Implementation of cell-free biological networks at steady state

Living cells maintain a steady state of biochemical reaction rates by exchanging energy and matter with the environment. These exchanges usually do not occur in in vitro systems, which consequently go to chemical equilibrium. This in turn has severely constrained the complexity of biological networks that can be implemented in vitro. We developed nanoliter-scale microfluidic reactors that exchange reagents at dilution rates matching those of dividing bacteria. In these reactors we achieved transcription and translation at steady state for 30 h and implemented diverse regulatory mechanisms on the transcriptional, translational, and posttranslational levels, including RNA polymerases, transcriptional repression, translational activation, and proteolysis. We constructed and implemented an in vitro genetic oscillator and mapped its phase diagram showing that steady-state conditions were necessary to produce oscillations. This reactor-based approach will allow testing of whether fundamental limits exist to in vitro network complexity.