title: Implementation of cell-free biological networks at steady state.
abstract: 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.
authors.affiliation: Institute of Bioengineering, School of Engineering, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
authors.forename: Henrike
authors.initials: H
authors.lastname: Niederholtmeyer
authors.forename: Viktoria
authors.initials: V
authors.lastname: Stepanova
authors.forename: Sebastian J
authors.initials: SJ
authors.lastname: Maerkl
mesh_terms: D002474:Cell-Free System / Q000502:physiology*; D031541:Fluorescence Resonance Energy Transfer; D053263:Gene Regulatory Networks / Q000502:physiology*; D005818:Genetic Engineering / Q000379:methods*; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D008856:Microscopy, Fluorescence; D014176:Protein Biosynthesis / Q000502:physiology*; D058615:Synthetic Biology / Q000379:methods; D014158:Transcription, Genetic / Q000502:physiology
keywords: cell-free protein synthesis; computational biology; minimal artificial cell; synthetic biology
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