title: Rapid cell-free forward engineering of novel genetic ring oscillators.
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 oscillations and 95% of 5-node oscillator cells oscillated for up to 72 hr. 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.
authors.affiliation: Institute of Bioengineering, School of Engineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
authors.forename: Henrike
authors.initials: H
authors.lastname: Niederholtmeyer
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States.
authors.forename: Zachary Z
authors.initials: ZZ
authors.lastname: Sun
authors.affiliation: Division of Engineering and Applied Science, California Institute of Technology, Pasadena, United States.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Hori
authors.affiliation: Division of Engineering and Applied Science, California Institute of Technology, Pasadena, United States.
authors.forename: Enoch
authors.initials: E
authors.lastname: Yeung
authors.affiliation: Institute of Bioengineering, School of Engineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
authors.forename: Amanda
authors.initials: A
authors.lastname: Verpoorte
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States.|Division of Engineering and Applied Science, California Institute of Technology, Pasadena, United States.
authors.forename: Richard M
authors.initials: RM
authors.lastname: Murray
authors.affiliation: Institute of Bioengineering, School of Engineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
authors.forename: Sebastian J
authors.initials: SJ
authors.lastname: Maerkl
mesh_terms: D002474:Cell-Free System; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D053263:Gene Regulatory Networks; D017930:Genes, Reporter; D008164:Luminescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D058615:Synthetic Biology / Q000379:methods*
chemical_list: D008164:Luminescent Proteins; D011994:Recombinant Proteins
keywords: E. coli; biochemistry; cell-free prototyping; computational biology; genetic networks; oscillators; synthetic biology; systems biology