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ACS Synth Biol. 2015 May 15;4(5):503-15. doi: 10.1021/sb400206c. Epub 2014 Mar 28.

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

Author information

1
†School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14850, United States.
2
‡CSHL Course in Synthetic Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, United States.
3
§Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, United States.
4
∥Department of Integrative Biology and Pharmacology, The University of Texas Medical School at Houston, Texas 77030, United States.
5
⊥Institute for Research in Immunology and Cancer (IRIC), Université de Montreal, Montreal, Quebec H3T 1J4, Canada.
6
∇Department of BioEngineering, University of Illinois, Chicago, Illinois 60607, United States.
7
#Department of Computer Science, Georgetown University, Washington, DC 20057, United States.
8
○School of Physics and Astronomy, University of Minnesota, Minneapolis, Minnesota 55455, United States.

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.

KEYWORDS:

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

PMID:
24621257
PMCID:
PMC4487224
DOI:
10.1021/sb400206c
[Indexed for MEDLINE]
Free PMC Article

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