title: Utilization of an unstable plasmid and the I-SceI endonuclease to generate routine markerless deletion mutants in Francisella tularensis.
abstract: We engineered an efficient system to make Francisella tularensis deletion mutations using an unstable, poorly maintained plasmid to enhance the likelihood of homologous recombination. For counterselection, we adapted a strategy using I-SceI, which causes a double-stranded break in the integrated suicide vector, forcing a second recombination to mediate allelic replacement.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Horzempa
authors.forename: Robert M Q
authors.initials: RM
authors.lastname: Shanks
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Brown
authors.forename: Brian C
authors.initials: BC
authors.lastname: Russo
authors.forename: Dawn M
authors.initials: DM
authors.lastname: O'Dee
authors.forename: Gerard J
authors.initials: GJ
authors.lastname: Nau
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D003850:Deoxyribonuclease I / Q000235:genetics / Q000378:metabolism*; D005604:Francisella tularensis / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D010957:Plasmids / Q000235:genetics*; D011995:Recombination, Genetic; D017384:Sequence Deletion; D014406:Tularemia / Q000382:microbiology
chemical_list: D001426:Bacterial Proteins; D003850:Deoxyribonuclease I