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Synthetic logic circuits using RNA aptamer against T7 RNA polymerase

Kim, Jongmin and Quijano, Juan F. and Yeung, Enoch and Murray, Richard M. (2014) Synthetic logic circuits using RNA aptamer against T7 RNA polymerase. . (Submitted) http://resolver.caltech.edu/CaltechAUTHORS:20140925-085129247

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Abstract

Recent advances in nucleic acids engineering introduced several RNA-based regulatory components for synthetic gene circuits, expanding the toolsets to engineer organisms. In this work, we designed genetic circuits implementing an RNA aptamer previously described to have the capability of binding to the T7 RNA polymerase and inhibiting its activity in vitro. Using in vitro transcription assays, we first demonstrated the utility of the RNA aptamer in combination with programmable synthetic transcription networks. As a step to quickly assess the feasibility of aptamer functions in vivo, a cell-free expression system was used as a breadboard to emulate the in vivo conditions of E. coli. We tested the aptamer and its three sequence variants in the cell-free expression system, verifying the aptamer functionality in the cell-free testbed. In vivo expression of aptamer and its variants demonstrated control over GFP expression driven by T7 RNA polymerase with different response curves, indicating its ability to serve as building blocks for both logic circuits and transcriptional cascades. This work elucidates the potential of RNA-based regulators for cell programming with improved controllability leveraging the fast production and degradation time scales of RNA molecules.


Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1101/008771DOISubmitted Article
ORCID:
AuthorORCID
Murray, Richard M.0000-0002-5785-7481
Additional Information:Funding: This work was supported by National Science Foundation award no. 0832824 (The Molecular Programming Project) and the Defense Advanced Research Projects Agency (DARPA/MTO) Living Foundries program, con- tract number HR0011-12-C-0065 (DARPA/CMO). The views and conclusions contained in this document are those of the authors and should not be interpreted as representing officially policies, either expressly or implied, of the Defense Advanced Research Projects Agency or the U.S. Government. Acknowledgments: The authors thank David Shis and Matthew Bennett for providing the pTara plasmid. Conflict of interest statement. None declared.
Funders:
Funding AgencyGrant Number
NSF0832824
Defense Advanced Research Projects Agency (DARPA)HR0011-12-C-0065
Subject Keywords:synthetic biology, logic circuits, cell-free breadboard, genelet, molecular programming, aptamer
Record Number:CaltechAUTHORS:20140925-085129247
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20140925-085129247
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:50017
Collection:CaltechAUTHORS
Deposited By: Jason Perez
Deposited On:14 May 2016 03:27
Last Modified:14 May 2016 03:27

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