Donor plasmids for phenotypically neutral chromosomal gene insertions in Enterobacteriaceae

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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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Abstract

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
Related URLs:	http://www.scopus.com/inward/record.url?...
Depositing User:	LivePure Connector
Date Deposited:	04 Dec 2020 00:45
Last Modified:	20 Apr 2023 19:33
URI:	https://ueaeprints.uea.ac.uk/id/eprint/77892
DOI:	10.1099/mic.0.000994

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