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An in vitro Biomolecular Breadboard for Prototyping Synthetic Biological Circuits

Citation

Sun, Zachary Zhipeng (2016) An in vitro Biomolecular Breadboard for Prototyping Synthetic Biological Circuits. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z9TB14TW. http://resolver.caltech.edu/CaltechTHESIS:10012015-221355676

Abstract

Biomolecular circuit engineering is critical for implementing complex functions in vivo, and is a baseline method in the synthetic biology space. However, current methods for conducting biomolecular circuit engineering are time-consuming and tedious. A complete design-build-test cycle typically takes weeks' to months' time due to the lack of an intermediary between design ex vivo and testing in vivo. In this work, we explore the development and application of a "biomolecular breadboard" composed of an in-vitro transcription-translation (TX-TL) lysate to rapidly speed up the engineering design-build-test cycle. We first developed protocols for creating and using lysates for conducting biological circuit design. By doing so we simplified the existing technology to an affordable ($0.03/uL) and easy to use three-tube reagent system. We then developed tools to accelerate circuit design by allowing for linear DNA use in lieu of plasmid DNA, and by utilizing principles of modular assembly. This allowed the design-build-test cycle to be reduced to under a business day. We then characterized protein degradation dynamics in the breadboard to aid to implementing complex circuits. Finally, we demonstrated that the breadboard could be applied to engineer complex synthetic circuits in vitro and in vivo. Specifically, we utilized our understanding of linear DNA prototyping, modular assembly, and protein degradation dynamics to characterize the repressilator oscillator and to prototype novel three- and five-node negative feedback oscillators both in vitro and in vivo. We therefore believe the biomolecular breadboard has wide application for acting as an intermediary for biological circuit engineering.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:synthetic biology, cell-free, TX-TL, biomolecular breadboard
Degree Grantor:California Institute of Technology
Division:Biology and Biological Engineering
Major Option:Biology
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Murray, Richard M.
Thesis Committee:
  • Elowitz, Michael B. (chair)
  • Baltimore, David L.
  • Murray, Richard M.
  • Noireaux, Vincent
Defense Date:22 June 2015
Record Number:CaltechTHESIS:10012015-221355676
Persistent URL:http://resolver.caltech.edu/CaltechTHESIS:10012015-221355676
DOI:10.7907/Z9TB14TW
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.3791/50762DOIArticle adapted for Ch. 2
http://dx.doi.org/10.1021/sb400131aDOIArticle adapted for Ch. 3
http://dx.doi.org/10.1101/019695DOIArticle adapted for Ch. 4
http://dx.doi.org/10.7554/eLife.09771DOIArticle adapted for Ch. 5
ORCID:
AuthorORCID
Sun, Zachary Zhipeng0000-0002-9425-2924
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:9191
Collection:CaltechTHESIS
Deposited By: Zachary Sun
Deposited On:08 Oct 2015 16:59
Last Modified:18 May 2017 19:50

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