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Edited by Jack W. Szostak, Howard Hughes Medical Institute and Massachusetts General Hospital, Boston, MA, and approved August 27, 2013 (received for review June 12, 2013)
Significance
Transcription and translation can be performed in vitro, outside of cells, allowing the assembly of artificial genetic networks. This bottom-up approach to engineering biological networks in a completely defined and minimal environment is instructive to define the rules and limitations of network construction. It is, however, still challenging to implement complex genetic networks in vitro because the reactions are usually performed in a batch format, where reaction products accumulate and synthesis rates decline over time. Here, we addressed this problem by developing a microfluidic device to perform in vitro transcription and translation reactions in continuous mode, where synthesis rates stay constant. This allowed us to build and implement a genetic oscillator that showed sustained oscillations for extended periods of times.
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.
Footnotes
- ↵1To whom correspondence should be addressed. E-mail: sebastian.maerkl{at}epfl.ch.
Author contributions: H.N. and S.J.M. designed research; H.N. and V.S. performed research; H.N. and S.J.M. analyzed data; and H.N. and S.J.M. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1311166110/-/DCSupplemental.
Freely available online through the PNAS open access option.
Cell-free genetic networks
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