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Implementation and System Identification of a Phosphorylation-Based Insulator in a Cell-Free Transcription-Translation System

Guo, Shaobin and Yeung, Enoch and Murray, Richard M. (2017) Implementation and System Identification of a Phosphorylation-Based Insulator in a Cell-Free Transcription-Translation System. . (Unpublished)

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An outstanding challenge in the design of synthetic biocircuits is the development of a robust and efficient strategy for interconnecting functional modules. Recent work demonstrated that a phosphorylation-based insulator (PBI) implementing a dual strategy of high gain and strong negative feedback can be used as a device to attenuate retroactivity. This paper describes the implementation of such a biological circuit in a cell-free transcription-translation system and the structural identifiability of the PBI in the system. We first show that the retroactivity also exists in the cell-free system by testing a simple negative regulation circuit. Then we demonstrate that the PBI circuit helps attenuate the retroactivity significantly compared to the control. We consider a complex model that provides an intricate description of all chemical reactions and leveraging specific physiologically plausible assumptions. We derive a rigorous simplified model that captures the output dynamics of the PBI. We performed standard system identification analysis and determined that the model is globally identifiable with respect to three critical parameters. These three parameters are identifiable under specific experimental conditions and we performed these experiments to estimate the parameters. Our experimental results suggest that the functional form of our simplified model is sufficient to describe the reporter dynamics and enable parameter estimation. In general, this research illustrates the utility of the cell-free expression system as an alternate platform for biocircuit implementation and system identification and it can provide interesting insights into future biological circuit designs.

Item Type:Report or Paper (Discussion Paper)
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URLURL TypeDescription DOIDiscussion Paper
Guo, Shaobin0000-0001-9736-4078
Yeung, Enoch0000-0001-7630-7429
Murray, Richard M.0000-0002-5785-7481
Additional Information:The copyright holder for this preprint is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. bioRxiv preprint first posted online Mar. 30, 2017. We would like to thank Kayzad Soli Nilgiriwala and Domitilla Del Vecchio of MIT for providing the original plasmids and helpful suggestions. We would like to thank Murray lab members for useful discussions and suggestions. This work was supported by Office of Naval Research (ONR) Multidisciplinary University Research Initiatives (MURI) Program (Grant number: N00014-13-1-0074) and Air Force Office of Scientific Research (Grant number: FA9550-14-1-0060). Author Contributions: S.G. and E.Y. contributed equally to this work. S.G., E.Y. and R.M.M. conceived the idea. S.G. and E.Y. designed the experiments, performed the experiments and subsequent data analysis and wrote the manuscript. R.M.M. revised the manuscript. R.M.M. has ownership in a company that commercializes the cell-free technology utilized in this paper. All the other authors claim no competing interest.
Funding AgencyGrant Number
Office of Naval Research (ONR)N00014-13-1-0074
Air Force Office of Scientific Research (AFOSR)FA9550-14-1-0060
Record Number:CaltechAUTHORS:20170705-103731007
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:78762
Deposited By: Tony Diaz
Deposited On:06 Jul 2017 21:39
Last Modified:11 Nov 2020 22:27

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