Donor plasmids for phenotypically neutral chromosomal gene insertions in Enterobacteriaceae. Issue 12 (23rd December 2020)

• Record Type: Journal Article
• Authors: Holden, Emma R.; Wickham, Gregory J.; Webber, Mark A.; Thomson, Nicholas M.; Trampari, Eleftheria
• Is Part Of: Microbiology. Volume 166:Issue 12(2020)
• Page Start: 1115
• Page End: 1120
• Publication Date: 2020-12-23
• Subjects: recombineering -- glmS -- complementation -- fluorescent -- gene doctoring -- tag; Microbiology -- Periodicals; 579
• DOI: 10.1099/mic.0.000994
• Languages: English
• ISSNs: 1350-0872

Abstract

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.