Yap W H, Thanabalu T, Porter A G
Institute of Molecular and Cell Biology, National University of Singapore.
J Bacteriol. 1994 May;176(9):2603-10. doi: 10.1128/jb.176.9.2603-2610.1994.
The influence of expression control sequences (ECSs; promoters and ribosome-binding sites [RBSs]), transcriptional terminators, and gene orientation on the expression of the Escherichia coli lacZ gene in the gram-negative microorganisms Caulobacter crescentus and E. coli was investigated. A series of broad-host-range expression vectors, based on the RK2 plasmid derivative pRK248, were constructed. The ECSs included the tac promoter, the promoter for the surface layer protein of C. crescentus, and promoters from a number of gram-positive bacteria together with their associated RBSs. In addition, synthetic ECSs were constructed by using different combinations of promoters and RBSs. lacZ expression was found to be dependent on the nature of the promoter and RBS and, to a lesser extent, on the presence of a transcriptional terminator and the orientation of the promoter-lacZ construct in pRK248. The relative efficiencies of the various ECSs in driving lacZ expression differed markedly in C. crescentus and E. coli. In C. crescentus, the ECS ptac1 (tac promoter and consensus RBS for C. crescentus mRNAs) appeared to be the most efficient, producing 12-fold-higher activity than did pSL (promoter for the surface layer protein of C. crescentus and its putative RBS). pSL was not transcribed in E. coli, whereas various promoters from gram-positive microorganisms were transcribed in both C. crescentus and E. coli. A number of ECSs were also used to drive mosquitocidal toxin gene expression in C. crescentus, and a correlation between toxin expression and lacZ expression was observed.
研究了表达控制序列（ECS；启动子和核糖体结合位点[RBS]）、转录终止子以及基因方向对革兰氏阴性微生物新月柄杆菌和大肠杆菌中大肠杆菌lacZ基因表达的影响。构建了一系列基于RK2质粒衍生物pRK248的广宿主范围表达载体。ECS包括tac启动子、新月柄杆菌表层蛋白的启动子、一些革兰氏阳性细菌的启动子及其相关的RBS。此外，通过使用启动子和RBS的不同组合构建了合成ECS。发现lacZ表达取决于启动子和RBS的性质，在较小程度上还取决于转录终止子的存在以及pRK248中启动子-lacZ构建体的方向。各种ECS驱动lacZ表达的相对效率在新月柄杆菌和大肠杆菌中差异显著。在新月柄杆菌中，ECS ptac1（tac启动子和新月柄杆菌mRNA的共有RBS）似乎是最有效的，其活性比pSL（新月柄杆菌表层蛋白的启动子及其推定的RBS）高12倍。pSL在大肠杆菌中不转录，而革兰氏阳性微生物的各种启动子在新月柄杆菌和大肠杆菌中均转录。还使用了一些ECS来驱动新月柄杆菌中杀蚊毒素基因的表达，并观察到毒素表达与lacZ表达之间的相关性。
