Methods

Here you can find a list of methods for working with Bacillus subtilis:

Media

• C minimal medium
• M9 minimal medium
• Spizizen minimal medium

A collection of mutant strains

• the complete mutant strain collection from the Koo et al. paper is now available: get access here

Toolboxes for the work with B. subtilis

• the biobrick box PubMed
• the biobrick box 2.0 PubMed
• a part toolbox to tune genetic expression PubMed

Analysis of protein-protein interactions

• Bacterial Two Hybrid: BACTH

• SPINE: A method to detect in vivo protein-protein interactions

• in vivo detection of protein-protein interactions using DivIVA and GFP PubMed

Analysis of membrane proteins

• application of a split green fluorescent protein reporter PubMed

Antibodies

• List of available Antibodies

Protein expression

• the LIKE system PubMed

Strain construction

• introduction of markerfree deletions: PubMed
• generation of markerfree mutations: PubMed
• an improved method for transformation: PubMed

RNA analysis

• Mechanical cell disruption and extraction of RNA from B. subtilis: Media:SOP-RNA.pdf

Genetic work with non-standard strains

• transformation of wild-type B. subtilis strains incl. NCIB3610: PubMed
• efficient electroporation of B. subtilis PubMed
• a novel transformation protocol for B. subtilis DB104 PubMed

General procedures

• Standard operation procedures (SOPs) of the SYSMO-BACELL consortium

Microscopic techniques

• Live cell imaging of B. subtilis cells using automated time-lapse microscopy PubMed
• Assay of gene expression dynamics using live cell imaging PubMed
• Visualization and quantification of gene expression heterogeneity in growing microbial cells PubMed
• GFP variants specifically optimizd for use in B. subtilis PubMed
• Quantitative analysis of cell types by comparing microscoy images PubMed
• Semi-automated single cell analysis using membrane or DNA staining PubMed

Transposon mutagenesis

• mariner transposon mutagenesis for random inducible-expression insertions and transcriptional reporter fusion insertions PubMed

Protein-DNA interactions

• ChAP-chip: A modified ChIP-chip protocol for the in vivo identification of binding sites of DNA-binding proteins PubMed

Genome engineering

• generalized bacterial genome editing using mobile group II introns and Cre-lox PubMed
• genome engineering using a synthetic gene circuit PubMed
• Ordered Gene Assembly in Bacillus subtilis (OGAB) PubMed
• construction of a super-competent strain PubMed
• use of the CRISPR-Cas system PubMed

• A review on genome engineering:

Kevin M Esvelt, Harris H Wang
Genome-scale engineering for systems and synthetic biology.
Mol Syst Biol: 2013, 9;641
[PubMed:23340847] [WorldCat.org] [DOI] (I p)

Visual descriptions of methods for B. subtilis in the Journal of Visual Experimentation

Lorena Stannek, Richard Egelkamp, Katrin Gunka, Fabian M Commichau
Monitoring intraspecies competition in a bacterial cell population by cocultivation of fluorescently labelled strains.
J Vis Exp: 2014, (83);e51196
[PubMed:24473333] [WorldCat.org] [DOI] (I e)

Samantha M Desmarais, Felipe Cava, Miguel A de Pedro, Kerwyn Casey Huang
Isolation and preparation of bacterial cell walls for compositional analysis by ultra performance liquid chromatography.
J Vis Exp: 2014, (83);e51183
[PubMed:24457605] [WorldCat.org] [DOI] (I e)

Ewa Król, Dirk-Jan Scheffers
FtsZ polymerization assays: simple protocols and considerations.
J Vis Exp: 2013, (81);e50844
[PubMed:24300445] [WorldCat.org] [DOI] (I e)

Elizabeth Anne Shank
Using coculture to detect chemically mediated interspecies interactions.
J Vis Exp: 2013, (80);e50863
[PubMed:24300024] [WorldCat.org] [DOI] (I e)

Juan C Garcia-Betancur, Ana Yepes, Johannes Schneider, Daniel Lopez
Single-cell analysis of Bacillus subtilis biofilms using fluorescence microscopy and flow cytometry.
J Vis Exp: 2012, (60);
[PubMed:22371091] [WorldCat.org] [DOI] (I e)

Imke G de Jong, Katrin Beilharz, Oscar P Kuipers, Jan-Willem Veening
Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy.
J Vis Exp: 2011, (53);
[PubMed:21841760] [WorldCat.org] [DOI] (I e)

Andrew D Klocko, Kaleena M Crafton, Brian W Walsh, Justin S Lenhart, Lyle A Simmons
Imaging mismatch repair and cellular responses to DNA damage in Bacillus subtilis.
J Vis Exp: 2010, (36);
[PubMed:20142799] [WorldCat.org] [DOI] (I e)

Key references

Regine Rahmer, Kambiz Morabbi Heravi, Josef Altenbuchner
Construction of a Super-Competent Bacillus subtilis 168 Using the P mtlA -comKS Inducible Cassette.
Front Microbiol: 2015, 6;1431
[PubMed:26732353] [WorldCat.org] [DOI] (P e)

Marian Wenzel, Josef Altenbuchner
Development of a markerless gene deletion system for Bacillus subtilis based on the mannose phosphoenolpyruvate-dependent phosphotransferase system.
Microbiology (Reading): 2015, 161(10);1942-1949
[PubMed:26238998] [WorldCat.org] [DOI] (I p)

