穿梭质粒pBC11的构建及性质研究

Construction and Characterization of the Shuttle Piasmid pBC11

质粒 pUB110与 pUC18分别册 EcoRI 酶切,T_4DNA 连接酶进行连接,得到重组质粒 pBC11。琼脂糖凝胶电泳分析,该质粒分子量为4.8×10~6道尔顿。pBC11是一种穿梭质粒,能转化大肠杆菌和枯草杆菌,它对大肠杆菌 C600、JM101的转化率分别为:4×10~5、1.5×10~5转化体/μgDNA.对枯草杆菌 BR151、QB1130的转化率分别为1.5×10~3、2.15×10~3转化体/μgDNA。在基因表达方面,pBC11上的 K_m^r 基因能在大肠杆菌和枯草杆菌中复制和表达,但 pBC11的 Ap^r 基因不能在枯草杆菌中表达。

A shuttle plasmid pBC11 was constructed by ligating the Ecokl fragments of Bacillus subtilis plasmid pUB110 and Escherichia coli plasmid pUC18 with T4 DNA ligase.The agarose gel electrophoretic pattern shows that the molecula weight of plasmid pBC11 is 7.2kb.As expected,there are two BamHl sites in the plasmid pBC11.The ligation orjentation of pUB110 and pUC18 was identified by the molecular weight of BamHl DNA fragments of pBC11.The shuttle plasmid pBC11 can transform both E.coli and B.subtilis.The trans- formation frequencies in E.coli C600 and JM101 are 4×10~3 and 1.5×10~3 transformants/ugDNA respectively.When plasmid DNA is transformed into B.subtilis BR151 and QB1130,the transformation frequencies are 1.2×10~3 and 2.15×10~3 transformants/ugDNA respectively.The plasmid pBC11 can replicate and cxpress Km^r gene in both E.coli and B.subtilis while the Ap^r gene from pUC18 in pBC11 is not expressed in B.subtilis.