Rapidly Characterizing the Fast Dynamics of RNA Genetic Circuitry with Cell-Free Transcription–Translation (TX-TL) Systems

TLDR

RNA regulators are emerging as powerful tools for engineering synthetic genetic networks, yet their potential for rapid signal propagation is constrained by the slow in vivo design‑build‑test cycle. The study adapts an E. coli‑based cell‑free transcription‑translation (TX‑TL) system to rapidly prototype RNA networks. Using this TX‑TL platform, the authors prototype RNA networks—including a single‑input module that temporally stages two genes in vivo.

Abstract

RNA regulators are emerging as powerful tools to engineer synthetic genetic networks or rewire existing ones. A potential strength of RNA networks is that they may be able to propagate signals on time scales that are set by the fast degradation rates of RNAs. However, a current bottleneck to verifying this potential is the slow design-build-test cycle of evaluating these networks in vivo. Here, we adapt an Escherichia coli-based cell-free transcription-translation (TX-TL) system for rapidly prototyping RNA networks. We used this system to measure the response time of an RNA transcription cascade to be approximately five minutes per step of the cascade. We also show that this response time can be adjusted with temperature and regulator threshold tuning. Finally, we use TX-TL to prototype a new RNA network, an RNA single input module, and show that this network temporally stages the expression of two genes in vivo.