IMPROVED SINGLE AND MULTICOPY LAC-BASED CLONING VECTORS FOR PROTEIN AND OPERON FUSIONS
IMPROVED SINGLE AND MULTICOPY LAC-BASED CLONING VECTORS FOR PROTEIN AND OPERON FUSIONS
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DOI：
10.1016/0378-1119(87)90095-3
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发表时间：
1987-01-01
期刊：
影响因子：
3.5
通讯作者：
KLECKNER, N
中科院分区：
文献类型：
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作者：
SIMONS, RW;HOUMAN, F;KLECKNER, N
我们描述了几个新的载体的操纵子和蛋白质融合的大肠杆菌lacZ基因的建设。体外构建利用多拷贝质粒，其含有位于上游转录终止子和下游lac操纵子区段之间的合适克隆位点，所述下游lac操纵子区段的lacZ基因保留或缺乏翻译起始信号。单拷贝.多拷贝构建体的原噬菌体形式可通过遗传学方法制备，而无需体外操作。新的载体，无论是单拷贝还是多拷贝，都得到了改进，因为它们具有非常低的背景lac基因表达水平，这使得非常弱的转录和翻译信号的容易检测和准确定量成为可能。开发这些载体用于分析IS 10转座酶基因的表达，该转座酶基因每代转录少于一次，并且其转录物平均每次翻译少于一次。单拷贝和多拷贝构建体也可用于选择影响融合表达的突变，并且在单拷贝构建体中分离的突变可遗传地杂交回到多拷贝质粒上用于进一步分析。
We describe several new vectors for the construction of operon and protein fusions to the Escherichia coli lacZ gene. In vitro constructions utilize multicopy plasmids containing suitable cloning sites located between upstream transcription terminators and downstream lac operon segments whose lacZ genes retain or lack translational start signals. Single-copy .lambda. prophage versions of multicopy constructs can be made genetically, without in vitro manipulation. The new vectors, both single and multicopy, are improved in that they have very low levels of background lac gene expression, which makes possible the easy detection and accurate quantitation of very weak transcriptional and translational signals. These vectors were developed for analysis of the expression of IS10''s transposase gene, which is transcribed less than once per generation, and whose transcripts are translated on average less than once each. Both single and multicopy constructs can also be used to select mutations affecting fusion expression, and mutations isolated in single-copy constructs can be crossed genetically back onto multicopy plasmids for further analysis.