FIG. 3.

Construction of the pldB deletion mutant. (A) After homologous pairing between the linearized targeting vector, pldB⁻ construct, and the genomic pldB locus, homologous recombination in the regions labeled X replaces the endogenous pldB sequence with an exogenous sequence containing the blasticidin resistance cassette (Bsr), resulting in a deletion of the pldB gene. The cell lines produced by this event are resistant to blasticidin. Black box, gene; white box, blasticidin resistance cassette. (B) PCR was performed on DNA prepared from wild-type Ax2 cells, the gene disruption construct (pldB⁻), and 9 blasticidin-resistant clones, and the resulting products were separated on an agarose gel. Primer pair 2-3 tests for the presence of the disruption construct in the genome, while primer pair 1-3 tests for proper insertion of the disruption construct in the pldB gene. Strains 1, 2, 3, 5, 6, 8, and 9 show proper integration of the disruption construct.