title: Improvement of pCVD442, a suicide plasmid for gene allele exchange in bacteria.
abstract: Allelic exchange experiments allow investigation of the functions of many unknown genes identified during the sequencing of entire genomes. Isogenic strains differing by only specific mutations can be constructed. Among other tools, suicide plasmids are widely used for this task. They present many advantages because they leave no scars on the chromosome, and therefore allow combining several mutations in the same genetic background. While using the previously described pCVD442 suicide plasmid [Infect. Immun. 59 (1991) 4310], we found untargeted recombination events due to the presence of an IS1 element on this plasmid. The plasmid was therefore improved by removal of the IS1 element. We also replaced the bla gene of pCVD442, conferring ampicillin resistance, by the cat gene conferring chloramphenicol resistance, leading to the new suicide plasmid pDS132. The plasmid was entirely sequenced. We demonstrate that this new vector can be easily used to introduce various types of mutations into different genetics backgrounds: removal of IS elements, introduction of point mutations or deletions. It can be introduced into bacterial strains by either transformation or conjugation.
authors.affiliation: Laboratoire Adaptation et Pathogénie des Microorganismes, CNRS UMR 5163, Université Joseph FOURIER, 460, rue de la piscine, 38400 Saint Martin d'Heres, France.
authors.forename: Nadège
authors.initials: N
authors.lastname: Philippe
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Alcaraz
authors.forename: Evelyne
authors.initials: E
authors.lastname: Coursange
authors.forename: Johannes
authors.initials: J
authors.lastname: Geiselmann
authors.forename: Dominique
authors.initials: D
authors.lastname: Schneider
mesh_terms: D000483:Alleles; D017931:DNA Primers; D004251:DNA Transposable Elements / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D041722:Genes, Transgenic, Suicide / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D014169:Transformation, Bacterial / Q000235:genetics
chemical_list: D017931:DNA Primers; D004251:DNA Transposable Elements