Construction of targeted single copy lac fusions using λ Red and FLP-mediated site-specific recombination in bacteria
Construction of targeted single copy lac fusions using λ Red and FLP-mediated site-specific recombination in bacteria
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DOI：
10.1016/s0378-1119(02)00551-6
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发表时间：
2002-05-15
期刊：
影响因子：
3.5
通讯作者：
Slauch, JM
中科院分区：
文献类型：
--
作者：
Ellermeier, CD;Janakiraman, A;Slauch, JM
本文描述了一种利用FLP介导的位点特异性重组构建lac操纵子靶向转录和翻译融合体的简单方法。构建了包含无启动子lacZY基因和两个方向的FLP识别靶点（FRT）位点的条件质粒，用于产生转录融合。同样，构建了用于创建翻译融合的质粒，其中去除了lacZ的内源性翻译起始。这些质粒可以转化为含有单个FRT位点的菌株，该位点先前使用X Red重组方法整合在感兴趣的启动子下游。由含有条件复制起始的辅助质粒产生的FLP蛋白促进FRT位点之间的位点特异性重组，从而导致lac与目标基因的整合融合。利用这种方法构建了鼠伤寒沙门菌基因sodCll和sitA的转录融合体，并显示出对各自调控基因rpoS和fur的突变有适当的反应。该方法还构建了平移融合体。在这种情况下，p -半乳糖苷酶的表达依赖于靶蛋白的翻译。鉴于FLP重组酶的功能不需要宿主因子，且该方法不需要进行分子克隆，因此该方法应适用于多种生物的基因表达分析。(C) 2002 Elsevier Science B.V.版权所有
A simple method for the construction of targeted transcriptional and translational fusions to the lac operon using FLP mediated site-specific recombination is described. Conditional plasmids containing promoterless lacZY genes and the FLP recognition target (FRT) site in both orientations were constructed for generating transcriptional fusions. Similarly, a plasmid used to create translational fusions was constructed in which the endogenous translational start of lacZ has been removed. These plasmids can be transformed into strains containing a single FRT site, which was previously integrated downstream of the promoter of interest using the X Red recombination method. The FLP protein produced from a helper plasmid that contains a conditional origin of replication promotes site-specific recombination between the FRT sites, resulting in an integrated lac fusion to the gene of interest. Transcriptional fusions to the Salmonella typhimurium genes sodCll and sitA were constructed using this method and shown to respond appropriately to mutations in the respective regulatory genes, rpoS and fur. Translational fusions were also constructed using this method. In this case, expression of P-galactosidase was dependent on translation of the target protein. Given that the FLP recombinase does not require host factors for function and that this method requires no molecular cloning, this method should be applicable for the analysis of gene expression in a variety of organisms. (C) 2002 Elsevier Science B.V. All rights reserved.