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High-Throughput Allelic Replacement Screening in Bacillus subtilis

  • Marie-Laure Diebold-Durand
  • Frank Bürmann
  • Stephan GruberEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2004)

Abstract

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.

Key words

Golden Gate assembly Bacillus subtilis Gene targeting High-throughput screening Cysteine scanning 

Notes

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Marie-Laure Diebold-Durand
    • 1
  • Frank Bürmann
    • 2
  • Stephan Gruber
    • 1
    Email author
  1. 1.Department of Fundamental MicrobiologyUniversity of LausanneLausanneSwitzerland
  2. 2.Structural StudiesMRC Laboratory of Molecular BiologyCambridgeUK

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