Suicide plasmids containing promoterless reporter genes can simultaneously disrupt and create fusions to target genes of diverse bacteria
Suicide plasmids containing promoterless reporter genes can simultaneously disrupt and create fusions to target genes of diverse bacteria
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DOI：
10.1016/s0378-1119(96)00778-0
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发表时间：
1997-03-25
期刊：
影响因子：
3.5
通讯作者：
Winans, SC
中科院分区：
文献类型：
--
作者：
Kalogeraki, VS;Winans, SC
我们描述了几种被设计用来在染色体或质粒编码的基因与LacZ、PhoA或GFP报告基因之间产生融合的质粒。这些质粒都含有R6K的营养起源，但缺乏R6K pir基因，因此不能在缺乏pir的菌株中复制。目的基因的片段被引入到这些质粒中，通过同源重组将整个质粒(Campbell-type)整合在一起，从而在一步内产生融合。含有目标基因5‘端及其启动子的完整5’端或基因完整3‘端的克隆片段保留了该基因的功能拷贝，而同时缺少目标基因5’端和3‘端的片段会导致基因中断。除了便于基因表达的测量外，一些质粒还可以产生与β-半乳糖苷酶或碱性磷酸酶的翻译融合，因此在研究目标蛋白质的膜拓扑结构方面是有用的。我们通过构建和测试根癌农杆菌的Virg基因和LacZ之间的两个操纵子融合和两个蛋白质融合来证明这些质粒的有效性。
We describe several plasmids that are designed to create fusions between chromosomal or plasmid-encoded genes and the lacZ, phoA or gfp reporter genes. These plasmids all contain the vegetative origin of R6K, but lack the R6K pir gene, and therefore fail to replicate in strains lacking pir. Fragments of target genes are introduced into these plasmids, and fusions are created in a single step as a consequence of (Campbell-type) integration of the entire plasmid by homologous recombination. Cloned fragments containing either an intact 5'-end of the target gene including its promoter or an intact 3'-end of the gene preserve a functional copy of that gene, while fragments lacking both 5'- and 3'-ends of the target gene cause a gene disruption. In addition to facilitating measurements of gene expression, some plasmids create translational fusions to beta-galactosidase or alkaline phosphatase and are therefore useful in studying the membrane topology of a target protein. We demonstrate the utility of these plasmids by constructing and testing two operon fusions and two protein fusions between the virG gene of Agrobacterium tumefaciens and lacZ.