Site-Directed Mutagenesis of FadR Protein

Site-directed mutagenesis was done as we recently described with little changes (Feng et al., 2014 (link)). The PCR reaction system (25 μL) consisted of the following components: 2.5 μL of 10× pfx buffer (Invitrogen), 0.5 μL of 40 mmol/L dNTP mix (10 mmol/L each), 1.0 μL of forward/reverse primers (10 pmol/μL), 1.0 μL of pET28-fadRec as template (5 ng/μL), 0.5 μL of Platinum pfx (2.5 U/μL, Invitrogen), and 17.5 μL of distilled sterilized H₂O. The reaction was performed using the program consisting of a denaturing cycle at 95°C for 5 min; 20 cycles comprised of 95°C for 50 s, 60°C for 50 s, and 68°C for 6 min and a final step of 8 min at 68°C. To remove the residual template plasmid pET28-fadRec, the gel purified PCR products were digested for 1 h with DpnI (20 U/μL, NEB) at 37°C. Subsequently, they were transformed into chemically-competent cells of DH5α and the inserts of purified plasmids were verified by direct DNA sequencing. The plasmids were transformed into BL21 (Tuner) to produce the FadR mutant proteins.