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

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

Research output: Contribution to journalArticlepeer-review

82 Scopus citations

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. (Figure Presented).

Original languageEnglish (US)
Pages (from-to)503-515
Number of pages13
JournalACS Synthetic Biology
Volume4
Issue number5
DOIs
StatePublished - May 15 2015

Keywords

  • RNA genetic circuits
  • RNA synthetic biology
  • TX-TL
  • cell-free
  • response time

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