High-Throughput Allelic Replacement Screening in Bacillus subtilis.

Details

Serval ID
serval:BIB_9C99FD65C519
Type
Article: article from journal or magazin.
Collection
Publications
Title
High-Throughput Allelic Replacement Screening in Bacillus subtilis.
Journal
Methods in molecular biology
Author(s)
Diebold-Durand M.L., Bürmann F., Gruber S.
ISSN
1940-6029 (Electronic)
ISSN-L
1064-3745
Publication state
Published
Issued date
2019
Peer-reviewed
Oui
Volume
2004
Pages
49-61
Language
english
Notes
Publication types: Journal Article
Publication Status: ppublish
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.
Keywords
Bacillus subtilis, Cysteine scanning, Gene targeting, Golden Gate assembly, High-throughput screening
Pubmed
Create date
17/06/2019 22:48
Last modification date
21/08/2019 6:37
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