3.2 Development of the random mutagenesis system
The above scheme was tested by direct evolution of Methyl Parathion
Hydrolase (MPH) to improve its activity toward chlorpyrifos, a pesticide
that often contaminates food and environment (Lu et al. 2013). To begin
with, secretion expression of MPH was realized through inserting MPH
expression cassette at the amyE locus of B. subtilis ; the
transcription of mpd is driven by promoterPcry3A and the secretion of MPH is mediated by
signal-peptide of AprE (SPAprE) (Figure 2a). Then, the
coding region of SPAprE and MPH was amplified by epPCR,
and another fragment of RF-cleavage site (Sac
I)-LF-ZeoR-Pcry3A was generated
via overlap PCR. After multimerization, the product was converted to
monomer of insertion construct (LF-ZeoR-Pcry3A-mpd-RF)
by Sac I digestion, which was confirmed by agarose gel electrophoresis.
As shown in Figure 2b, neither of the two fragments
(RF-LF-ZeoR-Pcry3A and epPCR
product) could be detected on gel after multimerization, indicating most
of the two fragments were transformed to multimers. Sac I digestion
product matched the size of insertion construct, which was further
verified by DNA sequencing. To enhance the transformation efficiency of
insertion construct, the amount of competent cell was increased for each
transformation reaction. As shown in Figure 2c, when 400 μL competent
cells were mixed with 100 ng DNA, the transformation efficiency was
highest reaching 2.71×105 CFU/μg DNA. Additionally,
the transformation efficiency was positive correlated with the length of
flanking region. The flanking region of 0.5, 1 and 1.5-kb at each side
led to the transformation efficiency of 1.24×105,
2.59×105 and 3.53×105 CFU/μg DNA
(Figure 2d), respectively. The flanking region of 1 kb at each side of
insertion construct was used to generate a library of mpdvariants. Ten clones were randomly selected, and DNA sequencing result
of SPAprE and MPH coding region shows that each mutant
harbored 1-5 mutations, with an overall mutation rate of 0.31%.