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.