大肠杆菌染色体上严谨型启动子的构建

Construction of promoters with tight regulation on chromosome of Escherichia coli

摘要:

【背景】启动子的渗漏表达是代谢工程和合成生物学较为关注的问题，探索严谨型启动子使之能像开关一样控制基因的表达有助于解决这一问题。【目的】为避免在质粒上研究启动子带来的弊端，本研究将在染色体上对严谨型启动子进行构建和评价。【方法】基于4种调控元件四环素tetO、乳糖lacO、阿拉伯糖araC和鼠李糖rhaR的序列，以及2种来源的启动子PL和Plac序列，设计和组合构建了6个启动子PtetO2、PtetO3、PlacO2、PlacO3、PlacO+ara和PlacO+rha。应用CRISPR/Cas9系统将这6个启动子序列整合到大肠杆菌ATCC 8739染色体上，利用绿色荧光蛋白(Green fluorescent protein，GFP)的表达，分析这6个启动子的相对表达强度和严谨型控制情况。【结果】GFP表达分析显示，启动子PlacO+rha为最佳严谨型启动子，在无诱导剂时表达为0.02，有诱导剂时最大表达强度为lacZ基因启动子的12倍，相对控制范围为600倍。【结论】研究结果将为代谢工程和合成生物学中的精确调控基因表达奠定良好的应用基础。

Abstract:

[Background] The leakage expression of promoters is a major concern in metabolic engineering and synthetic biology. To explore promoters with tight regulation like a switch would resolve this problem. [Objective] In order to avoid the shortcomings of using plasmid as research auxiliary, the aim of this study is to construct and assess promoter with tight regulation on chromosome. [Methods] Four elements including tetO, lacO, araC and rhaR regulated by TetR, LacI, AraC and RhaR respectively, were selected for designing six promoters including PtetO2, PtetO3, PlacO2, PlacO3, PlacO+ara and PlacO+rha. CRISPR/Cas9 system was applied to integrate these promoters into chromosome of Escherichia coli ATCC 8739. Promoter strength and relative expression with or without inducing agents were determined by the fluorescent quantities of GFP protein. [Results] Hybrid promoter PlacO+rha was the best one with tight regulation. The expression strength of PlacO+rha was 0.02 in absence of the inducer, and the maximum expression intensity was 12-fold higher than that of the induced lacZ promoter, of which the range of expression level was 600-fold. [Conclusion] This study would provide a good foundation for precisely regulating gene expression in metabolic engineering and synthetic biology.