Recombine Of The Genetically Engineered Bacteria Degrdning Creatinine To Sarcosine

Obejective:The plasmid of containing creatinine and creatinase were constructed respectively and transformed into Escherichia coli,studying its expression and enzyme activity.Methods:According to the Gen Bank data known of creatinine(stench fake single cell bacteria 1978) and creatinase(yellow Bacillus U-188) of gene sequence, by Shanghai Giquet bio limited for bio synthesis, get the gene fragment of creatinine a and creatinase b. According to creatinine and creatinase of gene sequence design primer,PCR amplification, double enzyme cutting recovery and purification, and clone it to GV296 to construct the recombinant plasmid GV296-a and GV296-b,which were transformed into E.coli DH5 a,meanwhile screening and identificating the transformant. Extract the recombination plasmid GV296-a and GV296-b,and colne it to E.coli BL21(DE3), bacterial liquid PCR identification, get the expression of engineering strain GV296-a/BL21(DE3) and GV296-b/BL21(DE3), joining the inducer IPTG-induced protein expression, and SDS-PAGE was conducted.Engineering bacteria of the GV296-a/BL21(DE3) was cultured at a particular concentration of creatinine acid in the culture medium for 24 hours, the concentration of creatinine acid in the culture fluid was determined;Engineering bacteria of the GV296-a/BL21(DE3) was cultured at a particular concentration of creatinase acid in the culture medium for 24 hours, the concentration of creatinase acid in the culture fluid was determined.Results:1.Construction of recombinant plasmid GV296-a and GV296-b successfully, these recombinant plasmids containing double digestion, electrophoresis revealed two restriction enzyme fragment size is about 0.783 Kb and 1.212 Kb and the theoretical values.2.The reconstructed plasmid GV296-a and GV296-b were transformed into E.coli BL21(DE3) competent cells. The bacterium was induced by IPTG and achieved efficient expression.The bacterium of GV296-a/BL21(DE3) expressed creatinine,which had activity, and its degradation incrased singnificantly, protein of molecular weight approximately 29 KD by SDS-PAGE;while the bacterium of GV296-b/BL21(DE3) expressed creatinase,which had activity,and its degradation incrased singnificantly,protein of molecular weight approximately 43 KD by SDS-PAGE.Conclusion:1.Construct recombinant plasmids of the creatinine and creatinase respectively successfully,by double restriction endonuclease and sequence analysis in line with theoretical values.2.Construct the expression of engineering strain GV296-a/BL21(DE3) and GV296-b/BL21(DE3) successfully,and efficiency induced the expression of the creatinine and creatinase,which both have the enzymes activity.