在大肠杆菌中利用SCLM系统进行高效率λ-Red基因敲除/整合的新策略

Novel efficient strategy for λ-Red-mediated gene knock-out/in in Escherichia coli using SCLM system

【目的】传统采用的λ-Red体系在大肠杆菌染色体上进行基因敲除/整合操作时存在操作繁琐、假阳性率高、多基因连续敲除/整合不稳定等问题。本研究基于上述问题建立一种便于基因构建、高筛选效率(100%)、具有统一技术步骤的λ-Red敲除/整合系统,为提高基因功能研究和代谢工程改造工作效率奠定基础。【方法】采用新的p SC101衍生复制起始位点消除假阳性;利用高拷贝数质粒和多克隆位点实现快速遗传构建操作;采用Cre/Lox P抗性消除位点便于多基因连续整合。选择一系列初级代谢重要基因靶点进行敲除/整合。【结果】构建了一套新型λ-Red质粒系统(SC101-Cre-Lox P-MCS,SCLM系统)。打靶片段经电转化受体细胞后在双抗性平板上筛选阳性克隆,基因敲除/整合的效率均可以达到100%。【结论】新建立的基因敲除与整合方法提高了基因重组效率,大幅度减少了相关操作的步骤,缩短了研究周期。该方法的建立为基因功能研究和构建新遗传特性的工程菌株提供了有力的工具。

[Objective] Generally, traditional λ-Red recombination system possessed low efficiency, complicated processes, inconsistent protocols, high false-positive rate and instability for multi-gene-knock-out/knock-in during manipulation on chromosome gene of Escherichia coli. In order to solve these problems, this study established a high efficiency and standard strategy of gene knock-out/in. [Methods] Based on λ-Red recombination system, new template plasmids were developed. A p SC101 derivative replication origin was used to diminish the false-positive problem. Convenient genetic manipulation was achieved by using high-copy-number plasmid and multiple cloning sites. New genetic marker was used to facilitate continuous multi-gene knock-out/in. A series of key targets within primary metabolite networks of E. coli were then knocked out/in using our methods. [Results] New λ-Red plasmids system, named SC101-Cre-Lox P-MCS system, was developed. The positive colonies were selected on two-resistance plate and 100% positive rate was achieved. [Conclusion] The efficiency of gene recombination was improved by the new method of gene knock-out/knock-in. This new system provides a rapid genetic manipulation. Our new strategy provides important insights into gene function research and genetic engineering bacteria with new genetic characteristics.