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).