FIG. 1.
Strategy for obtaining a pyrF deletion and PCR analysis of the pyrF deletion in the COM1 strain. (A) A diagram of the pyrF genome region is shown with the pyrF deletion plasmid having 1-kb regions from up- and downstream of pyrF for homologous recombination and also containing the Pgdh-hmg cassette for selection of simvastatin resistance. Homologous recombination can occur at the upstream or downstream pyrF flanking regions, integrating the plasmid into the genome and generating a strain that is simvastatin resistant. Selection on 5-FOA selects for loss of the plasmid along with deletion of the pyrF gene. Bent arrows depict primers used for verification of pyrF deletion. (B) Gel depicting PCR products of the pyrF genome region in the COM1 strain compared to the wild type, amplified by primers outside the up- and downstream regions used for homologous recombination (see bent arrows in panel A).