Engineered Genetic Oscillators

One defining goal of synthetic biology is the development of engineering-based approaches that enable the construction of gene-regulatory networks according to design specs generated from computational modeling. This approach provides a systematic framework for exploring how a given regulatory network generates a particular phenotypic behavior. This has resulted in the development of several fundamental gene circuits, such as toggle switches and oscillators, which have been applied in novel contexts such as triggered biofilm development and cellular population control. In this talk, I will describe an engineered genetic oscillator in Escherichia coli that is fast, robust, and persistent, with tunable oscillatory periods as fast as 20 minutes. The oscillator was designed using a previously modeled network architecture comprising linked positive and negative feedback loops. Using a microfluidic platform tailored for single-cell microscopy, we precisely control environmental conditions and monitor oscillations in individual cells through multiple cycles. Experiments show remarkable robustness and persistence of oscillations in the designed circuit; almost every cell exhibited large-amplitude fluorescence oscillations throughout observation runs. The period of oscillation can be tuned by altering inducer levels. Computational modeling reveals 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. In addition to increasing the size of this robust parameter space, the positive feedback loop increases the amplitude and regularizes the oscillations when stochastic effects are taken into consideration. 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. This oscillator is not as regular or tunable, thereby demonstrating the specific role of the positive feedback in the dynamics of the original oscillator.

Jeff Hasty, University of California, San Diego - Update title and link to abstract


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