Difference between revisions of "Team:SSTi-SZGD/Experiments"

Synthetic organophosphates (OPS) are a group of highly toxic chemicals widely used to control various agriculture pests. Accounting for 38％ of total pesticides used globally. OP pesticides are acetylcholinesterase (ACheE) inhibitors , and various clinical effects can occur from OP poisoning in humans. The growing public concern about their safety and the widespread use of OPs in modern agriculture has stimulated the development of effective and safe remediation strategies for detoxification of Ops.

Organophosphorus hydrolase (OPH), encoded by the opd gene of Pseudomonas diminuta MG and Flavobacterium sp. Strain ATCC27551, is a homodimeric phosphotriesterase that can hydrolyze a wide range of OPs (4, 5). Hydrolysis of OPs by OPH reduces their toxicity by several orders of magnitude (3). Practical applications of large-scale enzymatic degradation have always been limited by the cost of purification and stability of OPH. Although the use of whole cell as biocatalysts is an alternative strategy for treatment of OPs, the inaccessibility of the pesticides across the cell membrane reduces the overall catalytic efficiency (2, 6). This barrier of substrate transport can be overcome by displaying OPH on the cell surface. However, surface expression of OPH resulted in instability of the cell membrane and growth inhibition of the cells, arising from increased metabolic burden placed on the cell (2, 6).

Recently, an OP degradation gene (mpd) encoding methyl parathion hydrolase (MPH) was isolated from a methyl parathiondegrading bacterium Plesiomonas sp. strain M6, but it showed only 12% identity to opd gene at the amino acid level (17), suggesting significant novelty of the gene-enzyme system. More recently, we cloned the mpd gene (GenBank accession no. DQ677027) from a chlorpyrifos-degrading bacterium Stenotrophomonas sp. Strain YC-1 (18). In the present study, to enable secretion of MPH to the periplasm of E. coli, we used the twin-arginine signal peptide of trimethylamine N-oxide reductase (TorA) from E. coli (19). We apply light control system to express a gene that encodes hydrolase to degrade pesticides residues.

Bacterial strains and Plasmid Construction

The DH5 alpha E.coli strain was used to amplify plasmid and a negative expression control. The Luria Bertani broth medium used in this study was composed of 1.0 % (w/v) tryptone , 0.5% (w/v) yeast extract and 0.5% (w/v) NaCl in distilled water . the ph was adjusted to 7.0 with a NaOH solution-soid plates were obtained by adding 1.5% (w/v) agar . the antibiotic 1000ug/ml Penicillin and streptomycin (final concentration) was added during plate preparation after solution cooling or to the LB medium prior to culture.

The torA gene fragment encoding the signal peptide and the first four amino acid residues of the mature TorA was amplified by polymerase chain reaction (PCR) from plasmid pSCTorAGFP using primers P1 and P2. The PCR products were digested with EcoRI and BamHI, and then ligated into similarly digested pUC18 to generate pUT18. The mpd gene was amplified by PCR from plasmid pMDQ using primers P3 and P4. The PCR products were digested with BamHI and HindIII, and then ligated into similarly digested pUT18 to generate pUTM18. To construct a control plasmid expressing cytoplasmic MPH, the mpd gene was amplified by PCR from plasmid pMDQ usingprimers P5 and P4. The PCR products were digested with EcoRI and HindII I, and then ligated into similarly digested pUC18 to generate pUM18. All plasmid constructions were verified by DNA sequence analysis. Transformation of the plasmid into E. coli was carried out by using the below method.

Cell condition and Cell Fractionation

Sub-cultures were grown overnight to added suitable the concentration of antibiotics in 5ml LB and used to inoculate 30ml LB liquid to a starting OD600 of 0.5, cells were shaking culture at 37℃, when cultures reached a cell density of 1.0~2.0, used silver paper to make the culture be in dark ambient induce recombinant protein expression.

Cells were fractionated to yield cytoplasmic by the cell lysis solution ,and periplasmic samples by the Arginine buffer as follows, the E.coil strain cell medium by centrifugation 10000rpm, 5 min, and the 1 g cell added 10ml concentration of arginine buffer 0.4mol/L ,pH8.0, after extracting time 45 min on the ice by the centrifugation 10000rpm, the supernatant is periplasmic.

And we used “Soluble protein solution” to extract the cytoplasmic, used in this study was composed of 0.0625mol/L Tris- HCl, the ph was adjusted to 6.8 , 2% (w/v) SDS, 10%(w/v) glycerol and 5% 2-mercaptoethanol were obtained extract . Take the strain centrifugation 10000rpm,5min at 4℃, 15ml PB wash cell, centrifugation by the bacterium suspension, discard the supernatant, Repeat that steps twice. Added 200ul to soluble protein solution, to make the bacteria fully suspended and,10min boilingwater to make it inactive. Centrifugation 12000rpm,15min collect supernatant, keep at -20℃.

Periplasmic protein were (OD600=1.0) in 100 mM phosphate buffer (pH 7.4). MPH activity assay mixtures (1 mL, 3% methanol) contained 50 μg/mL methyl parathion (added from a 10 mg/mL methanol stock solution), 870 μL of 100 mM phosphate buffer (pH 7.4), and 100 μL of cells. The enzyme activity was measured using a UV/VIS spectrophotometer (UV-1601PC; Shimadzu Corp., Kyoto, Japan) at 30 C by monitoring the increases of linear optical density over time at 405 nm as methyl parathion was hydrolyzed to p-nitrophenol (ε405=17,700 M-1 cm-1). Activities were expressed as units (1 μmol of p-nitrophenol formed per minute) per OD600 whole cells.