Labes M, Pühler A, Simon R
Fakultät für Biologie, Lehrstuhl für Genetik, Universität Bielefeld, F.R.G.
Gene. 1990 Apr 30;89(1):37-46. doi: 10.1016/0378-1119(90)90203-4.
A series of broad-host-range expression and lac fusion vectors, based on RSF1010 derivatives, was constructed. The expression vectors contain various promoters (pNm, plac, ptac and pS1) for expression of foreign genes. The efficiency of the promoters was determined in Escherichia coli, Rhizobium meliloti, Rhizobium leguminosarum and Pseudomonas putida by beta-galactosidase activity measurements. Of the promoters assayed in E. coli, the most effective is the tac promoter, whereas in soil bacteria the appropriate promoter for overexpression of foreign genes is the NmR promoter. The GmR gene, serving as a selectable marker for the plasmids, was efficiently expressed in R. meliloti as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and thus, pGm was also used to construct an expression vector. The translational fusion vectors allow the identification and characterization of promoter-carrying cloned fragments on the translational level, whereas the transcriptional fusion vectors can be used to identify and to study promoters on cloned fragments. All lac fusion vectors contain the E. coli lacZ gene or the complete lac operon facilitating quantification of expression.
构建了一系列基于RSF1010衍生物的广宿主范围表达载体和lac融合载体。这些表达载体含有用于外源基因表达的各种启动子（pNm、plac、ptac和pS1）。通过β-半乳糖苷酶活性测定，在大肠杆菌、苜蓿根瘤菌、豌豆根瘤菌和恶臭假单胞菌中确定了这些启动子的效率。在大肠杆菌中检测的启动子中，最有效的是tac启动子，而在土壤细菌中，用于外源基因过表达的合适启动子是NmR启动子。作为质粒选择标记的GmR基因，经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示在苜蓿根瘤菌中高效表达，因此，pGm也用于构建表达载体。翻译融合载体允许在翻译水平上鉴定和表征携带启动子的克隆片段，而转录融合载体可用于鉴定和研究克隆片段上的启动子。所有lac融合载体都含有大肠杆菌lacZ基因或完整的lac操纵子，便于表达定量。
