Rapid cell-free forward engineering of novel genetic ring oscillators

TLDR

Synthetic gene networks are difficult to engineer because traditional in‑cell characterization leads to long design‑build‑test cycles and sparse data, and it is unclear whether cell‑free systems faithfully reproduce cellular behavior. The authors characterized a three‑node repressilator in a cell‑free system and then designed and rapidly tested novel three‑, four‑, and five‑gene ring oscillators using component characterization and full‑network analysis. In cells, the engineered 3‑node oscillators produced population‑wide rhythms, 95 % of 5‑node cells oscillated for up to 72 h, and oscillation periods matched the cell‑free predictions, demonstrating that cell‑free forward engineering can reliably predict cellular behavior.

Abstract

While complex dynamic biological networks control gene expression in all living organisms, the forward engineering of comparable synthetic networks remains challenging. The current paradigm of characterizing synthetic networks in cells results in lengthy design-build-test cycles, minimal data collection, and poor quantitative characterization. Cell-free systems are appealing alternative environments, but it remains questionable whether biological networks behave similarly in cell-free systems and in cells. We characterized in a cell-free system the ‘repressilator’, a three-node synthetic oscillator. We then engineered novel three, four, and five-gene ring architectures, from characterization of circuit components to rapid analysis of complete networks. When implemented in cells, our novel 3-node networks produced population-wide oscillations and 95% of 5-node oscillator cells oscillated for up to 72 hr. Oscillation periods in cells matched the cell-free system results for all networks tested. An alternate forward engineering paradigm using cell-free systems can thus accurately capture cellular behavior.