New Insight into Plasmid-Driven T7 RNA Polymerase in

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.