Super-competent strains of Bacillus subtilis
As discovered by John Spizizen in 1958 , Bacillus subtilis 168 can become transiently competent to take up DNA from its environment during early stationary phase. Competence is achieved by a minority population within a culture as a consequence of a developmental change known as the K-state, marked by growth cessation, arrest of septum formation, synthesis of DNA-uptake machinery, and activation of recombination-repair systems . Entry of cells into the K-state is under control of the master regulatory protein ComK . Over-expression of ComK leads to the phenomenon of super-competence, in which essentially every cell in a culture stops growing and takes up any DNA in its immediate environment. Removal of the inducer allows growth to resume.
The BGSC has two super-competent lines of B. subtilis. In the first, strain SCK6, the comK gene has been placed under the control of the xylose-inducible promoter PxylA . Addition of xylose to 1% (w/v) to B. subtilis cultures in LB allowed for plasmid DNA transformation frequencies of up to 10^7 with multimeric plasmid preps or 10^4 with ligated plasmid DNA. In the second, strain REG19, comK has been placed under the control of the mannitol-inducible promoter PmtlA, together with a second competence gene, comS . Transformation frequencies were slightly lower than those reported for SCK6, but still much higher than observed with the parental culture under an ordinary competence protocol.
These super-competent lines essentially eliminate technical difficulties associated with standard competence protocols, which generally require closely-monitored growth curves and extended incubation of cultures in two different minimal media. Their high levels of competence allow for simple alteration of chromosomal sequences via amplification fragments, for example by Gibson assembly . Some care must be taken not to allow non-competent mutants to take over stock cultures; super-competent cell lines tend to form smaller colonies on plates (Zhang, personal communication). But their availability should make complicated strain construction projects much simpler for many applications. Strain SCK6 is available from the BGSC under accession number 1A976. Strain REG19 is available under accession number 1A1276.
Strain Database is Now Updated!
Over the weekend we completed a bulk update of our online database. Included are each strain in the BKE knockout library, the CRISPRi knockdown library, the Bacillus subtilis gene expression toolbox, and many more. You should have a greatly increased ability to locate strains and plasmids using our online search engine.
You can enter string of three or more characters into the search box and find strains by their BGSC Code, Original Code, genotype, published reference, and in some cases a GenBank accession ID (we are working to update that last field).
For example: suppose you need a knockout of the B. subtilis xpaC gene. Simply enter xpaC in the search box, press enter, and you will discover that a knockout is available in strain BKE00250. Or suppose you are looking at a 1999 publication by Levin et al., Identification and characterization of a negative regulator of FtsZ ring formation in Bacillus subtilis. You could enter a phrase from the title (FtsZ ring formation) or the PMID for the article (10449747) and discover that we have two of the strains used in this research. Or suppose a BLAST search turns up a sequenced strain with GenBank accession number CP002905. Enter that number in the box, and you will discover we have the strain. Do we have the common lab strain PY79? Enter that name in the box, press enter, and you\'ll find that the answer is yes!
Of course we are always happy to answer inquiries about our holdings or to brainstorm with you about strains or plasmids that might work for your project. Write or call anytime!
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.
Some have reported recent problems with the online ordering system. When we receive an order, we always acknowledge it within 24 hours. If you do not hear back from us within that time period, please contact me (firstname.lastname@example.org) and let me know of the problem.
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.