A Method for Cost-effective and Rapid Characterization of Engineered T7-based Transcription Factors by Cell-free Protein Synthesis Reveals Insights into the Regulation of T7 RNA Polymerase-driven Expression

The T7 bacteriophage RNA polymerase (T7 RNAP) serves as a model for understanding RNA synthesis, as a tool for protein expression, and as an actuator for synthetic gene circuit design in bacterial cells and cell-free extract. T7 RNAP is an attractive tool for orthogonal protein expression in bacteria owing to its compact single subunit structure and orthogonal promoter specificity. Understanding the mechanisms underlying T7 RNAP regulation is important to the design of engineered T7-based transcription factors, which can be used in gene circuit design. To explore regulatory mechanisms for T7 RNAP-driven expression, we developed a rapid and cost-effective method to characterize engineered T7-based transcription factors using cell-free protein synthesis and an acoustic liquid handler. Using this method, we investigated the effects of the tetracycline operator's proximity to the T7 promoter on the regulation of T7 RNAP-driven expression. Our results reveal a mechanism for regulation that functions by interfering with the transition of T7 RNAP from initiation to elongation and validates the use of the method described here to engineer future T7-based transcription factors.