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 language | English |
---|---|
Article number | 000994 |
Pages (from-to) | 1115-1120 |
Number of pages | 6 |
Journal | Microbiology |
Volume | 166 |
Issue number | 12 |
Early online date | 23 Nov 2020 |
DOIs | |
Publication status | Published - Dec 2020 |
Keywords
- Complementation
- Fluorescent
- Gene doctoring
- Recombineering
- Tag
- glmS
Profiles
-
Mark Webber
- Norwich Medical School - Honorary Professor, Research Leader/Principal Investigator
Person: Honorary, Academic, Teaching & Research