Fig. 1.

a Standard SSM library construction using the one-step PCR approach with partially overlapping primers. A pair of partially overlapping mutagenic primers is used to perform the PCR reaction for incorporating mutations using plasmid pRSFDuet-1 harboring P450-BM3 gene as template; PCR reaction is performed to amplify the whole plasmid, and digestion with Dpn I is conducted to eliminate the parent template, followed by transformation into E. coli BL21 (DE3) and library harvesting. b New SSM library construction using the two-step PCR approach with a mutagenic primer and a non-mutagenic (silent) primer. A pair of primers consisting of one forward mutagenic primer and one reverse primer (or one forward primer and one reverse mutagenic prime, depending on the mutational position) is used to perform the PCR reaction for the purpose of incorporating designed mutations using pRSFDuet-1 harboring P450-BM3 gene as template. The amplified short DNA fragments (500 bp in length) containing mutations are recovered and subjected to the second PCR reaction as megaprimers. To amplify the whole plasmid, digestion with Dpn I is conducted to eliminate the parent template, followed by transformation into E. coli BL21 (DE3) and library harvesting