unknown genomes of <em>G. xylinus</em> ATCC 53582 and <em>G. xylinus</em> igem strains - the first genomes sequenced in the history of iGEM </li><br />
<li>Discovered four new plasmids capable of replication in <em>Gluconacetobacter</em> species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K, which replicate both in <em>G. xylinus</em> ATCC 53582 and igem strains as well as in <em>E. coli</em></li><br />
<li>Were the first in science to create transgenic cells of <em>G.xylinus</em> igem strain </li><br />
<li>Using our discovered plasmids, created a genetic toolbox consisting of 40 genes for <em>G. xylinus</em> engineering and expressed them in the ATCC 53582 and igem</li> <br />
<br />
<li>Developed a set of new and improved protocols for efficient genetic engineering of <em>G. xylinus</em></li><br />
<li>In summary, turned <em>G. xylinus</em> ATCC 53582 and igem strains into new model organisms and developed the necessary tools to create a powerful platform for the synthesis of new cellulose-based biomaterials and water filters</li><br />
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</section><br />
<section id="introduction"><br />
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<img class="image-full" src="https://static.igem.org/mediawiki/2014/f/f3/IC14_IMG_4132_RFP_glow.JPG"><br />
<figcaption> Genetically engineered <em>Gluconacetobacter xylinus</em> expressing RFP (top three) compared to wild-type (bottom). </figcaption><br />
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<h2>Introduction</h2><br />
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<p>Due to its high productivity, <em>G. xylinus</em> is the main species used for the production of bacterial cellulose based products. These range from superior wound dressings, artificial blood vessels, scaffolds for tissue engineering, high-quality speaker membranes, stronger paper, nata de coco and many others (Keshk 2014) .</p><br />
<p><em>G. xylinus</em> has been the subject of the majority of studies into production of bacterial cellulose. However, research into <em>G. xylinus</em> has primarily focused on the effects of different culture conditions (such as composition of growth media, aeration and agitation) on cellulose productivity, productivity of different strains and different post-processing methods. Few attempts have been made to improve cellulose productivity or the physical properties of cellulose through genetic engineering (but see Chien 2006). Consequently most of the genetic engineering methods and tools available for model organisms, such as <em>E.coli</em>, have not been developed for <em>G. xylinus</em>. These tools must first be developed, in order to begin serious efforts to genetically engineer <em>G. xylinus</em> strains capable of producing novel biomaterials, more cellulose, and at a lower cost.<br />
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</section><br />
<section id="aim"><br />
<h2>Aims</h2><br />
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<p>In addition to the lack of tools, the continuous cellulose production of <em>G. xylinus</em> introduces further problems for genetic engineering, as it results in a low growth rate (the division time of <em>G. xylinus</em> is 4 hours, which is 8 times slower than that of <em>E. coli</em>), formation of spontaneous cellulose non-producing mutants in agitated culture (detrimental for the engineering of high-producing strains) and difficulties in performing procedures such as transformation, plasmid DNA extraction, etc. due to the physically interfering cellulose pellicle.</p><br />
<p>Furthermore, although the highest cellulose-producing strain <em>G. xylinus</em> ATCC 53582 has been used in several studies, the genome sequence of this strain is still unknown, making it impossible to carry out targeted engineering of chromosomal genes, which is vital to achieve increased productivity.</p><br />
<p>We aim to solve all of these problems, by completing three major projects: sequencing the genomes of ATCC53582 and Kombucha-isolated strain and creating a large toolbox for <em>G. xylinus</em> genetic. We then aim to use these tools to increase and control cellulose productivity and create new biomaterials with wide-ranging properties, by incorporating proteins with different functions into the cellulose matrix (see <a href="https://2014.igem.org/Team:Imperial/Functionalisation">Functionalisation</a>).</p><br />
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</section><br />
<section id="genome_sequencing"><br />
<h2>Genome sequencing</h2><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/2/23/IC14_Alignment_example_output_file_2.jpg"><br />
<figcaption> Figure1. Example of alignment of reads from genome sequencing against Komagataeibacter xylinusE25 genome. Alignment done using Tablet genome browser. </figcaption><br />
