1. Department of Bioengineering, University of California, San Diego, La Jolla, California 92093, USA
2. Institute for Nonlinear Science, University of California, San Diego, La Jolla, California 92093, USA
3. These authors contributed equally to this work.

Correspondence to: Jeff Hasty^1,2 Correspondence and requests for materials should be addressed to J.H. (Email: hasty@bioeng.ucsd.edu).

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 modelling^{1, 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 oscillators^{8, 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 loops^{1, 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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