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 [1]. 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.

Super-competent strains of Bacillus subtilis

As discovered by John Spizizen in 1958 [1], 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 [2]. Entry of cells into the K-state is under control of the master regulatory protein ComK [3]. 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 [4]. 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 [5]. 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 [5]. 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.