Congratulations to IGEM 2016 Medalists!
We at the BGSC view supporting STEM education as one of our most important roles. For this reason I want to take a moment to congratulate two gold medalists at the 2016 iGEM competition. If you are not familiar with iGEM (International Genetically Engineered Machine), you should be! This year, over 5000 students in 42 nations participated at the high school, undergraduate, and overgraduate levels. The competition culminated in a jamboree held October 27-31 in Boson, Massachusetts, where over 3000 gathered to share and celebrate their achievements. The BGSC is proud to have supplied strains and advice to two gold medalists. (Please let me know if I am forgetting anyone!) Team Freiburg explored the use of Bacillus subtilis spore display for the targeted delivery of therapeutic drugs, with a test case of immune suppression therapy for ulcerative colitis. For more on their work, see their project website. Team UC Davis asked whether B. subtilis could be engineered to produce natural food colorants. Their proof of concept experiments suggested that cyanobacterial protein pigments could potentially replace Blue dye #1, or Brilliant Blue. For details, see the project website. A shout out to both teams! There are still plenty of Bacillus-related project for future IGEM competitions, and the BGSC is here to help.
Gene Expression Toolbox for B. subtilis
Sarah Guiziou, from the Jerome Bonnet lab at the University of Montpellier, has graciously donated a large set of plasmids and strains comprising a toolkit allowing tunable gene expression in Bacillus subtilis. The amyE integration vectors in the set contain various arrangements of natural promoters, optimized RBS sequences, and protein degradation tags. By fusing the constructs to sfGFP reporters, Guiziou et al. achieved a range of expression corresponding to an average number of GFP molecules per cell varying from 15 to 270000, a span of more than five orders of magnitude. (Some of the higher expression levels result in B. subtilis constructs that look distinctly bright yellow-green under ordinary room lighting!) A complete listing of the plasmids and B. subtilis strains in the set are beyond the scope of this news item, but I encourage you to read the paper. Supplementary data file 1, an Excel spreadsheet detailing expression levels, is especially helpful. We thank Guizhiou and colleagues for these valuable tools.
A Safe Simulant for Bacillus anthracis
Safe simulants that closely mimic the select agent Bacillus anthracis are needed both for laboratory and field studies. B. anthracis belongs to the Bacillus cereus group (BCG) of species. Members of the BCG are nearly identical is cell and spore morphology due to highly similar genome sequence and content. They differ primarily in toxins and virulence factors, many of which are encoded by megaplasmids that differ among isolates. B. thuringiensis (Bt) likewise belongs to the BCG. Some naturally insecticidal Bt strains have been safely used in agriculture for over half a century. Non-insecticidal derivatives of standard Bt strains would seem to make ideal simulants for B. anthracis. For this reason, Alistair Bishop and colleagues at the Defence Science and Technology Laboratory, Salisbury, Wiltshire, UK, have developed plasmid-cured derivatives of Bt kurstaki strain HD1. One of them, HD-1 Cry-, has been demonstrated to be particularly useful in studies of spore aerisolization, dispersal, and decontamination. We thank Dr. Bishop for depositing B. thuringiensis HD-1 Cry- in the BGSC. It is available as BGSC 4D24.
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New! Protease-free Bacillus subtilis host
Bacillus subtilis is widely used as a production platform for synthesizing enzymes, pharmaceuticals, and fine chemicals (1). Unfortunately, B. subtilis 168 secretes no fewer than seven proteases during vegetative growth and stationary phase. Strains in which multiple protease genes have been inactivated have proved to be superior to wild type strains in production of foreign proteins (2, 3). I have now constructed a seven-protease deletion strain that is free from antibiotic resistance genes or integrated plasmids. This strain, B. subtilis KO7, was generated from the commonly used laboratory host, PY79, by sequentially knocking out the coding sequences. At each step, I transformed the strain with the appropriate BKE cassette for knocking out one of the loci, removing the erythromycin resistance gene with the Cre-producing plasmid pDR244, and finally heat-curing the plasmid. All seven knockouts in KO7 were confirmed by sequencing to be marker-free, in-frame deletions with a 150-bp scar replacing the coding sequence. KO7 is prototrophic and grows rapidly in standard minimal media for B. subtilis. I am placing strain KO7 in the public domain and disclaim all downstream rights to any process or product that you develop with it. It will be available from the BGSC as accession number 1A1133. Standard user fees apply. Later this summer I hope to introduce further elaborations of KO7, such as restriction-negative and asporogenous variants. Feel free to put in requests for particular features!
