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

Melissa K. Takahashi, Clarmyra A. Hayes, James Chappell, Zachary Z. Sun, Richard M. Murray, Vincent Noireaux, Julius B. Lucks

Research output: Contribution to journalArticlepeer-review

64 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)60-72
Number of pages13
JournalMethods
Volume86
DOIs
StatePublished - Sep 15 2015

Bibliographical note

Funding Information:
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.

Publisher Copyright:
© 2015 Elsevier Inc..

Copyright:
Copyright 2015 Elsevier B.V., All rights reserved.

Keywords

  • Cell-free systems
  • Genetic networks
  • Rapid prototyping
  • Synthetic biology
  • Transcription-translation (TX-TL)

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