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Characterizing and prototyping genetic networks with cell-free transcription–translation reactions

Takahashi, Melissa K. and Hayes, Clarmyra A. and Chappell, James and Sun, Zachary Z. and Murray, Richard M. and Noireaux, Vincent and Lucks, Julius B. (2015) Characterizing and prototyping genetic networks with cell-free transcription–translation reactions. Methods, 86 . pp. 60-72. ISSN 1046-2023. https://resolver.caltech.edu/CaltechAUTHORS:20150608-135213453

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Abstract

A central goal of synthetic biology is to engineer cellular behavior by engineering synthetic gene networks for a variety of biotechnology and medical applications. The process of engineering gene networks often involves an iterative ‘design–build–test’ cycle, whereby the parts and connections that make up the network are built, characterized and varied until the desired network function is reached. Many advances have been made in the design and build portions of this cycle. However, the slow process of in vivo characterization of network function often limits the timescale of the testing step. Cell-free transcription–translation (TX–TL) systems offer a simple and fast alternative to performing these characterizations in cells. Here we provide an overview of a cell-free TX–TL system that utilizes the native Escherichia coli TX–TL machinery, thereby allowing a large repertoire of parts and networks to be characterized. As a way to demonstrate the utility of cell-free TX–TL, we illustrate the characterization of two genetic networks: an RNA transcriptional cascade and a protein regulated incoherent feed-forward loop. We also provide guidelines for designing TX–TL experiments to characterize new genetic networks. We end with a discussion of current and emerging applications of cell free systems.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1016/j.ymeth.2015.05.020DOIArticle
https://doi.org/10.1101/019620DOIDiscussion Paper
ORCID:
AuthorORCID
Takahashi, Melissa K.0000-0003-4937-2924
Sun, Zachary Z.0000-0002-9425-2924
Murray, Richard M.0000-0002-5785-7481
Noireaux, Vincent0000-0002-5213-273X
Additional Information:© 2015 Elsevier. Received 12 March 2015, Revised 20 May 2015, Accepted 21 May 2015, Available online 26 May 2015. V.N. thanks Ryan Marshall for providing data in Fig. 1. We thank the students of the 2013 Cold Spring Harbor Laboratory Synthetic Biology course (Vipul Singhal, Kevin J. Spring, Shaima Al-Khabouri, and Christopher P. Fall) for carrying out some of the experiments in Fig. 2. We also thank David Savage for design and performance of experiments regarding the autofluorescence of the TX–TL extract. This work was supported in part by: the National Science Foundation Graduate Research Fellowship Program (Grant No. DGE-1144153 to M.K.T.). The Defense Advanced Research Projects Agency Young Faculty Award (DARPA YFA) (N66001-12-1-4254 to J.B.L.). The Office of Naval Research Young Investigators Program Award (ONR YIP) (N00014-13-1-0531 to J.B.L.). The Office of Naval Research (N00014-13-1-0074 to V.N.). The Defense Advanced Research Projects Agency (DARPA/MTO) Living Foundries program (contract number HR0011-12-C-0065 to C.A.H., Z.Z.S., R.M.M.). J.B.L. is an Alfred P. Sloan Research Fellow. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing official policies, either expressly or implied, of the Defense Advanced Research Projects Agency or the U.S. Government.
Funders:
Funding AgencyGrant Number
NSF Graduate Research FellowshipDGE-1144153
Office of Naval Research (ONR)N66001-12-1-4254
Office of Naval Research (ONR)N00014-13-1-0531
Office of Naval Research (ONR)N00014-13-1-0074
Defense Advanced Research Projects Agency (DARPA)HR0011-12-C-0065
Alfred P. Sloan FoundationUNSPECIFIED
Subject Keywords:Cell-free systems; Transcription–translation (TX–TL); Rapid prototyping; Genetic networks; Synthetic biology
Record Number:CaltechAUTHORS:20150608-135213453
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20150608-135213453
Official Citation:Melissa K. Takahashi, Clarmyra A. Hayes, James Chappell, Zachary Z. Sun, Richard M. Murray, Vincent Noireaux, Julius B. Lucks, Characterizing and prototyping genetic networks with cell-free transcription–translation reactions, Methods, Volume 86, 15 September 2015, Pages 60-72, ISSN 1046-2023, http://dx.doi.org/10.1016/j.ymeth.2015.05.020. (http://www.sciencedirect.com/science/article/pii/S1046202315002261)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:58088
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:09 Jun 2015 14:01
Last Modified:11 Nov 2020 23:17

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