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.