Gene disruption in Escherichia coli: Tc R and Km R cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant

Abstract

Two cassettes with tetracycline-resistance (Tc R) and kanamycin-resistance (Km R) determinants have been developed for the construction of insertion and deletion mutants of cloned genes in Escherichia coli. In both cassettes, the resistance determinants are flanked by the short direct repeats ( FRT sites) required for site-specific recombination mediated by the yeast Flp recombinase. In addition, a plasmid with temperature-sensitive replication for temporal production of the Flp enzyme in E. coli has been constructed. After a gene disruption or deletion mutation is constructed in vitro by insertion of one of the cassettes into a given gene, the mutated gene is transferred to the E. coli chromosome by homologous recombination and selection for the antibiotic resistance provided by the cassette. If desired, the resistance determinant can subsequently be removed from the chromosome in vivo by Flp action, leaving behind a short nucleotide sequence with one FRT site and with no polar effect on downstream genes. This system was applied in the construction of an E. coli endA deletion mutation which can be transduced by P1 to the genetic background of interest using Tc R as a marker. The transductant can then be freed of the Tc R if required.