A T7 RNAP regulatory toolbox for cell-free network engineering and biosensing applications
2026-05-28
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 engineer a T7 RNAP regulatory toolbox consisting of programmable synthetic repressors, activators, and biosensors in a cell-free system. This toolbox enables scalable design of T7 RNAP-based gene regulatory networks and enables rapid, sensitive, and multiplexed detection of diverse biomolecules, including small-molecule drugs, antibodies, and proteins, in a simple one-pot reaction. By integrating a protein design pipeline, we generate biosensors using fully synthetic binders, demonstrating the potential for rapid development of protein-based sensors. We construct 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 demonstrates 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.