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%.