Figure 3.

The overlap PCR protocol used to generate a ybdT mutation in OKB105. Primers P1 and P2 amplified the 5′ region of ybdT gene in OKB105. Primers P5 and P6 amplified the 3′ region of ybdT gene. Primers P3 and P4 amplified the chloramphenicol resistance cassette from PDG1662. Primers P2 P3, P4 and P5 were designed with 18 bp overlap as shown in Table 4. PCR1, PCR2, and PCR3 are the primary PCR products with overlapping region as follows: the 3′end of PCR1 contains sequence for the upstream portion of the cat and the 5′ end of PCR3 has sequence for amino acids 149–151 encoded by ybdT (blue). The 5′end of PCR2 contains sequence for amino acids 214–226 encoded by cat and 3′end of PCR3 has sequence for amino acids 365–367 encoded by ybdT (red). The primary PCR products were then joined in a long PCR reaction to synthesize the PCR construct used for transforming competent OKB105 cells.