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)
BGSC Journal Club: June 2018
BGSC strains appeared in at least eight peer-reviewed journal articles in June 2018. We only have space here for the briefest of mentions. Check out the references for ideas about how our strains and genetic tools might be useful in your own research!
Peter Burby (University of Michigan) updated his detailed, very useful protocol for performing CRISPR/Cas9 genome editing in Bacillus subtilis using vectors pPB41 (BGSC No. ECE389) and pPB105 (BGSC ECE390).
Kim Harris (Yale University) used one of our inducible expression vectors to study an OLE (ornate, large, extremophilic) RNA in the moderate halophile Bacillus halodurans. This noncoding RNA and its two accessory proteins are required if the organism is to be tolerant to low temperatures or to short-chain alcohols in the growth medium.
Several articles explore the use of Bacillus and Paenibacillus as biocontrol organisms. Raida Zribi Zghal (University of Sfax) and colleagues investigated the potential of a local B. thuringiensis isolate to control mosquitoes. They used B. thuringiensis servor israelensis wild type (4Q2) and crystal-minus mutant (4Q7) strains from the BGSC for comparison studies.
Other researchers investigated antifungal Bacillus strains. Lamia Abdellaziz, along with colleagues at institutions in Algeria, France, and Belgium, characterized 16 antifungal isolates for their lipopeptide production. They used genome sequence data from two B. thuringiensis strains (BGSC 4BA1 and 4CC1) to assist them in designing screening primers. Ricardo Salvatierra-Martinez, together with colleagues at institutions in Chile and Mexico, likewise studied local antifungal isolates capable of colonizing roots. The used the proven biocontrol agent B. velezensis FZB42 (BGSC 10A6) and two of its mutants (BGSC 10A9 and 10A16) as comparison strains. Ambrin Sarwar and colleagues in Pakistan and Austria also used FZB42 and B. subtilis 168 (BGSC 1A1) in a mass spectrometry analysis of antifungal lipopeptides.
Paenibacillus polymyxa is a plant-growth promoting rhizobacterium. Elizabeth Finch (Queen\'s University Belfast) used BGSC 25A2 to demonstrate that P. polymyxa soil inoculation shifts the nematode population from plant-pathogenic species to predatory species, contributing to plant growth.
Finally, Patricia Calero and Pablo I. Nikel (Technical University of Denmark) reviewed the concept of the “bacterial chassis,” which they define as “the physical, metabolic and regulatory containment for plugging‐in and plugging‐out dedicated genetic circuits and regulatory devices” for the purpose of metabolic engineering. They focus on B. subtilis as a production platform and discuss the BGSC as a source of strains and genetic tools.
We congratulate these teams on their accomplishments and are happy that the BGSC could play a part!
New! Vectors for Spore Surface Display of Proteins
In recent years, there has been increasing interest in using the Bacillus subtlis endospore as a platform for immobilizing and displaying foreign proteins. The endospore coat is proteinaceus, and the outer layer self-assembles without requiring any transport across membranes. In theory, a very wide range of proteins could be anchored to the spore surface, including enzymes for biotechnology purposes or antigens for developing diagnostic tools. Now researchers in the Thorsten Mascher laboratory at the Technical University of Dresden have developed a set of vectors that greatly facilitate spore surface display . Each of the vectors can be manipulated in E. coli and then integrated into the B. subtilis amyE locus with selection for chloramphenicol resistance. The 12 vectors allow either N- or C-terminal fusions to be constructed with any of the six spore crust proteins, which include CotV, CotW, CotX, CotY, CotZ, and CgeA. Fusions are expressed under a strong sporulation promoter, PcotXY. We are grateful to Julia Bartels and her colleagues in the Mascher lab for donating this set of vectors to the BGSC, and we are excited to make them available to our user community. These vectors are accessioned in the collection under BGSC numbers ECE363-ECE374, inclusive. For more details, please consult the reference below.