3.1 Scheme for random mutagenesis by insertion of PCR products to the chromosome
As shown in Figure 1, error-prone PCR (epPCR) product is firstly assembled into an insertion construct consisting of left flanking region (LF), antibiotic resistant marker (AbR), epPCR product and right flanking region (RF); then, after the insertion construct is transformed into B. subtilis competent cells, epPCR product is inserted into the chromosome of B. subtilis through homologous recombination. Efficient assembly of insertion construct is the crucial step of this method. The plasmid multimerization method (Shafikhani et al. 1997; You et al. 2012) was employed to assemble insertion construct. First, LF, RF and AbR are fused together through overlap PCR (Shevchuk et al. 2004) generating fragment RF-LF-AbR.; notably, RF is put ahead of LF and a cleavage site of restriction enzyme is introduced between RF and LF. Gene of interest (GOI) is amplified by epPCR. The left end of GOI overlaps (40-50 bp) with the right end of RF-LF-AbR, while the right end of the GOI overlaps (40-50 bp) with the left end of RF-LF-AbR. Then, equal molar amount of GOI and RF-LF-AbR are mixed, the multimer of insertion construct (LF-AbR-GOI-RF) is generated by prolonged overlap extension PCR. Finally, the multimer is cut at the cleavage site between RF and LF, releasing the monomer of insertion construct.