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High-Throughput Allelic Replacement Screening in Bacillus subtilis.
Journal article

High-Throughput Allelic Replacement Screening in Bacillus subtilis.

  • Diebold-Durand ML Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland.
  • Bürmann F Structural Studies, MRC Laboratory of Molecular Biology, Cambridge, UK.
  • Gruber S Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland. stephan.gruber@unil.ch.
  • 2019-06-01
Published in:
  • Methods in molecular biology (Clifton, N.J.). - 2019
Bacillus subtilis
Cysteine scanning
Gene targeting
Golden Gate assembly
High-throughput screening
Alleles
Bacillus subtilis
Chromosomes
High-Throughput Screening Assays
Mutagenesis
Mutagenesis, Site-Directed
Point Mutation
English Site-directed mutagenesis is a key tool in the analysis of biological mechanisms. We have established an efficient and systematic gene targeting strategy for Bacillus subtilis based on the Golden Gate cloning methodology. Our approach permits the introduction of single or multiple point mutations or of heavily engineered alleles into the endogenous gene locus in a single step using a 96-well microtiter plate format. We have successfully applied this system for high-throughput functional screening of resized variants of the Structural Maintenance of Chromosome (Smc) protein and for exhaustive cysteine cross-linking mutagenesis. Here we describe, in detail, the experimental setup for high-throughput introduction of modifications into the B. subtilis chromosome. With minor modifications, the approach should be applicable to other bacteria and yeast.
Language
  • English
Open access status
closed
Identifiers
Persistent URL
https://folia.unifr.ch/global/documents/268473
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