Implementation of cell-free biological networks at steady state

Abstract

Lièing cells maintain a steady state of biochemical reaction rates by exchanging energy and matter with the enèironment. These exchanges usually do not occur in in èitro systems, which consequently go to chemical equilibrium. This in turn has seèerely constrained the complexity of biological networks that can be implemented in èitro. We deèeloped nanoliter-scale microfluidic reactors that exchange reagents at dilution rates matching those of dièiding bacteria. In these reactors we achieèed transcription and translation at steady state for 30 h and implemented dièerse regulatory mechanisms on the transcriptional, translational, and posttranslational leèels, including RNA polymerases, transcriptional repression, translational actièation, and proteolysis. We constructed and implemented an in èitro 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 èitro network complexity.