Snaith M R, Kilby N J, Murray J A
Institute of Biotechnology, University of Cambridge, UK.
Gene. 1996 Nov 21;180(1-2):225-7. doi: 10.1016/s0378-1119(96)00449-0.
We have developed an Escherichia coli system for testing the behaviour of plasmids carrying target sites for the F1p site-specific recombinase. The E. coli strain BL-FLP is described, which carries a chromosomally integrated bacteriophage T7 RNA polymerase gene expressed from a lac promoter, and harbours the plasmid pMS40.pMS40 has the features: (i) it carries the FLP recombinase gene under the control of a bacteriophage T7 promoter, (ii) it confers kanamycin resistance, and (iii) it uses an R6K origin of replication; these two latter features make it compatible with most conventional cloning vectors. Substrate plasmids carrying F1p-recognition targets (FRT) are transformed into BL-FLP, and the consequences of F1p-mediated recombination can be analysed after subsequent extraction of plasmid DNA. We show that this system is capable of base-perfect F1p-mediated recombination on plasmid substrates. We also present a corrected sequence of the commonly used F1p substrate plasmid, pNEO beta GAL (O'Gorman et al. (1991) Science 251, 1351-1355).
我们开发了一种大肠杆菌系统，用于测试携带F1p位点特异性重组酶靶位点的质粒的行为。描述了大肠杆菌菌株BL-FLP，它携带一个从lac启动子表达的染色体整合噬菌体T7 RNA聚合酶基因，并含有质粒pMS40。pMS40具有以下特征：（i）它在噬菌体T7启动子的控制下携带FLP重组酶基因，（ii）它赋予卡那霉素抗性，（iii）它使用R6K复制起点；后两个特征使其与大多数传统克隆载体兼容。携带F1p识别靶标（FRT）的底物质粒被转化到BL-FLP中，在随后提取质粒DNA后，可以分析F1p介导的重组的结果。我们表明，该系统能够在质粒底物上进行碱基完美的F1p介导的重组。我们还给出了常用的F1p底物质粒pNEO beta GAL的校正序列（奥戈尔曼等人（1991年）《科学》251卷，第1351 - 1355页）。
