Rapid cell-free forward engineering of novel genetic ring oscillators

  1. Henrike Niederholtmeyer
  2. Zachary Z Sun
  3. Yutaka Hori
  4. Enoch Yeung
  5. Amanda Verpoorte
  6. Richard M Murray
  7. Sebastian J Maerkl  Is a corresponding author
  1. École Polytechnique Fédérale de Lausanne, Switzerland
  2. California Institute of Technology, United States

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 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.

Article and author information

Author details

  1. Henrike Niederholtmeyer

    Institute of Bioengineering, School of Engineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
    Competing interests
    No competing interests declared.
  2. Zachary Z Sun

    Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States
    Competing interests
    Zachary Z Sun, ZZS has ownership in a company that commercializes the cell-free technology utilized in this paper..
  3. Yutaka Hori

    Division of Engineering and Applied Science, California Institute of Technology, Pasadena, United States
    Competing interests
    No competing interests declared.
  4. Enoch Yeung

    Division of Engineering and Applied Science, California Institute of Technology, Pasadena, United States
    Competing interests
    No competing interests declared.
  5. Amanda Verpoorte

    Institute of Bioengineering, School of Engineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
    Competing interests
    No competing interests declared.
  6. Richard M Murray

    Division of Biology and Bioengineering, California Institute of Technology, Pasadena, United States
    Competing interests
    Richard M Murray, RMM has ownership in a company that commercializes the cell-free technology utilized in this paper..
  7. Sebastian J Maerkl

    Institute of Bioengineering, School of Engineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
    For correspondence
    sebastian.maerkl@epfl.ch
    Competing interests
    No competing interests declared.

Copyright

© 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.

Metrics

  • 7,441
    views
  • 1,694
    downloads
  • 206
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Henrike Niederholtmeyer
  2. Zachary Z Sun
  3. Yutaka Hori
  4. Enoch Yeung
  5. Amanda Verpoorte
  6. Richard M Murray
  7. Sebastian J Maerkl
(2015)
Rapid cell-free forward engineering of novel genetic ring oscillators
eLife 4:e09771.
https://doi.org/10.7554/eLife.09771

Share this article

https://doi.org/10.7554/eLife.09771

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Lingzhi Gao, Dian Chen, Yu Liu
    Research Article

    Riboswitches represent a class of non-coding RNA that possess the unique ability to specifically bind ligands and, in response, regulate gene expression. A recent report unveiled a type of riboswitch, known as the guanidine-IV riboswitch, which responds to guanidine levels to regulate downstream genetic transcription. However, the precise molecular mechanism through which the riboswitch senses its target ligand and undergoes conformational changes remain elusive. This gap in understanding has impeded the potential applications of this riboswitch. To bridge this knowledge gap, our study investigated the conformational dynamics of the guanidine-IV riboswitch RNA upon ligand binding. We employed single-molecule fluorescence resonance energy transfer (smFRET) to dissect the behaviors of the aptamer, terminator, and full-length riboswitch. Our findings indicated that the aptamer portion exhibited higher sensitivity to guanidine compared to the terminator and full-length constructs. Additionally, we utilized Position-specific Labelling of RNA (PLOR) combined with smFRET to observe, at the single-nucleotide and single-molecule level, the structural transitions experienced by the guanidine-IV riboswitch during transcription. Notably, we discovered that the influence of guanidine on the riboswitch RNA’s conformations was significantly reduced after the transcription of 88 nucleotides. Furthermore, we proposed a folding model for the guanidine-IV riboswitch in the absence and presence of guanidine, thereby providing insights into its ligand-response mechanism.

    1. Biochemistry and Chemical Biology
    Marius Landau, Sherif Elsabbagh ... Joachim E Schultz
    Research Article

    The biosynthesis of cyclic 3′,5′-adenosine monophosphate (cAMP) by mammalian membrane-bound adenylyl cyclases (mACs) is predominantly regulated by G-protein-coupled receptors (GPCRs). Up to now the two hexahelical transmembrane domains of mACs were considered to fix the enzyme to membranes. Here, we show that the transmembrane domains serve in addition as signal receptors and transmitters of lipid signals that control Gsα-stimulated mAC activities. We identify aliphatic fatty acids and anandamide as receptor ligands of mAC isoforms 1–7 and 9. The ligands enhance (mAC isoforms 2, 3, 7, and 9) or attenuate (isoforms 1, 4, 5, and 6) Gsα-stimulated mAC activities in vitro and in vivo. Substitution of the stimulatory membrane receptor of mAC3 by the inhibitory receptor of mAC5 results in a ligand inhibited mAC5–mAC3 chimera. Thus, we discovered a new class of membrane receptors in which two signaling modalities are at a crossing, direct tonic lipid and indirect phasic GPCR–Gsα signaling regulating the biosynthesis of cAMP.