Site-Directed Mutagenesis of HepI Gene in E. coli

All materials, solvents, competent cells were obtained as previously reported [17 (link),18 (link),36 (link)]. The Q5 High-Fidelity PCR kit from New England Biolabs was used according to its instructions to perform mutagenesis using E. coli K-12 strain MB1760 960 bp HepI gene subcloned into pTOM-15b. New England Biolabs thermocycling conditions for routine PCR was used and the amplified DNA was then transformed into XL10-Gold competent cells, which were then incubated in a 5 mL LB/AMP overnight growth for plasmid DNA extraction by miniprep. Once the mutation was confirmed by sequence alignment with HepI wild-type DNA and the respective primers for each mutant, the purified plasmid was transformed into BL21-AI cells as per Agilent’s instructions.