Dual-feedback oscillator circuit in E. coli

The dual-feedback oscillator circuit was constructed by placing araC, lacI and yemGFP under the control of the hybrid P_lac/ara-1 promoter^{14 (link)} in three separate transcriptional cassettes. An ssrA degradation tag^{28 (link)} was added to each gene to decrease protein lifetime and increase temporal resolution. These transcriptional cassettes were placed on two modular plasmids^{14 (link)} and cotransformed into an ΔaraC ΔlacI E. coli strain. The negative feedback oscillator circuit was constructed by placing ssrA-tagged lacI and yemGFP under the control of the P_LlacO-1 promoter^{14 (link)} in two separate transcriptional cassettes, which were incorporated onto two modular plasmids and cotransformed into a ΔlacI strain. Cells were either grown in LB medium or minimal A medium with 2 g/L glucose. Oscillations were induced using arabinose (0.1–3%) and IPTG (0–30 mM). Single-cell microscopic data were collected by loading induced cells into PDMS-based microfluidic platforms that constrained the cells to a monolayer while supplying them with nutrients²⁹ , then supplying a constant source of medium and inducers and imaging GFP fluorescence every 2–3 min for at least 4–6 h. These data were further analyzed using ImageJ and custom-written MATLAB scripts to extract single-cell fluorescence trajectories. Flow cytometry was performed either by taking samples from a continuously grown and serially diluted culture or by growing multiple cultures in parallel for varying durations. In either case, samples were read directly from their growth medium and low-scatter noise was removed by thresholding. Flow cytometry oscillatory periods were defined as the time elapsed between the first and second fluorescence peaks. Details of the models discussed are presented in Supplementary Information. Stochastic simulations were performed using Gillespie’s algorithm²⁷ , and deterministic simulations were performed using custom MATLAB scripts.