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一步法快速灭活大肠杆菌中的单个或者多个基因

基因敲除实验经常用于基础和应用生物学研究。文中作者开发了一种基于整合辅助质粒的基因敲除系统，用于更高效、更快速地进行大肠杆菌工程学研究。整合辅助质粒 pCW611 含有两种重组酶，由两个独立的诱导系统反向表达。一个是由阿拉伯糖诱导系统控制的 Red 重组酶，利用线性基因敲除 DNA 片段诱导重组事件；另一个是由异丙基β-D-1-硫代吡喃半乳糖苷诱导系统控制的 Cre 重组酶，以获得无标记突变株。使用这种系统可以减少所需的时间和精力，因为不需要反复的转化和固化步骤。使用 pCW611，可以在三天内删除一个目的基因。为了验证该系统的实用性，作者进行了删除实验，分别敲除了四个目的基因（adhE、sfcA、frdABCD 和 ackA），并同时敲除了两个目的基因（adhE-aspA 和 sfcA-aspA）。此外，还成功地依次删除了四个目标基因（fumB、iclR、fumA 和 fumC），从而获得了富马酸生产菌株。这一成功开发和验证的快速高效基因操作系统将有助于大肠杆菌的代谢工程。

原文摘要

Rapid one-step inactivation of single or multiple genes in Escherichia coli

The functional analysis of individual proteins or of multiprotein complexes since the completion of several genome sequencing projects is in focus of current scientific work. Many heterologous proteins contain disulfide- bonds, required for their correct folding and activity, and therefore, need to be transported to the periplasm. The production of soluble and functional protein in the periplasm often needs target-specific regulatory genetic elements, leader peptides, and folding regimes. Usually, the optimization of periplasmic expression is a step-wise and time-consuming procedure. To overcome this problem we developed a dual expression system, containing a degPpromoter- based reporter system and a highly versatile plasmid set. This combines the differential protein expression with the selection of a target-specific expression plasmid. For the validation of this expression tool, two different molecular formats of a recombinant antibody directed to the human epidermal growth factor receptor and human 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) were used. By application of this expression system we demonstrated that the amount of functional protein is inversely proportional to the on-line luciferase signal. We showed that this technology offers a simple tool to evaluate and improve the yield of functionally expressed proteins in the periplasm, which depends on the used regulatory elements and folding strategies. 

Biotechnol J. 2013 Jul;8(7):776-84. doi: 10.1002/biot.201300153. Epub 2013 Jun 21.

PMID: 23653342