title: Positive and negative selection using the tetA-sacB cassette: recombineering and P1 transduction in Escherichia coli.
abstract: The two-step process of selection and counter-selection is a standard way to enable genetic modification and engineering of bacterial genomes using homologous recombination methods. The tetA and sacB genes are contained in a DNA cassette and confer a novel dual counter-selection system. Expression of tetA confers bacterial resistance to tetracycline (Tc(R)) and also causes sensitivity to the lipophillic chelator fusaric acid; sacB causes sensitivity to sucrose. These two genes are introduced as a joint DNA cassette into Escherichia coli by selection for Tc(R). A medium containing both fusaric acid and sucrose has been developed, in which, coexpression of tetA-sacB is orders of magnitude more sensitive as a counter-selection agent than either gene alone. In conjunction with the homologous recombination methods of recombineering and P1 transduction, this powerful system has been used to select changes in the bacterial genome that cannot be directly detected by other counter-selection systems.
authors.affiliation: Molecular Control and Genetics Section, Gene Regulation and Chromosome Biology, National Cancer Institute at Frederick, National Institutes of Health, Frederick, MD 21702, USA and Leidos Biomedical Research, Inc., Frederick National Laboratory, Frederick, MD 21702, USA.
authors.forename: Xin-Tian
authors.initials: XT
authors.lastname: Li
authors.forename: Lynn C
authors.initials: LC
authors.lastname: Thomason
authors.forename: James A
authors.initials: JA
authors.lastname: Sawitzke
authors.forename: Nina
authors.initials: N
authors.lastname: Costantino
authors.forename: Donald L
authors.initials: DL
authors.lastname: Court
mesh_terms: D017920:Antiporters / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics; D050939:Gene Fusion; D005818:Genetic Engineering / Q000379:methods*; D006602:Hexosyltransferases / Q000235:genetics*; D011995:Recombination, Genetic; D013395:Sucrose / Q000378:metabolism; D014161:Transduction, Genetic
chemical_list: D017920:Antiporters; D001426:Bacterial Proteins; D003470:Culture Media; D029968:Escherichia coli Proteins; C053139:sulA protein, E coli; C079051:tetA protein, Bacteria; D013395:Sucrose; D006602:Hexosyltransferases; C021513:levansucrase