T7 RNA polymerase (T7RNAP) and T7 promoter are powerful genetic components, thus a plasmid-driven T7 (PDT7) genetic circuit could be broadly applied for synthetic biology. However, the limited knowledge of the toxicity and instability of such a system still restricts its application. Herein, we constructed 16 constitutive genetic circuts of PDT7 and investigated the orthogonal effects in toxicity and instability. The T7 toxicity was elucidated from the construction processes and cell growth characterization, showing the importance of optimal orthogonality for PDT7. Besides, a protein analysis was performed to validate how the T7 system affected cell metabolism and led to the instability. The application of constitutive PDT7 in functional protein expressions, including carbonic anhydrase, lysine decarboxylase, and 5-ALA synthetase was demonstrated. Furthermore, PDT7 working as a genetic amplifier had been designed for E. coli cell-based biosensors, which illustrated the opportunities in the future of PDT7 used in synthetic biology.

中文翻译：

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大肠杆菌中质粒驱动的T7 RNA聚合酶的新见解，并用作生物传感器的遗传扩增子。

T7 RNA聚合酶（T7RNAP）和T7启动子是强大的遗传成分，因此质粒驱动的T7（PDT7）遗传电路可广泛应用于合成生物学。但是，对这种系统的毒性和不稳定性的了解有限，仍然限制了其应用。在本文中，我们构建了PDT7的16个本构遗传环，并研究了其毒性和不稳定性的正交效应。从构建过程和细胞生长特征中阐明了T7毒性，显示了PDT7最佳正交性的重要性。此外，进行了蛋白质分析以验证T7系统如何影响细胞代谢并导致不稳定性。证明了结构性PDT7在功能蛋白表达中的应用，包括碳酸酐酶，赖氨酸脱羧酶和5-ALA合成酶。