BACT online meeting April 2021
From Michel Gohar:
"The next BACT meeting, initially planned on September 2020, had been rescheduled to take place in Paris in April this year.
"Unfortunately, since the Covid19 pandemic is not expected to be fixed before summer in Europe, the physical meeting will be replaced by an online meeting, from April 26 to April 28 2021.
"Presentations from PhD students and postdoctoral fellows will be selected in priority for this meeting. Registrations and abstract submissions will open on February 3rd, on the BACT2021 website https://colloque.inrae.fr/bact2021.
"The regular meeting in Paris is not cancelled, but postponed to April 2022."
For those new to the field, the International Conference on Bacillus anthracis, B. cereus and B. thuringiensis (BACT) is a wonderful opportunity to learn from and network with other researchers who focus on species in the Bacillus Cereus Group. This would be a great opportunity for grad students and post docs to gain some visibility in this research community.
Upcoming changes to the BGSC
On March 1, 2021, the Bacillus Genetic Stock Center will transfer to a new earnings unit at The Ohio State University. In the next few weeks I will introduce a new collection manager. The website will also change, and there will be an improved ordering process. The stocks will still be available, and prices will remain stable the rest of the fiscal year. We intend the BGSC to maintain and distribute its stocks in perpetuity!
Because we will be unable to transfer legacy commitments to the new earnings unit, one practical consequence is that existing bulk purchase plans should be used up by 28 February. Please plan accordingly, and let me know if this requirement causes you any difficulties.
I will soon be retiring from Ohio State after 35 years of service and will begin a career as an independent consultant. You will still be able to reach me at firstname.lastname@example.org. Please feel free stay in touch. It has been a pleasure serving all of you!
Watch this spot for more details soon.
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 (email@example.com) 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.
Prophage-cured Bacillus subtilis strain Δ6
We are pleased to announce the availability of prophage-cured Bacillus subtilis strain Δ6 (BGSC 1A1299). This derivative of strain 168 was deleted of six prophage-like regions in the 168 chromosome, including SPβ, the defective phage PBSX, the skin element, and the prophage 1 and prophage 3 regions, together with the large polyketide synthesis operon (pks). Interestingly, mobile element ICEBs1 was later discovered to have been spontaneously cured (Reuss 2016). As a result of these deletions, the genome size of strain Δ6 has been reduced 8.1% relative to the 168 parent. This strain has demonstrated usefulness as a production platform (Commichau 2014, Juhas 2014, Van Dijl 2013) and as a host for phage studies (Willms 2016, Willms 2017). The genome sequence is publicly available at GenBank accession CP015975. We thank Jan Maarten van Dijl of the University of Groningen for donating strain Δ6 to the BGSC!
The Bacillus BioBrick Box 2.0
The BGSC is pleased to announce the availability of the Bacillus BioBrick Box 2.0 (Popp et al. 2017), a collection of standardized parts for assembling modules for B. subtilis. These tools include several new plasmid vectors, which are detailed below. They also include a collection of genes encoding fluorescent proteins that as a set can span the entire visible spectrum. This parts collection extends the highly successful BioBrick Box 1.0 (Radeck et al. 2013), which is also available from the BGSC. We thank the Thorsten Mascher lab at TU Dresden for donating this exciting collection of tools!
You can download the plasmid sequences in a zip file here.
Below is a general description of the items in this collection.
The following vectors replicate in E. coli with selection for ampicillin resistance. They contain an rfp gene in the multiple cloning site to facilitate screening for inserts.
General purpose shuttle vector
pBS0E, supplied in E. coi ECE732
Notes: Replicates in Bacillus from ori-1030 origin of replication with selection for MLS resistance.
Shuttle vectors with inducible promoters
pBS0EP liaI (V2) and pBS0EXylRP xylA (V2), supplied in E. coli ECE742 and ECE743
Notes: The pBS0E shuttle vector, with either the bacitracin-inducible or xylose-inducible promoters upstream from the multiple cloning site.
General purpose integration vectors
pBS1E, pBS1K supplied in E. coli ECE730 and ECE731
Notes: Integrate by double crossover events into the B. subtilis amyE locus with selection for MLS or kanamycin, respectively.
Integration vectors with inducible promoters
pBS2EP xylA (V2), pBS2EP liaI (V2), and pBS2EXylRP xylA (V2), supplied in E. coli ECE739, ECE740, and ECE741
Notes: Integrate by double crossover events into the B. subtilis lacA locus with selection for MLS; with either the bacitracin-inducible or xylose-inducible promoters upstream from the multiple cloning site.
Integration vectors with reporter genes
pBS3Klux and pBS3Elux supplied in E. coli ECE733 and ECE734
Notes: lux-reporter vectors; integrate into B. subtilis lacA locus with selection for kanamycin and MLS, respectively
pBS3Kcatlux and pBS3Ecatlux supplied in E. coli ECE735 and ECE736
Notes: lux-reporter vectors; integrate into B. subtilis lacA locus with selection for kanamycin and MLS, respectively; the promoterless cat gene, encoding chloramphenicol acetyl transferase, serves as a co-selection marker to evaluate the strength of promoters.
pBS1CαlacZ and pBS3Cαlux, supplied in E. coli ECE737 and ECE738
Notes: reporter vectors for evaluating ribosome binding sites for expression in B. subtilis; pBS1CαlacZ integrates into amyE and pBS3Cαlux integrates into sacA. Insertion of a functional RBS into the multiple cloning site, replacing the rfp gene, allows for red-blue-white color screening.
Fluorescent protein genes
The following parts are carried in E. coli plasmids with selection for resistance to chloramphenicol:
mTagBFP (codon usage for E. coli, excitation/emission 399/465) supplied in E. coli ECE744
mTagBFP_Bsu (codon optimized for B. subtilis, excitation/emission 399/465) supplied in E. coli ECE745
eCFP_Bsu (codon optimized for B. subtilis, excitation/emission 449/479) supplied in E. coli ECE746
sfGFP_Spn (codon optimized for S. pneumoniae, excitation/emission 481/511) supplied in ECE747 (RFC10) and ECE748 (RFC25)
GFPmut1 (codon usage for A. victoria, excitation/emission 483/513) supplied in E. coli ECE749
GFPmut1 (LT) (codon optimized for B. subtilis excitation/emission 483/513) supplied in E. coli ECE750
mEYFP (codon usage for E. coli, excitation/emission 500/530) supplied in E. coli ECE751
mEYFP_Bsu (codon optimized for B. subtilis, excitation/emission 500/530 supplied in E. coli ECE752
SYFP2 (codon usage for E. coli, excitation/emission 500/530) supplied in E. coli ECE753 (RFC10) and ECE754 (RFC25)
mCherry (codon usage for E. coli, excitation/emission 585/615) supplied in E. coli ECE755
mCherry_Bsu (codon optimized for B. subtilis excitation/emission 585/615) supplied in E. coli ECE756 (RFC10) and ECE757 (RFC25)