</figure><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of G. xylinus is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.<br />
<br />
In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction). We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9,063,619 reads with 98.71% reads identified; see Figure1 for an example output of read alignment against Komagateibacter xylinus E25 reference genome). We performed the quality control of the reads using FasQC and Trim Galore and completed the genome assembly with the assistance of Imperial Bioinformatics Support service using their in-house assembly pipeline BugBuilder. We are currently in the process of completing the bioinformatics analyses of the genomes, and will publish the full genome sequences soon.<br />
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</section><br />
<section id="toolbox"><br />
<h2>Creating a genetic toolbox for <em>G. xylinus</em></h2><br />
<p>Despite the great potential <em>G. xylinus</em> holds for new biomaterial production, very few tools for genetic engineering had been developed so far. Thus, in parallel to sequencing the genomes, we began building a new system in order to equip G.xylinus with all of the necessary tools and to turn the ATCC 53582 and KI strains into systems for genetically engineered biomaterials production. <br />
We first characterized ATCC 53582 and KI strains, and created a set of new and improved protocols necessary for their genetic engineering (see <a href="https://2014.igem.org/Team:Imperial/Protocols">G.xylinus protocols</a>). As only a few vectors are available for G. xylinus genetic engineering (this is due to various reasons including patents, materials-transfer agreements, and retired or deceased researchers) we first created a set of new biobrick-compatible plasmid backbones capable of replication both in Gluconacetobacter and E.coli. In order to create the genetic toolkit, we decided to test out and modify already existing Biobricks in the parts registry (rather than creating a set of new parts) in order to retain the already existing familiarity genetic engineers have with these genes and make the toolkit more user-friendly. None of these parts had been used in Gluconacetobacter before however, so it was necessary to determine if they can be used in G.xylinus in the first place. Finally, we used this toolkit to clone Vitreoscilla hemoglobin gene Vhb (which is known to increase cellulose productivity and growth rate of G.xylinus (Chien et al. 2006)) behind a strong Anderson promoter, and express it in G.xylinus ATCC 53582 and G.xylinus KI strains to increase cellulose productivity and decrease its cost.</p><br />
<div class="accordion"><br />
<h3>Characterization of G.xylinus ATCC 53582 and KI strains</h3><br />
<div><br />
<p> Parts: <br />
-<a href="http://parts.igem.org/Part:BBa_K1321305">BBa_K1321305</a><br />
-<a href="http://parts.igem.org/Part:BBa_K1321306">BBa_K1321306</a><br />
</p><br />
<p> Cellulose productivity of G.xylinus ATCC 53582 and KI on HS-glucose</p> <br />
<p> G.xylinus cellulose productivity depends strongly on the carbon feedstocks used. In HS-glucose media grown for 7 days at 30degC standing using 50ml HS medium in 250ml conical flasks (sealed with foam buns), cellulose productivity of the KI strain is approximately one fifth of that of ATCC 53582 strain (see Figure X for comparison of ATCC53582 and KI strains and experimental details). ATCC 53582 strain produces around 0.5-0.6g of pure cellulose after 5 days of incubation in these conditions. As 50ml HS medium contains 1g of glucose, ATCC 53582 strain converts glucose to cellulose with around 50-60% efficiency. However, it seems for KI cellulose productivity, HS-glucose, is not optimal, because in HS-glycerol media and HS-sucrose media the cellulose productivity is increased. Importantly, in HS-sucrose medium, cellulose productivity is higher than that of ATCC 53582, which is of industrial significance due to the high availability and low cost of sucrose (see Feedstocks for G.xylinus KI). <br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/b/b3/IC14-ATCC_and_KI_cellulose_productivity_in_HS_correct.jpg"><br />
<figcaption> Figure 3. Cellulose production of G.xylinus ATCC 53582 and KI strains in HS-glucose medium. Cultures were inoculated using 50µl of 48h-grown seed culture, and grown in 50ml HS in 250ml conical flasks, sealed with foam buns at 30degC standing. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD. </figcaption><br />
</figure><br />
</p> <br />
<p>Cellulose productivity on different carbon feedstocks </p><br />
