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Gene Circuit Performance Characterization and Resource Usage in a Cell-Free “Breadboard”

Siegal-Gaskins, Dan and Tuza, Zoltan A. and Kim, Jongmin and Noireaux, Vincent and Murray, Richard M. (2014) Gene Circuit Performance Characterization and Resource Usage in a Cell-Free “Breadboard”. ACS Synthetic Biology, 3 (6). pp. 416-425. ISSN 2161-5063. http://resolver.caltech.edu/CaltechAUTHORS:20140414-094313495

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Abstract

The many successes of synthetic biology have come in a manner largely different from those in other engineering disciplines; in particular, without well-characterized and simplified prototyping environments to play a role analogous to wind-tunnels in aerodynamics and breadboards in electrical engineering. However, as the complexity of synthetic circuits increases, the benefits—in cost savings and design cycle time—of a more traditional engineering approach can be significant. We have recently developed an in vitro “breadboard” prototyping platform based on E. coli cell extract that allows biocircuits to operate in an environment considerably simpler than, but functionally similar to, in vivo. The simplicity of this system makes it a promising tool for rapid biocircuit design and testing, as well as for probing fundamental aspects of gene circuit operation normally masked by cellular complexity. In this work, we characterize the cell-free breadboard using real-time and simultaneous measurements of transcriptional and translational activities of a small set of reporter genes and a transcriptional activation cascade. We determine the effects of promoter strength, gene concentration, and nucleoside triphosphate concentration on biocircuit properties, and we isolate the specific contributions of essential biomolecular resources—core RNA polymerase and ribosomes—to overall performance. Importantly, we show how limits on resources, particularly those involved in translation, are manifested as reduced expression in the presence of orthogonal genes that serve as additional loads on the system.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1021/sb400203pDOIArticle
http://pubs.acs.org/doi/abs/10.1021/sb400203pPublisherArticle
http://pubs.acs.org/doi/suppl/10.1021/sb400203pPublisherSupporting Information
ORCID:
AuthorORCID
Murray, Richard M.0000-0002-5785-7481
Additional Information:© 2014 American Chemical Society. Received: December 8, 2013. Publication Date (Web): March 26, 2014. The authors thank Eduardo Sontag and members of the Murray Group for useful discussions and S. C. Livingston, P. Rovo, and G. Smith for helpful comments on the manuscript. This research is funded in part by the Gordon and Betty Moore Foundation through Grant GBMF2809 to the Caltech Programmable Molecular Technology Initiative, and by the Defense Advanced Research Projects Agency (DARPA/MTO) Living Foundries program, contract number HR0011-12-C- 0065. Z.A.T. was partially supported by grants TAMOP-4.2.1- B-11/2/KMR-2011-0002, TAMOP-4.2.2./B-10/1-2010-0014, and OTKA NF 104706. 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
Gordon and Betty Moore FoundationGBMF2809
DARPA/MTO Living Foundries programHR0011-12-C-0065
TAMOP-4.2.1-B-11/2/KMR-2011-0002 UNSPECIFIED
TAMOP-4.2.2./B-10/1-2010-0014UNSPECIFIED
OTKANF 104706
Subject Keywords:cell-free systems; biological circuit prototyping; crosstalk; in vitro synthetic biology; RNA aptamer
Record Number:CaltechAUTHORS:20140414-094313495
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20140414-094313495
Official Citation:Gene Circuit Performance Characterization and Resource Usage in a Cell-Free “Breadboard” Dan Siegal-Gaskins, Zoltan A. Tuza, Jongmin Kim, Vincent Noireaux, and Richard M. Murray ACS Synthetic Biology 2014 3 (6), 416-425
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:44912
Collection:CaltechAUTHORS
Deposited By: Jason Perez
Deposited On:14 Apr 2014 20:15
Last Modified:09 Nov 2017 00:43

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