title: Markerless Deletion System for Escherichia coli Using Short Homologous Sequences and Positive-Negative Selectable Cassette.
abstract: Red homologous recombination has been extensively used in recombineering. Because foreign sequences, such as antibiotic resistance genes, FRT-sites, or loxP-sites, are often unwanted in mutant Escherichia coli, we established a markerless deletion system containing short homologous sequences, a positive-selectable marker (kan), and a negative-selectable marker (sacB) for E. coli. For markerless deletion of a specific region of the E. coli genome, a two-step recombination procedure using two different PCR fragments, which were amplified from pUC57-kan-sacB and pUC57-298, was performed. The generation of a pheA-tyrA deficient mutant demonstrated that this markerless deletion system was a simple and efficient method to generate markerless chromosomal deletions in E. coli.
authors.affiliation: Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Sciences, Jilin University, 5333 Xi'an Road, Changchun, 130062, People's Republic of China.
authors.forename: Fuwang
authors.initials: F
authors.lastname: Chen
authors.forename: Jie
authors.initials: J
authors.lastname: Jiang
authors.forename: Hongsheng
authors.initials: H
authors.lastname: OuYang
authors.forename: Teng
authors.initials: T
authors.lastname: Ma
authors.forename: Zhiyuan
authors.initials: Z
authors.lastname: Peng
authors.forename: Yunzhi
authors.initials: Y
authors.lastname: Ma
authors.forename: Xinrong
authors.initials: X
authors.lastname: Chen
authors.forename: Daxing
authors.initials: D
authors.lastname: Pang
authors.forename: Songyi
authors.initials: S
authors.lastname: Lin
authors.forename: Linzhu
authors.initials: L
authors.lastname: Ren
mesh_terms: D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics*; D005819:Genetic Markers; D059765:Homologous Recombination / Q000235:genetics; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000235:genetics; D017384:Sequence Deletion; D017385:Sequence Homology
chemical_list: D005819:Genetic Markers