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.

In parallel to sequencing the genome, we began building a large toolbox for G. xylinus genetic engineering. Only a few vectors have been developed for G. xylinus, 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 E. coli promoters and proteins have been shown to work in Gluconacetobacter or Acetobacter species (Setyawati, 2007; Chien et al., 2006), and similarly to E.coli, G. xylinus is a Gram- species, we decided to test out and modify already existing Biobricks in the parts registry in G. xylinus. Therefore, the toolkit consists of:

• New, Biobrick compatible shuttle vectors for Gluconacetobacter and E. coli
  • pSEVA321
  • pSEVA331
  • pSEVA351
  • pBAV1K
• Characterization and optimization of parts from the Registry, including:
  • Fluorescent proteins, GFP, RFP, YFP, CFP
  • Chromoproteins
  • Vitreoscilla haemoglobin
  • The entire Anderson promoter library
  • Widely used gene expression control proteins AraC and TetR.

We aim to increase cellulose productivity by inserting the Vitreoscilla hemoglobin gene into G. xylinus and finding the optimum expression level by expressing Vitreoscilla hemoglobin behind different Anderson promoters. (Vitreoscilla haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).

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.

In parallel to sequencing the genome, we began building a large toolbox for G. xylinus genetic engineering. Only a few vectors have been developed for G. xylinus, 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 E. coli promoters and proteins have been shown to work in Gluconacetobacter or Acetobacter species (Setyawati, 2007; Chien et al., 2006), and similarly to E.coli, G. xylinus is a Gram- species, we decided to test out and modify already existing Biobricks in the parts registry in G. xylinus. Therefore, the toolkit consists of:

• New, Biobrick compatible shuttle vectors for Gluconacetobacter and E. coli
  • pSEVA321
  • pSEVA331
  • pSEVA351
  • pBAV1K
• Characterization and optimization of parts from the Registry, including:
  • Fluorescent proteins, GFP, RFP, YFP, CFP
  • Chromoproteins
  • Vitreoscilla haemoglobin
  • The entire Anderson promoter library
  • Widely used gene expression control proteins AraC and TetR.

We aim to increase cellulose productivity by inserting the Vitreoscilla hemoglobin gene into G. xylinus and finding the optimum expression level by expressing Vitreoscilla hemoglobin behind different Anderson promoters. (Vitreoscilla haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).

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.

In parallel to sequencing the genome, we began building a large toolbox for G. xylinus genetic engineering. Only a few vectors have been developed for G. xylinus, 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 E. coli promoters and proteins have been shown to work in Gluconacetobacter or Acetobacter species (Setyawati, 2007; Chien et al., 2006), and similarly to E.coli, G. xylinus is a Gram- species, we decided to test out and modify already existing Biobricks in the parts registry in G. xylinus. Therefore, the toolkit consists of:

• New, Biobrick compatible shuttle vectors for Gluconacetobacter and E. coli
  • pSEVA321
  • pSEVA331
  • pSEVA351
  • pBAV1K
• Characterization and optimization of parts from the Registry, including:
  • Fluorescent proteins, GFP, RFP, YFP, CFP
  • Chromoproteins
  • Vitreoscilla haemoglobin
  • The entire Anderson promoter library
  • Widely used gene expression control proteins AraC and TetR.

We aim to increase cellulose productivity by inserting the Vitreoscilla hemoglobin gene into G. xylinus and finding the optimum expression level by expressing Vitreoscilla hemoglobin behind different Anderson promoters. (Vitreoscilla haemoglobin was shown to increase cellulose productivity and growth rate by Chien 2006).