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)

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.

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.

A Safe Simulant for Bacillus anthracis

Safe simulants that closely mimic the select agent Bacillus anthracis are needed both for laboratory and field studies. B. anthracis belongs to the Bacillus cereus group (BCG) of species. Members of the BCG are nearly identical is cell and spore morphology due to highly similar genome sequence and content. They differ primarily in toxins and virulence factors, many of which are encoded by megaplasmids that differ among isolates. B. thuringiensis (Bt) likewise belongs to the BCG. Some naturally insecticidal Bt strains have been safely used in agriculture for over half a century. Non-insecticidal derivatives of standard Bt strains would seem to make ideal simulants for B. anthracis. For this reason, Alistair Bishop and colleagues at the Defence Science and Technology Laboratory, Salisbury, Wiltshire, UK, have developed plasmid-cured derivatives of Bt kurstaki strain HD1. One of them, HD-1 Cry-, has been demonstrated to be particularly useful in studies of spore aerisolization, dispersal, and decontamination. We thank Dr. Bishop for depositing B. thuringiensis HD-1 Cry- in the BGSC. It is available as BGSC 4D24.

ordering issues

Some have reported recent problems with the online ordering system. When we receive an order, we always acknowledge it within 24 hours. If you do not hear back from us within that time period, please contact me ( and let me know of the problem.


New! Protease-free Bacillus subtilis host

Bacillus subtilis is widely used as a production platform for synthesizing enzymes, pharmaceuticals, and fine chemicals (1). Unfortunately, B. subtilis 168 secretes no fewer than seven proteases during vegetative growth and stationary phase. Strains in which multiple protease genes have been inactivated have proved to be superior to wild type strains in production of foreign proteins (2, 3). I have now constructed a seven-protease deletion strain that is free from antibiotic resistance genes or integrated plasmids. This strain, B. subtilis KO7, was generated from the commonly used laboratory host, PY79, by sequentially knocking out the coding sequences. At each step, I transformed the strain with the appropriate BKE cassette for knocking out one of the loci, removing the erythromycin resistance gene with the Cre-producing plasmid pDR244, and finally heat-curing the plasmid. All seven knockouts in KO7 were confirmed by sequencing to be marker-free, in-frame deletions with a 150-bp scar replacing the coding sequence. KO7 is prototrophic and grows rapidly in standard minimal media for B. subtilis. I am placing strain KO7 in the public domain and disclaim all downstream rights to any process or product that you develop with it. It will be available from the BGSC as accession number 1A1133. Standard user fees apply. Later this summer I hope to introduce further elaborations of KO7, such as restriction-negative and asporogenous variants. Feel free to put in requests for particular features!

BGSC Accession: 1A1133

Original Code: Bacillus subtilis KO7

Reference: Zeigler DR, unpublished

Genotype: ΔnprE ΔaprE Δepr Δmpr ΔnprB Δvpr Δbpr

Description: Free of secreted proteases; marker-free deletions in PY79 genetic background; prototrophic