FIGURES LEGENDS
FIGUER 1 The scheme for insertion of epPCR products to the chromosome of B. subtilis. Firstly, fragment RF-LF-AbR is generated by overlap PCR and fragment GOI is amplified by error-prone PCR (epPCR). Secondly, the DNA multimer is formed by prolonged overlap extension PCR using fragments RF-LF-AbR and GOI. Thirdly, the DNA multimers is digested to monomer (LF-AbR-GOI-RF) at the cleavage site introduced between RF and LF. Finally, the insertion construct is transformed into competent cells. LF, left flanking region; AbR, antibiotic resistant marker; GOI, gene of interest; RF, right flanking region; scissors, restriction endonuclease digestion.
FIGUER 2 Development of the random mutagenesis system.(a) Construction of MPH secretion strain BPC1. PromoterPcry3A controls transcription of mpd and the signal-peptide of AprE (SPAprE) mediates the secretion of the Methyl Parathion Hydrolase (MPH). (b) Analysis of insertion construct assembly process by agarose gel electrophoresis. The arrow indicates the position of the target DNA. (c) The effect of competent cell number on transformation efficiency of insertion construct. The amount of the insertion construct was 100 ng and contained 1-kb flanking region. (d) The effect of flanking region size on transformation efficiency of insertion construct. The amount of the insertion construct was 100 ng and the volume of competent cells is 400 μL. All data were collected from at least three biological replicates and are shown as the mean ± SD. Bars indicated by the same letter are not significantly different (P > 0.05, evaluated by Duncan’s test).
FIGUER 3 Screening of the library of MPH variants. (a) Screening of the library on LB plate containing 50 mg/L chlorpyrifos. Frist, transformants were selected on LB plates containing Zeocin. Then, the colonies were transferred to LB plates containing 50 mg/L chlorpyrifos using sterile toothpick. When chlorpyrifos are hydrolyzed by MPH, a transparent halo forms around the colony. (b) The extracellular activities of MPH in the supernatant of strain BPC1 and its mutants. All data were collected from at least three biological replicates and are shown as the mean ± SD.
FIGUER 4 Identification of the effect of each mutation on MPH . (a) Base mutations in mpd variants. The vertical line indicates the base mutations in the variants. (b) The extracellular activities of MPH in the supernatant of strain BPC1 and six mutants with single mutation. (c) Specific activity of wild-type MPH and the five variants with single mutation. (d) SDS-PAGE analysis of extracellular MPH in the supernatant of the strain BPC1 and its mutants. (e) SDS-PAGE analysis of extracellular MPH in the supernatant of the mutants MT-C2 and MT-C3. Mutant MT-C2 harbors mutations T47C and T806A, and mutant MT-C3 contains mutations of T47C, T806A and G938A. The strains were grown on 2×SR medium and incubated at 37°C with shaking at 200 rpm for 36 h. Equal amounts (20 μl) of culture supernatant were loaded into each lane. (f) The extracellular activities of MPH in the supernatant of strain BPC1, MT-C2 and MT-C3. M, protein markers. The arrow indicates the position of the target band. All data were collected from at least three biological replicates and are shown as the mean ± SD. Bars indicated by the same letter are not significantly different (P > 0.05, evaluated by Duncan’s test).