利用不相容质粒共转化大肠杆菌对Cre重组酶体内重组活性的可视检测

源自噬菌体P1的Cre重组酶可以识别 34bp的靶DNA序列loxP ,进行位点特异性的重组反应。为了简便地检测Cre酶在大肠杆菌中的重组活性 ,分别将cre基因和上下游带有loxP的绿色荧光蛋白基因 (gfp)克隆到具有不同抗性的两种不相容质粒中 ,然后将构建的原核表达载体pET30a Cre和pET2 3b loxGFP电击共转化大肠杆菌BL2 1(DE3) ,利用卡那霉素和氨苄青霉素双抗生素抗性进行筛选。通过直接观察转化子的绿色荧光 ,便可以显示Cre酶的体内重组活性 ,并进一步通过SDS PAGE分析、质粒酶切鉴定进行了验证。结果表明 :以gfp为报告基因、通过两种不相容质粒共转化大肠杆菌可以为研究和改进Cre loxP重组系统提供一种简便直观的检测方法

A simple and visible assay for cre recombinase activity in Escherichia coli by using two incompatible plasmids

The Cre/loxP system derived from bacteriophage P1 is widely used to carry out complex manipulations of DNA molecules both in vitro and in vivo. In order to further characterize and modify the Cre/loxP system, a convenient method for assaying the recombination efficiency is needed. A simple and visible assay is described, in which two incompatible plasmids, separately carrying the cre gene and loxP-flanked gfp gene, were co-transferred into E.coli. The cre gene was inserted into a kanamycin-resistant bacterial expression vector, designated pET30a-Cre. The gfp gene, flanked by directly repeated loxP sites, was cloned into an ampicillin-resistant expression vector to generate pET23b-loxGFP. E.coli BL21(DE3) was cotransformed with pET30a-Cre and pET23b-loxGFP, and cultured in the presence of both ampicillin and kanamycin. Under UV illumination, the Cre-mediated recombination events can be easily detected. The fidelity of recombination was verified by SDS-PAGE analysis and restriction analysis followed by DNA sequencing. Thus, this cotransformation method provides a straightforward assay that can be used to modify the Cre/loxP system.