Donor plasmids for phenotypically neutral chromosomal gene insertions in Enterobacteriaceae.

Holden ER,Wickham GJ,Webber MA,Thomson NM,Trampari E. (2020)

Microbiology (Reading, England)

Recombineering using bacteriophage lambda Red recombinase (?-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 ?-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.

Microbiology (Reading, England)

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