Kenji Tsuge, Yukari Sato, Yuka Kobayashi, Maiko Gondo, Masako Hasebe, Takashi Togashi, Masaru Tomita, Mitsuhiro Itaya
Method of preparing an equimolar DNA mixture for one-step DNA assembly of over 50 fragments.
Sci Rep: 2015, 5;10655
[PubMed:25990947] [WorldCat.org] [DOI] (I e)

Ting Shi, Guanglu Wang, Zhiwen Wang, Jing Fu, Tao Chen, Xueming Zhao
Establishment of a markerless mutation delivery system in Bacillus subtilis stimulated by a double-strand break in the chromosome.
PLoS One: 2013, 8(11);e81370
[PubMed:24282588] [WorldCat.org] [DOI] (I e)

Peter J Enyeart, Steven M Chirieleison, Mai N Dao, Jiri Perutka, Erik M Quandt, Jun Yao, Jacob T Whitt, Adrian T Keatinge-Clay, Alan M Lambowitz, Andrew D Ellington
Generalized bacterial genome editing using mobile group II introns and Cre-lox.
Mol Syst Biol: 2013, 9;685
[PubMed:24002656] [WorldCat.org] [DOI] (I p)

Wout Overkamp, Katrin Beilharz, Ruud Detert Oude Weme, Ana Solopova, Harma Karsens, Ákos T Kovács, Jan Kok, Oscar P Kuipers, Jan-Willem Veening
Benchmarking various green fluorescent protein variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for live cell imaging.
Appl Environ Microbiol: 2013, 79(20);6481-90
[PubMed:23956387] [WorldCat.org] [DOI] (I p)

Arturo Rodríguez-Banqueri, Lukasz Kowalczyk, Manuel Palacín, José Luis Vázquez-Ibar
Assessment of membrane protein expression and stability using a split green fluorescent protein reporter.
Anal Biochem: 2012, 423(1);7-14
[PubMed:22285978] [WorldCat.org] [DOI] (I p)

Jonathan W Young, James C W Locke, Alphan Altinok, Nitzan Rosenfeld, Tigran Bacarian, Peter S Swain, Eric Mjolsness, Michael B Elowitz
Measuring single-cell gene expression dynamics in bacteria using fluorescence time-lapse microscopy.
Nat Protoc: 2011, 7(1);80-8
[PubMed:22179594] [WorldCat.org] [DOI] (I e)

Eric R Pozsgai, Kris M Blair, Daniel B Kearns
Modified mariner transposons for random inducible-expression insertions and transcriptional reporter fusion insertions in Bacillus subtilis.
Appl Environ Microbiol: 2012, 78(3);778-85
[PubMed:22113911] [WorldCat.org] [DOI] (I p)

Imke G de Jong, Katrin Beilharz, Oscar P Kuipers, Jan-Willem Veening
Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy.
J Vis Exp: 2011, (53);
[PubMed:21841760] [WorldCat.org] [DOI] (I e)

Chong Zhang, Xiaohui Zhang, Zhengying Yao, Yaping Lu, Fengxia Lu, Zhaoxin Lu
A new method for multiple gene inactivations in Bacillus subtilis 168, producing a strain free of selectable markers.
Can J Microbiol: 2011, 57(5);427-36
[PubMed:21542786] [WorldCat.org] [DOI] (I p)

Reindert Nijland, J Grant Burgess, Jeff Errington, Jan-Willem Veening
Transformation of environmental Bacillus subtilis isolates by transiently inducing genetic competence.
PLoS One: 2010, 5(3);e9724
[PubMed:20300532] [WorldCat.org] [DOI] (I e)

Haojie Yu, Xin Yan, Weiliang Shen, Yujia Shen, Ji Zhang, Shunpeng Li
Efficient and precise construction of markerless manipulations in the Bacillus subtilis genome.
J Microbiol Biotechnol: 2010, 20(1);45-53
[PubMed:20134232] [WorldCat.org] (P p)

Takuya Morimoto, Katsutoshi Ara, Katsuya Ozaki, Naotake Ogasawara
A new simple method to introduce marker-free deletions in the Bacillus subtilis genome.
Genes Genet Syst: 2009, 84(4);315-8
[PubMed:20057169] [WorldCat.org] [DOI] (P p)

A Nicole Edwards, Jason D Fowlkes, Elizabeth T Owens, Robert F Standaert, Dale A Pelletier, Gregory B Hurst, Mitchel J Doktycz, Jennifer L Morrell-Falvey
An in vivo imaging-based assay for detecting protein interactions over a wide range of binding affinities.
Anal Biochem: 2009, 395(2);166-77
[PubMed:19698693] [WorldCat.org] [DOI] (I p)

Christina Herzberg, Lope Andrés Flórez Weidinger, Bastian Dörrbecker, Sebastian Hübner, Jörg Stülke, Fabian M Commichau
SPINE: a method for the rapid detection and analysis of protein-protein interactions in vivo.
Proteomics: 2007, 7(22);4032-5
[PubMed:17994626] [WorldCat.org] [DOI] (P p)

Shu Ishikawa, Yoshitoshi Ogura, Mika Yoshimura, Hajime Okumura, Eunha Cho, Yoshikazu Kawai, Ken Kurokawa, Taku Oshima, Naotake Ogasawara
Distribution of stable DnaA-binding sites on the Bacillus subtilis genome detected using a modified ChIP-chip method.
DNA Res: 2007, 14(4);155-68
[PubMed:17932079] [WorldCat.org] [DOI] (P p)