title: An efficient method of selectable marker gene excision by Xer recombination for gene replacement in bacterial chromosomes.
abstract: A simple, effective method of unlabeled, stable gene insertion into bacterial chromosomes has been developed. This utilizes an insertion cassette consisting of an antibiotic resistance gene flanked by dif sites and regions homologous to the chromosomal target locus. dif is the recognition sequence for the native Xer site-specific recombinases responsible for chromosome and plasmid dimer resolution: XerC/XerD in Escherichia coli and RipX/CodV in Bacillus subtilis. Following integration of the insertion cassette into the chromosomal target locus by homologous recombination, these recombinases act to resolve the two directly repeated dif sites to a single site, thus excising the antibiotic resistance gene. Previous approaches have required the inclusion of exogenous site-specific recombinases or transposases in trans; our strategy demonstrates that this is unnecessary, since an effective recombination system is already present in bacteria. The high recombination frequency makes the inclusion of a counter-selectable marker gene unnecessary.
authors.affiliation: Cobra Biomanufacturing Plc, The Science Park, Keele, Staffordshire ST5 5SP, United Kingdom.
authors.forename: Alexandra E
authors.initials: AE
authors.lastname: Bloor
authors.forename: Rocky M
authors.initials: RM
authors.lastname: Cranenburgh
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics / Q000254:growth & development; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002876:Chromosomes, Bacterial / Q000235:genetics*; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D005798:Genes, Bacterial; D005819:Genetic Markers; D005821:Genetic Techniques; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005819:Genetic Markers; C473360:XerC protein, E coli; C121793:codV protein, Bacillus subtilis; D019426:Integrases; C473361:XerD protein, E coli