BGSC to re-open 3 June 2020!
After a nearly 11-week lockdown, the BGSC will be reopening on Wednesday, 3 June 2020. Initally we will be onsite for 20 hours a week. We will begin shipping orders right away. There are a large number of backorders, so it may take us a few weeks to get totally caught up. Please do not hesitate to begin placing orders. We will process them just as soon as we possibly can!
Subtillery - a virtual Bacillus conference
The BGSC remains under lockdown conditions. We hope to resume operations on or around June 1, 2020. We pass on updates as they become available.
In the meantime, we are happy to announce that Prahathees Eswara of the University of South Florida is organizing a virual conference called Subtillery, with the stated aim of "distilling and understanding the biology of Bacillus subtilis and related organisms." The plan is to have nearly 40 twenty-minute trainee talks presented on Zoom between June 8-12 (10am-1:30pm US Eastern Time). A registration form can be found at Subtillery - Registration
COVID-19 and the BGSC
As most of your probably realize, the Bacillus Genetic Stock Center is hosted on campus at The Ohio State University in Columbus, Ohio. Like most educational institutions around the US and worldwide, Ohio State is grappling with ways to mitigate the severity of the current coronavirus pandemic. The situation is fluid and changing rapidly. Currently, face-to-face classroom instruction has been suspended, and university personnel who are able to work remotely are required to do so. There is a high likelihood that other measures, including a lock down of laboratories, will be mandated in the near future. For this reason, the BGSC will have limited functionality for an extended time. It will be possible to reach us by email (firstname.lastname@example.org) and by telephone (1-614-292-5550) during regular working hours, 8 am - 5 pm EDT (UTC-04:00). If you are open to receive shipments and are able to make purchases, it may be possible for us to distribute strains. However, I cannot guarantee our continued ability to do so. Please inquire by email before attempting to make a purchase. Thanks for your understanding. Wishing you health and happiness in the days ahead, Daniel
pminiMAD, a tool for markerless allele replacement
What makes Bacillus subtilis 168 such a powerful model system? Many factors: a world-wide community of investigators and a 60-year history of focused enquiry; a carefully annotated genome sequence with associated proteomic and transcriptomic data; a suite of useful curated databases making all of this information discoverable and accessible. But what sets B. subtilis apart from most other microbial model systems is a genetic toolbox of great sophistication and variety. One important set of tools are designed to introduce marker-free mutations to the B. subtilis genome. Several technologies are available, including Cre-lox marker loop-out and CRISPR methodologies. But an older technique--allele replacement by temperature-sensitive plasmids--remains highly useful. And the method is potentially extensible to any Gram-positive bacterium with an available plasmid-transformation system, whether by natural competence of by physical methods such as electroporation.
One such vector is pminiMAD2 (also called simply pminiMAD in the research literature). Originally constructed by Patrick and Kearns (2008), this shuttle vector replicates normally in E. coli with selection for ampicillin resistance and in temperature sensitive fashion in Bacillus, where selection is for erythromycin resistance. In Gram-positive hosts, plasmid replication is permissive at normal room temperature but restricted at 37°C. In practice, one simply inserts a fragment from the target chromosome, altered with either a point mutation, deletion, or insertion. The plasmid construct is introduced into the host by transformation, and then selection is maintained as the temperature is raised to 37°C. Single crossovers produce Campbell-type insertion events, where the entire vector is integrated into the chromosome at the target locus, flanked on either side by a normal and mutated copy of the insert. Transformants are next cultured at the permissive temperature in the absence of selection. Several generations of growth, usually at room temperature overnight, allow the plasmid to excise from the genome by hommologous recombination. In many cells, the plasmid will be cured spontaneously, leaving behind either a wild type or mutated form of the target locus in the chromosome. A simple screening step by PCR and sequencing can identify the desired mutant.
The PubMed Central database lists over 40 publications that use pminiMAD2 (or pminiMAD). At least 10 were published during the last two years; they are listed in the article citations below. All describe work in B. subtilis 168 or its wild type ancestor NCIB 3610 with one exception. Spacapan et al. (2018) used pminiMAD2 to introduce a marker-free deletion into an environmental isolate of B. subtilis, PS216 (BGSC accession 3A36). In principle, however, the vector could be used with any mesophilic isolate from Bacillus or related genera.
We thank the Dan Kearns laboratory at Indiana University for donating pminiMAD2 to the BGSC. It is available in an E. coli host as our catalog number ECE765.