基于T7系统与原核宿主正交的转录元件构建及应用

构建基因线路模块是现代分子生物学技术和合成生物研究中发展最迅速的领域，但是基因线路模块的通用性、高效性和可调节性是面临的重要问题，构建各种基因线路模块的关键是其中的转录元件要具有通用性。本课题拟利用反义RNA调控噬菌体T7表达系统的方法，构建与宿主菌正交的转录元件，用以解决基因线路的通用性、高效性和可调节性问题。研究首先测定大肠杆菌DE3溶源菌中T7 RNAP的非致死表达量；然后利用报告基因(gfp)构建具有通用性、高效性和可调节性的与宿主正交的正交转录元件，研究其在模式工业菌株(大肠杆菌、枯草芽孢杆菌、假单胞菌、乳酸菌)中的调控规律；最后将该正交转录元件应用于重要工业酶的高活性表达和工业菌的代谢网络构建研究。本研究能为基因线路模块的研究，以及野生菌的基因工程改造提供重要的理论数据和有效工具，具有重要的理论意义和应用价值。

Synthetic gene circuits are rapidly becoming core components in synthetic biology and biotechnology. However, it was embarrassed by the consistency, effectiveness and regulable ability of synthetic gene circuits, of which the transcriptional element played a critical role. In the present project, the strategy of regulation of T7 expression system with small RNAs was applied to construct an orthogonal transcriptional element to achieve the consistency, effectiveness and regulable ability. The present studies include the determination of the lethal dosage of T7 RNAP in E. coli（DE3）strains, feasible examination of T7 gene circuits with gfp report gene in different model bacterium (E. coli, B. subtilis, P. putida, L. lactis ) and industrial application of protein soluble expression and metabolic network reconstruction with T7 gene circuits. The project and its results are invaluable for the theory research and industrial application of metabolic engineering and synthetic biology.