Introduction

Due to its high productivity, G. xylinus 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) .

G. xylinus has been the subject of the majority of studies into production of bacterial cellulose. However, research into G. xylinus 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 E.coli, have not been developed for G. xylinus. These tools must first be developed, in order to begin serious efforts to genetically engineer G. xylinus strains capable of producing novel biomaterials, more cellulose, and at a lower cost.

Aims

In addition to the lack of tools, the continuous cellulose production of G. xylinus introduces further problems for genetic engineering, as it results in a low growth rate (the division time of G. xylinus is 4 hours, which is 8 times slower than that of E. coli), 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.

Furthermore, although the highest cellulose-producing strain G. xylinus 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.

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 G. xylinus 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 Functionalisation).

Genome sequencing

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. 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 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.

Creating a genetic toolbox for G.xylinus

Despite the great potential G.xylinus 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. We first characterized ATCC 53582 and KI strains, and created a set of new and improved protocols necessary for their genetic engineering (see G.xylinus protocols). 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 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 genes had been expressed 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.

Engineering

Modelling

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Testing

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Results

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.

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).

References