Genetic Modification of the Escherichia coli Strain DH5 to Allow the Selection of Plasmids Carrying Complementary Yeast Genes

The Escherichia coli strain DH5α is used extensively in recombinant DNA technology. This is due in part to the endA gene mutation, which results in the reduction of endogenous levels of nuclease activity and increases the yield and quality of purified plasmid molecules. However, DH5α lacks the pyrF, leuB, and trpC mutations and is therefore not convenient for the selection of plasmids carrying the yeast LEU2, TRP1 and URA3 genes that are commonly used in the two-hybrid system. A novel method of disrupting E. coli genes was used to create pyrF, leuB, and trpC gene disruptions in the strain DH5α. The method utilizes the polymerase chain reaction (PCR) to create a gene disruption cassette. This cassette contains the kanamycin resistance gene flanked by FRT (FLP recognition target) sites, in turn flanked by DNA sequences homologous to the gene targeted for disruption. The gene disruption cassette was transformed into DH5α cells containing the helper plasmid pKD46, which has the λ Red recombinase functions for stimulation of homologous recombination at the target site. Gene disruptions were selected by kanamycin resistance and identified by screening for uracil, leucine, or tryptophan auxotrophy. The kanamycin resistance gene was then removed by induction of recombination between the flanking FRT sites through expression of the FLP recombinase from a second helper plasmid pCP20. This procedure allowed the sequential disruption of the pyrF, leuB and trpC genes in DH5α creating a new strain, MG107. This new strain can be used to select for plasmids containing the yeast URA3, LEU2, and TRP1 genes and produces high quality plasmid DNA preparations.