群体感应调控的GFP报告质粒构建与表达

Construction and Expression of Green Fluorescent Protein Reporter Plasmid Regulated by Quorum-Sensing

根据重叠区延伸基因拼接法(SOEing)设计特别的引物,运用PCR分别从pLuxI-aiiA质粒和pEGFP-N1质粒上扩增海洋费氏弧菌(Vibrio fischeri)荧光素酶操纵子的启动子pLuxI和绿色荧光蛋白基因EGFP。然后利用SOEing法将这两个独立片段拼接得到pLuxI-EGFP融合基因,接着使用AatⅡ、HindⅢ限制性酶双酶切pLuxI-EGFP融合基因和载体pluxCcdB3,再用T4 DNA连接酶进行连接,接着转化DH5α感受态细菌。挑取长出的单克隆培养并抽提质粒,经测序鉴定质粒构建成功。以GFP报告质粒和pLuxRI2质粒共转化DH5α细菌,流式细胞计检测共转化菌有明显的荧光信号。

Recombinant gene SOEing was used to construct the fusion gene of the bioluminescent marine bacterium Vibrio fischeri promoter pLuxI and EGFP. pLuxI-EGFP Fusion gene and pluxCcdB3 vector were double digested with Aat Ⅱ, Hind Ⅲ restriction enzyme, then made ligation reaction with T4 DNA ligase, and then transformed DH5α competent cell. Afterwards,several single clones were picked up for culture, then plasmid was extracted. Finally,DNA sequenceing verified the correctness of construction. In order to test the GFP expression,GFP reporter plasmid was mixed with pLuxRI2 plasmid to co-transform DH5α cell, and strong fluorescent sigal in co-transformed cell was detected by flow cytometer.