A T7 RNAP regulatory toolbox for cell-free network engineering and biosensing applications

T7 RNA polymerase is ubiquitously used in the fields of synthetic biology and biotechnology. Yet the ability to precisely and modularly regulate T7 RNAP remains surprisingly limited. Here, we developed a T7 RNAP regulatory toolbox consisting of programmable synthetic repressors, activators, and biosensors in a cell-free system. This toolbox enables the scalable design of T7 RNAP based gene regulatory networks and enables the rapid and sensitive detection of diverse biomolecules, including small-molecule drugs, antibodies, and proteins. By integrating a protein design pipeline, we generated biosensors using fully synthetic binders, demonstrating the potential for rapid development of novel protein-based sensors. We constructed a diagnostic cell-free system combining SARS-CoV-2 Spike protein sensing, gene regulatory based amplification, enzymatic amplification, and glucose based detection demonstrating the potential for point-of-care detection with high sensitivity. This work establishes a flexible and expandable framework for constructing gene circuits responsive to a wide range of biomolecules and demonstrates the potential for engineering point-of-care cell-free diagnostic assays.