FIG 2.

Targeted gene deletion at the orotidine 5′-monophosphate decarboxylase locus (OMPDC). (A) Strategy for deletion and validation of the endogenous OMPDC locus via targeted integration of the HXGPRT marker at the OMPDC locus of the Pru Δku80 Δhxgprt strain. Approximate locations of PCR products used to validate genotypes are depicted. The Pru Δku80 Δhxgprt parental strain was positive for the PCR 1 product, while the targeted OMPDC knockout was positive for PCR 2 and PCR 3 (see Materials and Methods). (B) Strategy for cleanup of the deleted OMPDC locus. The HXGPRT marker was removed from the disrupted OMPDC locus using the depicted strategy that utilized the ability to negatively select against the HXGPRT marker using 6TX selection. Successfully cleaned up strains with HXGPRT deleted were validated using PCR 4 (see Materials and Methods).