含双乳糖启动子质粒pLJ3的改建

用EcoRI部分酶切质粒pLJ3,将其粘末端变成平末端,进行平末端连接;再用EcoRI完全酶切,进行粘末端连接,最后我们消去了pLJ3质粒Lacuv5启动子上游的EcoRI酶切位点,得到了一种新质粒pFGI。pFGI含有两个串联的Lacuv5启动子片段,其中包括Lacuv5启动子,操纵基因及LacZ基因的SD序列和一个四环素抗性基因,紧挨LacZ基因的SD序列后,有一EcoRI酶切位点,可方便地插入外源基因,为外源基因在大肠杆菌中的表达提供有效的启动子和核糖体结合所需的SD序列。

Reconstruction of Plasmid pLJ3 That Contains Two Copies of Lacuv5 Promotor

Using a simple, quick and efficient method, namely, partially digesting of pLJ3 DNA with EcoRI, converting protruding termini of DNA to blunt ends, joining blunt cnds; cutting with EcoRI again, and joining together DNA moleculet with compatible cohesive termini, we eliminated a EcoRI site upstream from lacuv5 promotor of pLJ3 and got a new plasmid pFG1. pFG1 contains two copies of lacuv 5 promoter (including Lacuv5 promotor, operator gene and the SD sequence of LacZ gene). In pFG1 there is a EcoRI site at the downstream of Lacuv 5 promotor and just two bases before the point of translation of the lacz gene, so that we could insert exogenous gene at the EcoRI site conveniently. The PFGI provides efficient promotors and SD sequence for exogenous gene expression in E. coli.