Experimental Study Of Transforming Gene Frc Of Oxalobacter Formigenes To Escherichia Coli And Expression

Objective to study cloning oxalate-degredation gene Frc of Oxalobacter formigenes (OF)into expression vector PGEX-4T-2,and transformed it into probiotic E. coli Nissle 1917 (EcN), aim to observe the stable expression of oxalate-degredation associated enzyme- Formyl-CoA Transferase(FCoAT) and evaluate the potential of oxalate metabolism of EcN-Frc.Methods to culture Oxalobacter formigenes in modified MRS medium under YQX-II-type anaerobic workstation. Identified OF by G Gram stain, bacterial morphology and specific nucleic acid sequences detection. PCR amplification of target gene Frc, using OF genome as a template .Cloned it into vector pMD? 19-T to construct vector pMD ? 19-T-Frc. Transformed into Escherichia coli JM109, selected monoclonal one. Verified by sequencing, then compare it to GenBank (U82167.1) in BLAST .Select the exactly monoclonal one and cultured it .Restriction enzyme digestion pMD ? 19-T-Frc, and the prokaryotic expression vector PGEX-4T-2 individually by BamHⅠ/ EcoRⅠ,after the products were purified , the Frc was extracted and inserted into the prokaryotic expression vector PGEX-4T-2 by T4 ligase in 16℃for 12 hours to construct the recombinant plasmid PGEX-4T-2-Frc,and transformed into Escherichia coli JM109, picked monoclonal, enzyme electrophoresis analysis ,verified by sequencing. Finally the recombinant expression plasmid PGEX-4T-2-Frc was transformed into E.coli Nissle 1917, selected by AMP (100mg/L) LB plate. The new bacteria was called E. coli Nissle 1917-Frc (E.coli Nissle 1917-Frc, EcN-Frc),which induced in a final concentration of 1.0mmol/L IPTG ,at 0,2,4,6,8,10 hours .Bacteria medium and bacteria was collected,Extract the cell protein of EcN,GST-FCoAT was detected culture medium and bacteria by ELISA. Furthermore, induce EcN-Frc in a final concentration of 1.0mmol/L IPTG at 0,1,2,3,4,5, 6 hours.Collect 50ug total protein to identify in western-blot. Results Successly culture OF in modified MRS medium at anaerobic condition, and culture EcN in LB medium. Electrophoresis Gene amplification OF genome: appears Frc specific band in 1300bp position. Sequence analysis proved that the Frc was correct to GenBank (U82167.1) Restriction enzyme digest prokaryotic expression plasmid PGEX-4T-2-Frc, and electrophoresis was appered 1300bp and 4900 specific band, the former is Frc, the latter is linear PGEX-4T-2, which is consistent with sequencing results. Western blot: induce EcN in 1.0mmol / L IPTG, 75KD protein band was visible, which was GST-FCoAT. The ELISA results showed that it was expressed in EcN-Frc supernatant and biomass. And expression level was higher in the supernatant than the deposit of bacteria. Conclusion Construct the prokaryotic expression vector PGEX-4T-2-Frc was success. E.coli Nissle 1917-Frc is a new type of oxalate-degradation probiotic bacteria, successfully carried gene Frc and express FCoAT, which will provide materials of the next experimental in vitro and vivo of oxalate metabolism, and is expected to be used in hyperoxaluria and prevention of oxalate stones.