Donor plasmids for phenotypically neutral chromosomal gene insertions in Enterobacteriaceae

Emma R. Holden, Gregory J. Wickham, Mark A. Webber, Nicholas M. Thomson, Eleftheria Trampari

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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.

Original languageEnglish
Article number000994
Pages (from-to)1115-1120
Number of pages6
JournalMicrobiology
Volume166
Issue number12
Early online date23 Nov 2020
DOIs
Publication statusPublished - Dec 2020

Keywords

  • Complementation
  • Fluorescent
  • Gene doctoring
  • Recombineering
  • Tag
  • glmS

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