A fast, robust and tunable synthetic gene oscillator

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. 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 switches and oscillators, and these have been applied in new contexts such as triggered biofilm development and cellular population control. 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. 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.

Figure 1.

Oscillations in the dual-feedback circuit. a, Network diagram of the dual-feedback oscillator. A hybrid promoter P_lac/ara-1 drives transcription of araC and lacI, forming positive and negative feedback loops. b, Single-cell fluorescence trajectories induced with 0.7% arabinose and 2 mM IPTG. Points represent experimental fluorescence values, and solid curves are smoothed by a Savitsky-Golay filter (for unsmoothed trajectories, see Supplementary Fig. 3). The trajectory in red corresponds to the density map above. c–h, Single-cell density trajectories for various IPTG conditions (c, 0 mM IPTG; d, 0.25 mM; e, 0.5 mM; f, 1 mM; g, 2 mM; h, 5 mM). X-axes are in min.

Figure 2.

Robust oscillations. a–c, Oscillatory periods on transects along 0.7% arabinose (a), 2 mM IPTG (b), or both with varying temperature (c). Mean periods from single-cell microscopy (red diamonds ± s.d.) or flow cytometry (green circles) are shown. Black curves are trend lines, or in c, the theoretical prediction based on reference values at 30°C (see Supplemental Information). Samples are grown in LB or minimal medium (×). d, Oscillatory period and cell division time increase monotonically as the growth temperature decreases. Symbols are as above, and the black line is a linear regression of samples grown in LB.

Figure 3.

An oscillator with no positive feedback loop. a, Network diagram of the negative feedback oscillator. This oscillator is similar to the dual-feedback oscillator except that the P_LlacO-1 promoter driving the components gives expression in the absence of LacI or in the presence of IPTG without requiring an activator. b, Single-cell density trajectories for cells containing this oscillator (see Supplementary Movie 11 and Supplementary Fig. 5).

Figure 4.

Modeling the genetic oscillator. a, Intermediate processes are explicitly modeled in the refined oscillator model. b–c, Simulation results from Gillespie simulations (b) or deterministic modeling (c) at 0.7% arabinose and 2 mM IPTG. AraC dimers (green), LacI tetramers (red), and lacI mRNA (black) are shown. d–e, Comparison of modeling and experiment for oscillation period at 0.7% arabinose (d) or 2 mM IPTG (e). Values from deterministic modeling (blue curve), stochastic simulations (gray symbols, Supplementary Fig. 18), and microscopy (red diamonds) or flow cytometry (green circles) are shown.