Rapid cell-free forward engineering of novel genetic ring oscillators

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Abstract

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 synchronized 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.

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Henrike Niederholtmeyer

Institute of Bioengineering, School of Engineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

Competing interests
The authors declare that no competing interests exist.
Zachary Sun

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States

Competing interests
The authors declare that no competing interests exist.
Yutaka Hori

Division of Engineering and Applied Science, California Institute of Technology, Pasadena, United States

Competing interests
The authors declare that no competing interests exist.
Enoch Yeung

Division of Engineering and Applied Science, California Institute of Technology, Pasadena, United States

Competing interests
The authors declare that no competing interests exist.
Amanda Verpoorte

Institute of Bioengineering, School of Engineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

Competing interests
The authors declare that no competing interests exist.
Richard M Murray

Division of Biology and Bioengineering, California Institute of Technology, Pasadena, United States

Competing interests
The authors declare that no competing interests exist.
Sebastian J Maerkl

Institute of Bioengineering, School of Engineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

For correspondence
sebastian.maerkl@epfl.ch

Competing interests
The authors declare that no competing interests exist.

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