甲基对硫磷降解菌GFP标记菌株的构建

CONSTRUCTION OF GFP-LABELLED PSEUDOMONAS PUTIDA DLL-1,A METHYLPARATHION-DEGRADING BACTERIUM

用Sau3AⅠ酶切甲基对硫磷降解菌DLL 1总DNA ,与BamHⅠ酶切的启动子探针载体 pRobe -GFP酶连 ,转化E .coliDH5α ,在选择性平板LB(Ampr)上从大约 1× 10 4个菌落筛选到 5 0个含启动子片断的阳性克隆 .挑选其中一个阳性克隆 ,将启动子片断克隆到pIJ2 92 5和 pBBR -MCS2上构建成重组质粒 pIJGFP和广宿主标记载体pB BRGFP .然后将 pIJGFP载体上的启动子片断克隆到广宿主载体 pTR10 2上 ,获得第二个广宿主标记载体pTRGFP .将pTRGFP和pBBRGFP通过三亲接合分别标记于DLL 1得到两株标记菌 ,即DLLTR和DLLBR .通过激光共聚焦显微镜观察和软件Lasersharpversion 3.2分析发现 pTRGFP在E .coli中表达很强而在DLL 1中表达很弱 ,而 pBBRGFP正好相反 .图 5表 2参

The total DNA of DLL-1 was digested by Sau3AⅠand ligated to pRobe-GFP that was digested by BamHⅠ, and the product was transformed to the E.coli DH5αcompetent cells. Fifty positive clones that could emit green fluorescence under UV were selected from 100 000 clones under selective plates with ampicillin. One selected clone with a promoter fragment was cloned into pIJ2925 and pBBR-MCS2,and then constructed the recombinant pIJGFP and labeled vector pBBRGFP. The promoter fragment from pIJGFP was subcloned into broad host vector pTR102, and then pTRGFP was obtained. pTRGFP and pBBRGFP were transformed into methylparathion-degrading bacterium Pseudomonas putida DLL-1 by tri-parents conjugation, respectively. The two resulted trans-conjugants were DLLTR and DLLBR. pTRGFP expressed well in E.coli and weakly in DLL-1 through confocal laser scanning microscopy and software lasersharp version 3.2. But pBBRGFP expressed inversely. Fig 5, Tab 2, Ref 12