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Strict regulation of gene expression from a high-copy plasmid utilizing a dual vector system

机译：利用双重载体系统严格控制高拷贝质粒的基因表达

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摘要

High-copy plasmids are useful for producing large quantities of plasmid DNA, but are generally inadequate for tightly regulating gene expression. Attempts to suppress expression of genes on high-copy plasmids often results in residual or "leaky" production of protein. For stringent regulation of gene expression, it is often necessary to excise the gene of interest and subclone it into a low-copy plasmid. Here, we report a dual plasmid technique that enables tight regulation of gene expression driven by the lac promoter in a high-copy vector. A series of plasmids with varying copies of the lacl(q) gene have been constructed to permit titration of the Lacl protein. When a high-copy plasmid is transformed along with the appropriate lacl(q)-containing plasmid, tight gene regulation is achieved, thus eliminating the need to subclone genes into low-copy plasmids. In addition, we show that this dual plasmid technique enables high-copy gene expression of a protein lethal to Escherichia coli, the ccdB protein. In principle, this technique can be applied to any high-copy plasmid containing the popular pUC replication of origin and provides an easier means of obtaining rigid control over gene expression. (c) 2008 Elsevier Inc. All rights reserved.
机译：高拷贝质粒可用于产生大量质粒DNA，但通常不足以严格调节基因表达。试图抑制高拷贝质粒上基因的表达通常会导致蛋白质残留或“渗出”。为了严格调节基因表达，通常需要切除目的基因并将其亚克隆到低拷贝质粒中。在这里，我们报道了一种双重质粒技术，该技术能够在高拷贝载体中严格控制由lac启动子驱动的基因表达。已经构建了一系列具有不同lacl（q）基因拷贝的质粒，以允许滴定Lacl蛋白。当将高拷贝质粒与适当的含lacl（q）的质粒一起转化时，可实现严格的基因调节，因此无需将基因亚克隆到低拷贝质粒中。此外，我们证明了这种双重质粒技术能够使对大肠杆菌致死的蛋白质ccdB蛋白实现高拷贝基因表达。原则上，该技术可应用于任何包含流行的pUC复制起点的高拷贝质粒，并提供了一种获得基因表达刚性控制的简便方法。 （c）2008 Elsevier Inc.保留所有权利。

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