<p>G.xylinus is commonly grown on HS-glucose. However, it can readily use other carbon feedstocks, including sucrose and glycerol. We have found that ATCC 53582 cellulose productivity is highest when grown on glycerol (see Figure Y.), although the effects of routine culturing of ATCC 53582 on HS-glycerol media are unknown. Suprisingly however, KI strain has a higher cellulose productivity in HS-sucrose than G.xylinus ATCC 53582 (see Figure 2 for comparison of ATCC and KI strains and details of he experiment). This could be due to the niche KI strain has evolved into, as the Kombucha tea contains high concentrations (commonly around 100g/L) of sucrose. </p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/5/5c/IC14_ATCC_and_KI_carbon_feedstocks_experimentII_with_acetate_and_negative.jpg"><br />
<figcaption> Figure 4. Cellulose productivity of G.xylinum ATCC 53582 and KI strains in HS medium containing different carbon feedstocks. Glycerol seems to result in highest productivity, however the effects of glycerol on routine culture are not known. All carbon feedstocks were normalized to 2% (w/v). Cells were cultured in 50ml Corning tubes, filled with 20ml HS medium standing at 30C for 10 days, cultured with loose caps to allow diffusion of air, and kept at 4degC until measurement. Cellulose was washed twice with dH20, treated with 0.1M NaOH for 4 hours at 60degC, washed twice with dH20 and dried at 60degC for 48h before measuring cellulose pellicle weight. N=3, error bars denote SD.</figcaption><br />
</figure><br />
</p> <br />
<p> Natural antibiotics resistance of ATCC 53582 and KI strains </p><br />
<p></p><br />
<figure class="content-image image-left"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/4/4c/IC14_XYZNatural_antibiotics_resistance_of_ATCC_53582_and_KI_strains.jpg"><br />
<figcaption> Figure 5. Natural antibiotics resistance of G.xylinus ATCC 53582 and KI strains. Due to small colony diameter and large clustering, colonies were rounded to the nearest hundred, if more than a hundred colonies were present. Plates with over 500 colonies often formed a lawn. Plates were seeded with 50µl of ATCC and KI seed culture normalized to equal OD600, and grown inverted at 30degC for 5 days until colony counting. N=3, error bars denote SD. </figcaption><br />
</figure><br />
<p>Using optimal antibiotics concentrations is critical for selection of transformed cells while not inhibiting the cell growth. G.xylinus ATCC 53582 is not resistant to kanamycin and ampicillin in concentrations normally used for E.coli (50µg/ml and 100µg/ml respectively), however is resistant to chloramphenicol (at 35µg/ml; see Figure 5 for an experiment of ATCC 53582 natural antibiotic concentrations, experimental details, and comparison of ATCC and KI strain). However, when using high cell densities for plating (after transformations), antibiotic -resistant colonies of ATCC 53582 appear at up to 3x concentrations. Thus, the recommended antibiotic concentrations for HS-agar plates are 200µg/ml kanamyicn, 400µg/ml ampicillin and 140µg/ml chloramphenicol.</p> <br />
<p>G.xylinus KI is naturally resistant to kanamycin, ampicillin and chloramphenicol concentrations normally used for E.coli. Also, we have found that the frequency of appearance of antibiotic-resistant colonies is dependent on the number of cells used for plating - with higher cell numbers, resistant colonies can be found on higher antibiotics concentrations. We have found that the KI strain forms antibiotic-resistant colonies up to 6x antibiotic concentrations used for E.coli, thus the recommended amounts of antibiotics for transformations are 350µg/ml kanamycin, 700µg/ml ampicillin or 245µg/ml chloramphenicol.</p><br />
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<h3>Plasmid backbones</h3><br />
<div><br />
<figure class="content-image"><br />
<img class="image-full" src="https://static.igem.org/mediawiki/2014/3/31/IC14-Plasmid_backbones_in_G.xylinus.jpg"><br />
<figcaption>Figure 1. PCR confirmation of replication of pSEVA321, pSEVA331, pSEVA351, pBla and pBAV1K in G.xylinus. DNA source is miniprepped plasmid DNA from G.xylinus KI after transformation and culturing (we had similar results with the ATCC 53582 strain.) L- NEB 2-log ladder, Ctr- Negative control, roman numerals denote replicates. Positive controls were miniprepped from E.coli prior to the experiment. Expected band sizes of positive results: pSEVA321-351: 328bp, pBla approximately 700bp, pBAV1K-985bp. 1% agarose gel, run at 100V, 20 min.</figcaption><br />
</figure><br />
