2.3 Generation of MPH mutant library
The right flanking region (RF) of amyE locus was amplified from
strain BPC1 using primer pair P1/P5. Another fragment containing left
flanking region (LF), ZeoR andPcry3A was amplified from strain BPC1 using
primer pair P6/P2. These two fragments were fused by overlap PCR
(Shevchuk et al. 2004) using primer pair P1/P2, resulting in fragment
RF-LF-ZeoR-Pcry3A . The cleavage
site Sac I was introduced between RF and LF.
The coding region of SPaprE and MPH was amplified by
error-prone PCR with primer pair P3/P4 using the Starmut Random
Mutagenesis Kit (GenStar, Beijing, China). The PCR reaction solution
with a total volume of 50 μL contained 0.02 ng/μL template, 25 μL
2×buffer, 3 μL StarMut Enhancer, 0.4 mM primer pair P3/P4. The PCR was
conducted as follows: initial denaturation at 95 °C for 2 min and
subsequent steps of 94 °C for 30 s, annealing at 56 °C for 1 min, and
extension at 72 °C for 1 min for 30 cycles total. The overlap of 47 bp
between the left end of the epPCR product and the right end of
RF-LF-ZeoR-Pcry3A was
introduced through primers P4 and P1, and the overlap of 45 bp between
the right end of the epPCR product and the left end of
RF-LF-ZeoR-Pcry3A was
introduced through primers P3 and P2.
To generate the multimer of fragment
LF-ZeoR-Pcry3A -mpd -RF,
each 50 μL reaction system contained 0.2 mM dNTP, equal molar amount of
RF-LF-ZeoR-Pcry3A and epPCR
product (~30 pmol for each fragment), and 0.04 U/μL
Phusion polymerase without the addition of primers. The prolonged
overlap extension PCR was conducted as follows: denaturation at 98 °C
for 30 s; 30 cycles of denaturation at 98 °C for 15 s, annealing at 60
°C for 15 s, and extension at 72 °C at a rate of 2 kb/min; followed by
72 °C extension for 10 min. The multimer product was digested with Sac I
to release the monomer of fragment
LF-ZeoR-Pcry3A -mpd -RF
which was transformed into the competent cells of strain SCK6 according
to following procedure.