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)