<p>We tested 9 different plasmid backbones previously not known to replicate in G.xylinus - pSEVA311, pSEVA321, pSEVA331, pSEVA341, pSEVA351, pBla-Vhb-122 (from Chien et al. 2006), pBAV1K-T5-sfgfp, pSB1C3 and pBca1020 (part number BBa_J61002) in order to create a set of plasmid backbones with different copy numbers for G.xylinus genetic engineering. We used pBla-Vhb-122 as the positive control, as it has been previously shown to replicate in G.xylinus (see Chien et al. 2006). In order to test for replication, we prepared two different sets of electrocompetent cells from both ATCC 53582 and Ki strains (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Preparing electrocompetent cells</a>) and transformed using 33µl, 50µl and 100µl of electrocompetent cells and 50µg, 200µg and 500µg of DNA (see <a href="https://2014.igem.org/Team:Imperial/Protocols">Transformation of G.xylinus using electroporation</a>). Transformants were plated out on 4 different antibiotic concentrations for each transformation: 0.5x, 1x, 2x and 6x concentrations (this refers to concentrations of antibiotics commonly used for E.coli, which are 50µg/ml kanamycin, 100µg/ml ampicillin and 35µg/ml chloramphenicol). Colonies were then inoculated into HS-cellulase medium and grown for 3 days shaking at 180RPM with appropriate antibiotics, after which plasmid DNA was miniprepped using the QiaPrep Spin Miniprep kit, according to manufacturer's instructions. Miniprepped DNA was then used as a template for PCR to determine plasmid-containing colonies. Primers used for PCR had been previously verified with pure plasmid DNA. DNA sequence of successfully amplified plasmids were confirmed by Sanger sequencing.</p><br />
<p>We discovered that 5 out of the 9 tested plasmids - pSEVA321, pSEVA331, pSEVA351, pBAV1K and pBla-Vhb-122 (as the positive control)- were capable of replication in both G.xylinus KI and ATCC 53583 (See figure X). No positive results were obtained for other plasmids, indicating that the rest can not replicate in G.xylinus, or were not transformed due to other reasons. We converted pSEVA321 and pSEVA331 into a biobrick-compatible format by inserting the biobrick prefix and suffix in place of the original multiple cloning site using PCR with mutagenic primers and are currently in the process of converting pSEVA351 and pBla-Vhb-122 (converting them into biobrick format has required more time, as they contain several forbidden restriction enzyme sites requiring multiple steps of mutagenesis). pBAV1K was already engineered to be biobrick compatible by authors. We have also verified replication of all of these plasmids in E.coli. Thus, we have determined 5 new plasmid backbones that can act as shuttle vectors for G.xylinus genetic engineering: </p> <br />
<ul><br />
<li> pSEVA321-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321301">BBa_K1321301</a> </li> <br />
<li> pSEVA331-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321300">BBa_K1321300</a> <br />
<li> pSEVA351-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321307">BBa_K1321307</a> <br />
<li> pBla-Vhb-122-Bb - part <a href="http://parts.igem.org/Part:BBa_K1321308">BBa_K1321308</a> <br />
<li> pBAV1K - part <a href="http://parts.igem.org/Part:BBa_K1321309">BBa_K1321309</a> </li> <br />
</ul><br />
</div><br />
<h3>Anderson promoters</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
</div><br />
<h3>Inducible promoters</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p> <br />
</div><br />
<h3>Vitreoscilla hemoglobin</h3><br />
<div><br />
<p>We cloned the Vitreoscilla hemoglobin gene (<em> Vhb </em>) behind a medium-to-strong Anderson promoter <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> in order to increase growth rate and cellulose productivity of <em>G. xylinus</em>. We chose J23101 in order not to overburden the cell with protein expression, while still providing a large amount of Vhb as it is not hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
</div><br />
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</section><br />
<section id="engineering"><br />
<h2>Engineering</h2><br />
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<div class="accordion"><br />
<h3>Sequencing the genomes of <em>G. xylinus</em> ATCC 53582 and Kombucha-isolated <em>G. xylinus</em> strains</h3><br />
<div><br />
<p>Although genome sequencing has become widely available due to the improvements in next-generation sequencing, even with smaller bacterial genomes (the genome of <em>G. xylinus</em> is approximately 3.5Mbp in size), genome sequencing is still unavailable for small to mid-sized research groups due to high costs. We performed a survey of prices offered by all commercially available service providers using the Illumina MiSeq next-generation sequencing platform (which is the most cost-effective method for bacterial genome sequencing) in Europe, and found that the average costs of fully sequencing a 3.5Mbp genome (which includes library preparation, sequencing, bioinformatics and gap filling) is approximately £3000.</p><br />
<p>In order to be able to sequence the genome with the limited budget available to our team, we did not use a commercial sequencing service, and decided to perform the full sequencing cycle ourselves, despite having no previous experience in genome sequencing. <br />