BGSC Accession: 1A1133
Original Code: Bacillus subtilis KO7
Reference: Zeigler DR, unpublished
Genotype: ΔnprE ΔaprE Δepr Δmpr ΔnprB Δvpr Δbpr
Description: Free of secreted proteases; marker-free deletions in PY79 genetic background; prototrophic
B. subtilis essential gene knockdown library
The BGSC is excited to announce the availability of a new collection of Bacillus subtilis 168 mutants designed to explore the functions of 289 essential genes in this organism. The paper describing the construction of this library and its initial characterization will be released online today (26 May 2016) and will appear in the June 2 edition of Cell. The paper is a collaboration among labs at the University of California, San Francisco, Stanford University, University of California, Berkeley, and McMaster University, Hamilton, Ontario. The co-first authors are Jason M. Peters of UCSF and Alexandre Colavin and Handuo Shi of Stanford.
The library uses a CRISPR interference (CRISPRi) strategy to created a tunable “knockdown” of individual essential genes. Every strain in the library has a Streptococcus pyogenes dcas9 gene integrated into the B. subtilis lacA locus, where it has been placed under control of the xylose-inducible Pxyl promoter. Each strain also has a single-guide RNA (sgRNA) targeting a specific essential gene. The sgRNA coding sequence is integrated into B. subitlis amyE, where it has been placed under the control of the strongly constitutive Pveg promoter. The dCas9 protein lacks nuclease activity. But when dCas9 is present, the sgRNA enables it to bind to the 5’ end of the target gene, where it effectively blocks transcription via steric hindrance. Basal level expression of dcas9 in the absence of xylose knocks down expression of the targeted essential gene about 3-fold. This reduction creates subtle phenotypes, such as increased sensitivity to specific antibiotics and chemical inhibitors, but allows for essentially normal growth under standard laboratory conditions. Full induction of dcas9 with xylose (1%) reduces expression of the essential gene ~150-fold, with drastic consequences for cell morphology and viability. Varying the concentration of xylose between 0.001% and 0.1% allows tunable expression of the essential gene. Peters et al. have not only reported the construction of the library, but have demonstrated its power for analyzing essential genes. They used chemical genomics, for example, to reveal the essential gene network of B. subtilis, revealing interesting connections between seemingly unrelated processes.
These strains provide an invaluable tool for a systematic study of essential genes in a bacterial model system. We thank Jason Peters, Carol Gross, and the entire consortium for donating the library to the BGSC, and we look forward to supplying strains from it to scientists from the B. subtilis research community and beyond. For a complete list of the genes targeted in the library, please see the Peters et al. publication. Summaries of what has been learned previously about most of these genes can be accessed at SubtiWiki. It will take a little while for us to update the BGSC online database to include these strains, but they are available for immediate distribution. But their naming convention is simple. The numeric portion of the gene’s locus tag is appended to the prefix “BEC” to produce the strain name. Hence the knockdown strain for the essential gene ligA, which encodes DNA ligase and carries the locus tag BSU06620, is BCE06620. The full genotype of this strain is lacA::Pxyl-dcas9 amyE::Pveg-sgRNA(ligA) trpC2, and it carries resistance markers for erythromycin and chloramphenicol. Users may request these strains by giving us the targeted gene name or locus tag. Standard user fees apply.
pLIKE vector sequences
Over the past two years, there has been considerable interest in the pLIKE vectors from the Thorsten Mascher lab at Ludwig-Maximilians-University (LMU) Munich. These expression vectors feature a bacitracin-inducible promoter, available both in a replicative plasmid (our ECE255) and an amyE-integration vector (ECE256). For the convenience of our users, we have uploaded maps and DNA sequences of these plasmids onto our website. You can download them here. Thanks to Prof. Mascher for providing this document.
Low-Noise Fluorescent Protein Vectors
Some of the most useful tools for Bacillus geneticists are integrative vectors designed to fuse a promoter of interest to a coding sequence specifying a fluorescent protein. Strains bearing these vectors are among the most widely requested items in the BGSC collection, and improved versions offering some advantage over first generation vectors have regularly appeared on this page. Such is the case today, as we feature the pXFP_Star reporter system, developed and kindly donated by Stephanie Trauth and Ilka B. Bischofs in the Bischofs lab at the University of Heidelberg. These vectors, which come in green, cyan, and yellow “flavors,” feature extremely low noise. A B. subtilis strain with an empty pXFP_Star vector integrated into its chromosome has no greater background fluorescence than the host strain alone. This significant improvement was achieved by including a strong transcription terminator just upstream of the promoter-cloning site. The pXFP_Star vectors will be useful for studying all kinds of promoters, but should be especially advantageous for analyzing those that are weakly expressed. As a bonus, they also are designed to allow ligation independent cloning (LIC). The vectors are available in the following BGSC strains. Note that the NCBI sequence files may not be released for a few weeks, but in the meantime are available from the BGSC upon request.
ECE295 | pGFP_Star | KJ411636
ECE296 | pYFP_Star | KJ411637
ECE297 | pCFP_Star | KJ411638