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A fast, robust and tunable synthetic gene oscillator

Nature volume 456, pages 516–519 (2008)Cite this article

Abstract

One defining goal of synthetic biology is the development of engineering-based approaches that enable the construction of gene-regulatory networks according to ‘design specifications’ generated from computational modelling1,2,3,4,5,6. This approach provides a systematic framework for exploring how a given regulatory network generates a particular phenotypic behaviour. Several fundamental gene circuits have been developed using this approach, including toggle switches7 and oscillators8,9,10, and these have been applied in new contexts such as triggered biofilm development11 and cellular population control12. Here we describe an engineered genetic oscillator in Escherichia coli that is fast, robust and persistent, with tunable oscillatory periods as fast as 13 min. The oscillator was designed using a previously modelled network architecture comprising linked positive and negative feedback loops1,13. Using a microfluidic platform tailored for single-cell microscopy, we precisely control environmental conditions and monitor oscillations in individual cells through multiple cycles. Experiments reveal remarkable robustness and persistence of oscillations in the designed circuit; almost every cell exhibited large-amplitude fluorescence oscillations throughout observation runs. The oscillatory period can be tuned by altering inducer levels, temperature and the media source. Computational modelling demonstrates that the key design principle for constructing a robust oscillator is a time delay in the negative feedback loop, which can mechanistically arise from the cascade of cellular processes involved in forming a functional transcription factor. The positive feedback loop increases the robustness of the oscillations and allows for greater tunability. Examination of our refined model suggested the existence of a simplified oscillator design without positive feedback, and we construct an oscillator strain confirming this computational prediction.

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Figure 1: Oscillations in the dual-feedback circuit.
Figure 2: Robust oscillations.
Figure 3: An oscillator with no positive feedback loop.
Figure 4: Modelling the genetic oscillator.

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Acknowledgements

We thank H. Bujard, C. Yang, and Z. Zhang for gifts of reagents, and D. Volfson and M. Simpson for discussions. This work was supported by grants from the National Institutes of Health (GM69811-01) and the US Department of Defense.

Author Contributions J.S. and J.H. designed the oscillator circuits, and J.S. constructed the circuits. S.C. performed the microscopy experiments, and J.S. and S.C. performed the flow cytometry experiments. S.C., L.S.T. and J.H. performed the single-cell data analysis. M.R.B., W.H.M. and L.S.T. performed the computational modelling. All authors wrote the manuscript.

Author information

Author notes

  1. Jesse Stricker, Scott Cookson and Matthew R. Bennett: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Bioengineering, University of California, San Diego, La Jolla, California 92093, USA,

    Jesse Stricker, Scott Cookson, Matthew R. Bennett, William H. Mather & Jeff Hasty

  2. Institute for Nonlinear Science, University of California, San Diego, La Jolla, California 92093, USA ,

    Matthew R. Bennett, Lev S. Tsimring & Jeff Hasty

Authors
  1. Jesse Stricker
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  2. Scott Cookson
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  3. Matthew R. Bennett
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  6. Jeff Hasty
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Corresponding author

Correspondence to Jeff Hasty.

Supplementary information

Supplementary Information

This file contains Supplementary Materials and Methods, Supplementary Figures 1-22 and Supplementary References (PDF 9422 kb)

Supplementary Movie 1

Supplementary Movie file 1 shows a timelapse microscopy of JS011 cells continuously induced with 0.7% arabinose and 2 mM IPTG at 37 C. The brightfield image is shown in grey, and fluorescence is shown in green. Total time of movie is 228 min with a sampling rate of one image every 3 min. (MOV 1787 kb)

Supplementary Movie 2

Supplementary Movie file 2 shows a timelapse microscopy of JS011 cells continuously induced with 0.7% arabinose and 0 mM IPTG at 37 C. The brightfield image is shown in grey, and fluorescence is shown in green. Total time of movie is 219 min with a sampling rate of one image every 3 min. (MOV 3754 kb)