We created the gDNA library using Illumina Nextera kit, by modifying the original Illumina protocol to be amenable for small sample number (see gDNA library preparation protocol). During gDNA library preparation, we discovered that cellulose production of G.xylinus resulted in the presence of a persistent contaminant in DNA preparation, which persisted even after four consecutive DNA purification methods. This contamination interfered with effective library preparation, retarding the project several weeks, and ultimately prompted us to create a new and specific protocol for G.xylinus DNA extraction (see G.xylinus DNA extraction).<br />
We performed the sequencing run (multiplexed sequencing of ATCC 53582 and KI in single run, paired-end 250bp reads using standard flow cell) on the Illumina MiSeq present at Imperial College’s Genomics Laboratory. Despite problems with gDNA library preparation, the sequencing yielded good results (9063619 reads with 98.71% reads identified). We completed the genome assembly with the support of Imperial Bioinformatics Support service using their in-house assembly pipeline. </p><br />
</div><br />
<h3>Creating a toolbox for Gluconacetobacter genetic engineering</h3><br />
<div><br />
<p>In parallel to sequencing the genome, we began building a large toolbox for <em>G. xylinus genetic engineering</em>. Only a few vectors have been developed for <em>G. xylinus</em>, and most of them were not available for us for various reasons (including patents, materials-transfer agreements, and retired or deceased researchers). For this reason, we first tested out a set of new plasmids, and engineered them to be Biobrick compatible. As some <em>E. coli</em> promoters and proteins have been shown to work in <em>Gluconacetobacter</em> or <em>Acetobacter</em> species (Setyawati, 2007; Chien et al., 2006), and similarly to <em>E.coli</em>, <em>G. xylinus</em> is a Gram- species, we decided to test out and modify already existing Biobricks in the parts registry in <em>G. xylinus</em>. Therefore, the toolkit consists of:</p><br />
<ul><br />
<li>New, Biobrick compatible shuttle vectors for <em>Gluconacetobacter</em> and <em>E. coli</em><br />
<ul><br />
<br />
<li><em>pSEVA321</em><br />
</li><br />
<li><em>pSEVA331</em><br />
</li><br />
<li><em>pSEVA351</em><br />
</li><br />
<li><em>pBAV1K</em><br />
</li><br />
<br />
</ul><br />
</li><br />
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<li>Characterization and optimization of parts from the Registry, including:<br />
<ul><br />
<li>Fluorescent proteins, GFP, RFP, YFP, CFP<br />
</li><br />
<li>Chromoproteins</li><br />
<li>Vitreoscilla haemoglobin<br />
</li><br />
<li>The entire Anderson promoter library<br />
</li><br />
<li>Widely used gene expression control proteins AraC and TetR.<br />
</li><br />
<br />
<br />
</ul><br />
<br />
</li><br />
</ul><br />
</div><br />
<h3>Engineering a high cellulose-producing ATCC 53582 strain</h3><br />
<div><br />
<p>We aim to increase cellulose productivity by inserting the <em>Vitreoscilla</em> hemoglobin gene into <em>G. xylinus</em> and finding the optimum expression level by expressing <em>Vitreoscilla</em> hemoglobin behind different Anderson promoters. (<em>Vitreoscilla</em> haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).</p><br />
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</section><br />
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<section id="results"><br />
<h2>Results</h2><br />
<p>We have isolated a new strain of Acetobacter from kombucha, and determined its growth rate, cellulose productivity, and natural antibiotic resistance. We have also developed a set of methods for genetic engineering of this strain and have determined five new plasmids capable of replication in this strain. Thus we are the first to genetically engineer it.</p><br />
<p>We have discovered four new plasmids capable of replication in Acetobacter species - pSEVA321, pSEVA331, pSEVA351 and pBAV1K - both in G.xylinus ATCC 53582 strain, and in the strain we isolated from Kombucha. We have biobricked these plasmids, with the exception of pBAV1K, which had been originally engineered to be biobrick compatible (however, pBAV1K can not be uploaded to parts registry due to binding materials-transfer agreements).<br />
</p><br />
</section><br />
<br />
<section id="references"><br />
<h3>References</h3><br />
<ol><br />
<li></li><br />
<li></li><br />
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{{:Team:Imperial/Templates:footer}}</div>Mf1512http://2014.igem.org/File:IC14-mass_production2.pngFile:IC14-mass production2.png2014-10-18T00:13:25Z<p>Mf1512: Workflow of the processing steps for making cellulose shapes and dyed flexible cellulose.</p>
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<div>Workflow of the processing steps for making cellulose shapes and dyed flexible cellulose.</div>Mf1512