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Rapidly characterizing the fast dynamics of RNA genetic circuitry with cell-free transcription-translation (TX-TL) systems

By Melissa K. Takahashi, James Chappell, Clarmyra A Hayes, Zachary Z. Sun, Vipul Singhad, Kevin J Spring, Shaima Al-Khabouri, Christopher P Fall, Vincent Noireaux, Richard M. Murray, Julius B. Lucks

Posted 17 Mar 2014
bioRxiv DOI: 10.1101/003335 (published DOI: 10.1021/sb400206c)

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 timescales 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 response 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. Now published as: ACS Synthetic Biology doi:10.1021/sb400206c

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