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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. Access through your institution Buy or subscribe This is a preview of

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OSCILLATOR IN NON-MICROFLUIDIC ENVIRONMENTS Article Open access 13 May 2023 FROM RESONANCE TO CHAOS BY MODULATING SPATIOTEMPORAL PATTERNS THROUGH A SYNTHETIC OPTOGENETIC OSCILLATOR Article

Open access 23 August 2024 INDEPENDENT CONTROL OF AMPLITUDE AND PERIOD IN A SYNTHETIC OSCILLATOR CIRCUIT WITH MODIFIED REPRESSILATOR Article Open access 11 January 2022 REFERENCES * Hasty,

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

AND AFFILIATIONS * 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 * Institute for Nonlinear Science, University of California, San Diego, La Jolla, California 92093, USA , Matthew R. Bennett, Lev S. Tsimring & Jeff Hasty Authors *

Jesse Stricker View author publications You can also search for this author inPubMed Google Scholar * Scott Cookson View author publications You can also search for this author inPubMed 

Google Scholar * Matthew R. Bennett View author publications You can also search for this author inPubMed Google Scholar * William H. Mather View author publications You can also search for

this author inPubMed Google Scholar * Lev S. Tsimring View author publications You can also search for this author inPubMed Google Scholar * Jeff Hasty View author publications You can also

search for this author inPubMed Google Scholar 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) POWERPOINT SLIDES POWERPOINT SLIDE FOR FIG. 1 POWERPOINT SLIDE FOR FIG. 2 POWERPOINT SLIDE FOR FIG. 3 POWERPOINT

SLIDE FOR FIG. 4 RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Received: 09 July 2008 * Accepted: 05 September 2008 * Published: 29 October 2008 *

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