氨基甲酸乙酯降解酶的基因克隆

Gene clone of urethane degradation enzyme

提取氨基甲酸乙酯降解菌株基因组DNA,以27f和1492r为引物PCR扩增并测定目的菌株16Sr DNA序列,基因组DNA以Mbo I进行酶切,与BamH I酶切的pUC18连接形成重组载体,将载体转化入大肠杆菌BL21中,构建基因组文库。利用溴麝香草酚蓝筛选培养基上筛选含有氨基甲酸乙酯降解酶基因的重组菌。通过本实验,鉴定出氨基甲酸乙酯降解酶菌株属于蜡样芽孢杆菌。阳性重组菌株可通过含有1%溴麝香草酚蓝的培养基(pH=6.5)筛选出来。

Ethyl carbamate was extracted to degrade genomic DNA, then 27f and 1492r as PCR primer was used to amplify and measure strains 16s rDNA sequences. This genomic DNA was digested with the restriction endonuclease MboI, then connected with pUC 18 which was digested by Barn/-/I to form recombinant vector. Recombinant vector was transformed into Escherichia coli BL21 cartier to construct genomic libraries. Recom- binant bacteria containing urethane hydrolase gene can be screened by bromothymol blue screening culture medium. According to the result, urethane degrading enzyme strains belonged to Bacillus cereus, the positive recombinant strains can be screened out from medium（pH=6.5） which contained 1% bromothymol blue.