Supplementary Movie 3

Supplementary Movie file 3 shows a timelapse microscopy of JS011 cells continuously induced with 0.7% arabinose and 0.25 mM IPTG at 37 C. The brightfield image is shown in grey, and fluorescence is shown in green. Total time of movie is 222 min with a sampling rate of one image every 3 min. (MOV 2176 kb)

Supplementary Movie 4

Supplementary Movie file 4 shows a timelapse microscopy of JS011 cells continuously induced with 0.7% arabinose and 0.5 mM IPTG at 37 C. The brightfield image is shown in grey, and fluorescence is shown in green. Total time of movie is 210 min with a sampling rate of one image every 3 min. (MOV 1033 kb)

Supplementary Movie 5

Supplementary Movie file 5 shows a timelapse microscopy of JS011 cells continuously induced with 0.7% arabinose and 0.75 mM IPTG at 37 C. The brightfield image is shown in grey, and fluorescence is shown in green. Total time of movie is 268 min with a sampling rate of one image every 2 min. (MOV 3717 kb)

Supplementary Movie 6

Supplementary Movie file 6 shows a timelapse microscopy of JS011 cells continuously induced with 0.7% arabinose and 1 mM IPTG at 37 C. The brightfield image is shown in grey, and fluorescence is shown in green. Total time of movie is 176 min with a sampling rate of one image every 2 min. (MOV 2029 kb)

Supplementary Movie 7

Supplementary Movie file 7 shows a timelapse microscopy of JS011 cells continuously induced with 0.7% arabinose and 5 mM IPTG at 37 C. The brightfield image is shown in grey, and fluorescence is shown in green. Total time of movie is 246 min with a sampling rate of one image every 3 min. (MOV 1647 kb)

Supplementary Movie 8

Supplementary Movie file 8 shows a timelapse microscopy of JS011 cells continuously induced with 0.7% arabinose and 10 mM IPTG at 37 C. The brightfield image is shown in grey, and fluorescence is shown in green. Total time of movie is 204 min with a sampling rate of one image every 3 min. (MOV 1538 kb)

Supplementary Movie 9

Supplementary Movie file 9 shows a timelapse microscopy of JS011 cells continuously induced with 0.7% arabinose and 2 mM IPTG at 25 C. The phase contrast image is shown in grey, and fluorescence is shown in green. Total time of movie is 702 min with a sampling rate of one image every 3 min. (MOV 3568 kb)

Supplementary Movie 10

Supplementary Movie file 10 shows a timelapse microscopy of JS011 cells upon initiation of induction with0.7% arabinose and 2 mM IPTG at 37 C. The brightfield image is shown in grey, and fluorescenceis shown in green. Total time of movie is 75 min with a sampling rate of one image every 3 min. Note the initial synchrony of the fluorescence response. (MOV 1078 kb)

Supplementary Movie 11

Supplementary Movie file 11shows a timelapse microscopy of JS013 cells continuously induced with 0.6mM IPTG at 37 C. The phase-contrast image is shown in grey, and fluorescence is shown in green. Total time of movie is 210 min with a sampling rate of one image every 3 min. (MOV 2084 kb)

Supplementary Movie 12

Supplementary Movie file 12 shows a timelapse microscopy of MG1655Z1/pZE12-yemGFP-ssrA cells continuously induced with 2 mM IPTG at 37 C. These cells express GFP from the pLlacO-1 promoter and express LacI constitutively. There is no feedback control of GFP expression in this strain. The phase-contrast image is shown in grey, and fluorescence is shown in green. Total time of movie is 252 min with a sampling rate of one image every 3 min. (MOV 2966 kb)

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Stricker, J., Cookson, S., Bennett, M. et al. A fast, robust and tunable synthetic gene oscillator. Nature 456, 516–519 (2008). https://doi.org/10.1038/nature07389

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