luxAB发光基因标记的铜绿假单胞菌株的构建

目的构建表达luxAB发光基因的铜绿假单胞基因工程菌。方法用BglⅡ酶切pUC-luxAB质粒,回收luxAB片段与BamHⅠ酶切的质粒载体pBBR1MCS-5连接形成重组质粒pBBR-luxAB,再转化E.coliDH5α感受态细胞,经庆大霉素抗性、氯霉素抗性、发光检测多重筛选含有pBBR-luxAB重组质粒的的阳性克隆,并设立对照菌株。抽提pBBR-luxAB质粒、酶切、凝胶电泳,验证质粒构建的正确性。通过二亲本杂交方式将pBBR-luxAB质粒导入铜绿假单胞菌(Pseudomonas aeruginosa),构建基因工程菌铜绿假单胞菌(pBBR-luxAB)并对其进行质粒传代稳定性、发光动力学曲线以及发光度和活菌数关系进行测定。结果成功构建pBBR-luxAB重组质粒并且确定其成功转入铜绿假单胞菌中,连续转接4次后质粒保持率仍可达93%。在加入底物20min后,重组菌发光强度趋于稳定水平(1.32mV/ml),其发光强度与活菌量呈显著正相关(r=0.96,P<0.05)。结论该研究成功构建luxAB发光基因标记的铜绿假单胞菌(pBBR-luxAB)。

Construction of luxAB-labelled Pseudomonas aeruginosa

Objective To construct a genetically engineered Pseudomonas aeruginosa expressing luxAB luciferase gene.Methods Use the BglⅡ enzyme to cut the plasmid pUC-luxAB,then recovered luxAB segment,and concatenate the plasmid carrier cut by the enzyme BamHⅠto form the reconstructed plasmid pBBR-luxAB,then transform E.coli DH5α competent cells.The positive clones that contain the reconstructed plasmid pBBR-luxAB experience the multiple screening by gentamycin(Gm),chloromycin(Cm) and luminescence detection,and at the same time,set up the control bacteria.Extract the plasmid pBBR-luxAB,then cut by enzyme,and then gel electrophoresis,and validate the rightness of the plasmid construction.By the mean of 2-generation mating,introduce the plasmid pBBR-luxAB into Pseudomonas aeruginosa,construct the gene engineered bacteria P.aeruginosa(pBBR-luxAB),and then measure the reproductive stableness of the plasmid,bioluminescence kinetic curves and relationship between luminescence and plate counting.Results Successfully construct the pBBR-luxAB reconstructed plasmid,and surely,it is successfully introduced to P.aeruginosa.After 4 successively transfer,the pBBR-luxAB retaining rate still can keep at 93%.After the addition of substrates for 20min,the luminous intensity of the reconstructed bacteria tends to be stable(1.32 mV /ml).There is a significantly positive correlation between luminous intensity and viable cell counting(r = 0.96,P < 0.05).Conclusion This research successfully constructed luxAB labelled P.aeruginosa,providing a useful means to study the survival,migration and damage of P.aeruginosa.