CFPU: A Cell-Free Processing Unit for High-Throughput, Automated In Vitro Circuit Characterization in Steady-State...

Forward engineering synthetic circuits are at the core of synthetic biology. Automated solutions will be required to facilitate circuit design and implementation. Circuit design is increasingly being automated with design software, but innovations in experimental automation are lagging behind. Microfluidic technologies made it possible to perform in vitro transcription-translation (tx-tl) reactions with increasing throughput and sophistication, enabling screening and characterization of individual circuit elements and complete circuit designs. Here, we developed an automated microfluidic cell-free processing unit (CFPU) that extends high-throughput screening capabilities to a steady-state reaction environment, which is essential for the implementation and analysis of more complex and dynamic circuits. The CFPU contains 280 chemostats that can be individually programmed with DNA circuits. Each chemostat is periodically supplied with tx-tl reagents, giving rise to sustained, long-term steady-state conditions. Using microfluidic pulse width modulation (PWM), the device is able to generate tx-tl reagent compositions in real time. The device has higher throughput, lower reagent consumption, and overall higher functionality than current chemostat devices. We applied this technology to map transcription factor-based repression under equilibrium conditions and implemented dynamic gene circuits switchable by small molecules. We expect the CFPU to help bridge the gap between circuit design and experimental automation for in vitro development of synthetic gene circuits.