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Rapid cell-free forward engineering of novel genetic ring oscillators

Niederholtmeyer, Henrike and Sun, Zachary Z. and Hori, Yutaka and Yeung, Enoch and Verpoorte, Amanda and Murray, Richard M. and Maerkl, Sebastian J. (2015) Rapid cell-free forward engineering of novel genetic ring oscillators. eLife, 4 . Art. No. e09771. ISSN 2050-084X. PMCID PMC4714972. doi:10.7554/eLife.09771.

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While complex dynamic biological networks control gene expression in all living organisms, the forward engineering of comparable synthetic networks remains challenging. The current paradigm of characterizing synthetic networks in cells results in lengthy design-build-test cycles, minimal data collection, and poor quantitative characterization. Cell-free systems are appealing alternative environments, but it remains questionable whether biological networks behave similarly in cell-free systems and in cells. We characterized in a cell-free system the 'repressilator,' a three-node synthetic oscillator. We then engineered novel three, four, and five-gene ring architectures, from characterization of circuit components to rapid analysis of complete networks. When implemented in cells, our novel 3-node networks produced population-wide oscillations and 95% of 5-node oscillator cells oscillated for up to 72 hours. Oscillation periods in cells matched the cell-free system results for all networks tested. An alternate forward engineering paradigm using cell-free systems can thus accurately capture cellular behavior.

Item Type:Article
Related URLs:
URLURL TypeDescription CentralArticle
Niederholtmeyer, Henrike0000-0002-1375-0287
Sun, Zachary Z.0000-0002-9425-2924
Hori, Yutaka0000-0002-3253-4985
Yeung, Enoch0000-0001-7630-7429
Murray, Richard M.0000-0002-5785-7481
Maerkl, Sebastian J.0000-0003-1917-5268
Additional Information:© 2015 Niederholtmeyer et al. This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited. Received June 29, 2015. Accepted October 1, 2015. Published October 2, 2015. We thank Yin He, Transcriptic, Inc. and Holly Rees for cloning assistance, Jan Kostecki and Stephen Mayo for protein purification and size exclusion chromatography assistance, Rohit Sharma and Marcella Gomez for initial testing and modeling of oscillators in vitro, Kyle Martin for laboratory assistance, Adam Abate, Tanja Kortemme, and Charles Craik for laboratory space and equipment, Matthieu Delincé, Joachim De Jonghe, Marc Spaltenstein, John McKinney and Jin Park for mother machine material and assistance, Tim Chang and Benjamin Alderete for CellASIC assistance, and Michael Elowitz for insights and scientific support. This work was supported in part by EPFL and the Defense Advanced Research Projects Agency (DARPA/MTO) Living Foundries program, contract number HR0011-12-C-0065 (DARPA/CMO). Z.Z.S. is also supported by a UCLA/Caltech Medical Scientist Training Program fellowship, Z.Z.S and E.Y by a National Defense Science and Engineering Graduate fellowship, and Y.H. by a JSPS Fellowship for Research Abroad. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing officially policies, either expressly or implied, of the Defense Advanced Research Projects Agency or the U.S. Government.
Funding AgencyGrant Number
Defense Advanced Research Projects Agency (DARPA)HR0011-12-C-0065
UCLA/Caltech Medical Scientist Training ProgramUNSPECIFIED
National Defense Science and Engineering Graduate (NDSEG) FellowshipUNSPECIFIED
Japan Society for the Promotion of Science (JSPS)UNSPECIFIED
PubMed Central ID:PMC4714972
Record Number:CaltechAUTHORS:20151013-090251954
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:61028
Deposited By: Tony Diaz
Deposited On:15 Oct 2015 04:12
Last Modified:10 Nov 2021 22:43

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