Protocol for Constructing Glycine Oxidase Mutant Libraries by Random Mutagenesis

Experiment Summary

Glyphosate, a broad spectrum herbicide widely used in agriculture all over the world, inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, and glycine oxidase (GO) has been reported to be able to catalyze the oxidative deamination of various amines and cleave the C-N bond in glyphosate. Here, in an effort to improve the catalytic activity of the glycine oxidase that was cloned from a glyphosate-degrading marine strain of Bacillus cereus (BceGO), we used a bacteriophage T7 lysis-based method for high-throughput screening of oxidase activity and engineered the gene encoding BceGO by directed evolution.

Materials and Reagents

Bacillus cereus HYC-7
Escherichia coli (E.coli) DH5α strain, bacteriophage T7
Glyphosate
Tryptone
Yeast extract
Ampicillin
o-Dianisidine dihydrochloride
Horseradish peroxidase
Protein expression vector of pGEX-6P-1
Recombinant plasmid pGEX-GO contains encoding gene of glycine oxidase from Bacillus cereus HYC-7
The nucleotide sequence (1,110 bp) was submitted to the NCBI Genbank and gained the accession number (KC203486.1).
Taq DNA polymerase
Restriction Enzyme (BamHI and XhoI)
dATP, dTTP, dCTP, dGTP
Luria-Bertani medium

Equipment

96 deep-well plates
Gel purification column
Spectrum plate reader
Cyclers
Ultrasonic processor

Procedure

A. Random mutagenesis

Prepare the amplification mixture (100 µl) as follows:

10 µl of 10x Taq buffer (Mg2+ plus)

5 µl of 10 mM Mn2+

2 µl of 10 mM dGTP and dCTP

1 µl of 10 mM dATP and dTTP

2 µl of 100 nM oligonucleotide primer F

2 µl of 100 nM oligonucleotide primer R

1 µl of recombinant plasmid pGEX-GO as template

2 µl of Taq DNA polymerase

Add ddH₂O to a final volume of 100 μl

2. The error-prone PCR procedure was performed using the following parameters:

The error-prone PCR procedure was performed using the following parameters

3. Check product by electrophoresis of 5 μl of error-prone PCR product on 1% agarose gel.

4. Error-prone PCR products were purified, digested with BamHI and XhoI, cloned into pGEX-6P-1, and transformed into E.coli DH5α to construct the random mutant library.

Fig. 1 Agarose gel electrophoresis of PCR products by the first round error-prone PCR and recombinant plasmids.

B. Screening

The resulting library of BceGO mutants were expressed into 96 deep-well plates (containing 0.6 ml Luria-Bertani medium) and transferred onto Luria-Bertani agar plates as corresponding copies, followed by an overnight growth (37 °C, 300 rpm).
When the cultures grew to saturation, both IPTG (at a final concentration of 0.1 mM) and the bacteriophage T7 (above 100 particles per cell) were added into 96 deep-well plates to synchronize the induction of recombinant mutants with the release of the lysis of the host E.coli DH5α at 37 °C with shaking for 6 h.
The enzyme-coupled colorimetric assay (200 µl) was performed as follows:

159 µl of lysis cell extracts

20 µl of 50 mM glyphosate (at a decreasing substrate concentration gradient in sequential rounds of screening system)

20 µl of 0.32 mg/ml o-dianisidine dihydrochloride

1 µl of 5 unit/ml horseradish peroxidase

Then incubated at 25 °C for 8 h

4. The absorbance change at 450 nm for each well in the microtiter plates was measured and compared with the control (harboring wild-type BceGO or containing the empty vector pGEX-6P-1). Mutants that outperformed the wild-type were chosen for further activity analysis.

Fig. 2 The screening process of glycine oxidase mutant library.

* For research use only. Not intended for any clinical use.

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Protocol for Constructing Glycine Oxidase Mutant Libraries by Random Mutagenesis

Experiment Summary

Materials and Reagents

Equipment

Procedure

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