Donor plasmids for phenotypically neutral chromosomal gene insertions in Enterobacteriaceae

Holden, Emma R., Wickham, Gregory J., Webber, Mark A., Thomson, Nicholas M. and Trampari, Eleftheria (2020) Donor plasmids for phenotypically neutral chromosomal gene insertions in Enterobacteriaceae. Microbiology, 166 (12). pp. 1115-1120. ISSN 1350-0872

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Recombineering using bacteriophage lambda Red recombinase (λ-Red) uses homologous recombination to manipulate bacterial genomes and is commonly applied to disrupt genes to elucidate their function. This is often followed by the introduction of a wild-type copy of the gene on a plasmid to complement its function. This is often not, however, at a native copy number and the introduction of a chromosomal version of a gene can be a desirable solution to provide wild-type copy expression levels of an allele in trans. Here, we present a simple methodology based on the λ-Red-based 'gene doctoring' technique, where we developed tools used for chromosomal tagging in a conserved locus downstream of glmS and found no impact on a variety of important phenotypes. The tools described provide an easy, quick and inexpensive method of chromosomal modification for the creation of a library of insertion mutants to study gene function.

Item Type: Article
Additional Information: Acknowledgements: The authors gratefully acknowledge the support of the Biotechnology and Biological Sciences Research Council (BBSRC); E. T., N. T. and M. A. W. were supported by the BBSRC Institute Strategic Programme Microbes in the Food Chain BB/R012504/1 and its constituent project BBS/E/F/000PR10349. E. R. H. is supported by a PhD studentship funded by Quadram Institute Biosciences. The pDOC-K vector was kindly gifted by Professor Stephen Busby (University of Birmingham).
Uncontrolled Keywords: complementation,fluorescent,gene doctoring,recombineering,tag,glms,microbiology ,/dk/atira/pure/subjectarea/asjc/2400/2404
Faculty \ School: Faculty of Medicine and Health Sciences > Norwich Medical School
UEA Research Groups: Faculty of Medicine and Health Sciences > Research Centres > Norwich Institute for Healthy Aging
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Depositing User: LivePure Connector
Date Deposited: 04 Dec 2020 00:45
Last Modified: 20 Apr 2023 19:33
DOI: 10.1099/mic.0.000994


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