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Synthetic Regulation of Eukaryotic Gene Expression by Noncoding RNA


D'Espaux, Leopold Daniel (2013) Synthetic Regulation of Eukaryotic Gene Expression by Noncoding RNA. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z9CR5RBP.


Synthetic biological systems promise to combine the spectacular diversity of biological functionality with engineering principles to design new life to address many pressing needs. As these engineered systems advance in sophistication, there is ever-greater need for customizable, situation-specific expression of desired genes. However, existing gene control platforms are generally not modular, or do not display performance requirements required for robust phenotypic responses to input signals. This work expands the capabilities of eukaryotic gene control in two important directions.

For development of greater modularity, we extend the use of synthetic self-cleaving ribozyme switches to detect changes in input protein levels and convey that information into programmed gene expression in eukaryotic cells. We demonstrate both up- and down-regulation of levels of an output transgene by more than 4-fold in response to rising input protein levels, with maximal output gene expression approaching the highest levels observed in yeast. In vitro experiments demonstrate protein-dependent ribozyme activity modulation. We further demonstrate the platform in mammalian cells. Our switch devices do not depend on special input protein activity, and can be tailored to respond to any input protein to which a suitable RNA aptamer can be developed. This platform can potentially be employed to regulate the expression of any transgene or any endogenous gene by 3’ UTR replacement, allowing for more complex cell state-specific reprogramming.

We also address an important concern with ribozyme switches, and riboswitch performance in general, their dynamic range. While riboswitches have generally allowed for versatile and modular regulation, so far their dynamic ranges of output gene modulation have been modest, generally at most 10-fold. We address this shortcoming by developing a modular genetic amplifier for near-digital control of eukaryotic gene expression. We combine ribozyme switch-mediated regulation of a synthetic TF with TF-mediated regulation of an output gene. The amplifier platform allows for as much as 20-fold regulation of output gene expression in response to input signal, with maximal expression approaching the highest levels observed in yeast, yet being tunable to intermediate and lower expression levels. EC50 values are more than 4 times lower than in previously best-performing non-amplifier ribozyme switches. The system design retains the modular-input architecture of the ribozyme switch platform, and the near-digital dynamic ranges of TF-based gene control.

Together, these developments suggest great potential for the wide applicability of these platforms for better-performing eukaryotic gene regulation, and more sophisticated, customizable reprogramming of cellular activity.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:biosensing; cellular information processing; ligand-controlled gene regulation; programming cellular function; riboswitches; ribozyme switches; RNA devices; gene circuits; amplifier
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemical Engineering
Minor Option:Biology
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Smolke, Christina D.
Thesis Committee:
  • Tirrell, David A. (chair)
  • Shan, Shu-ou
  • Hay, Bruce A.
  • Smolke, Christina D.
Defense Date:30 May 2013
Non-Caltech Author Email:leodespaux (AT)
Record Number:CaltechTHESIS:06062013-121826969
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:7852
Deposited By: Leopold d'Espaux
Deposited On:20 Sep 2016 16:47
Last Modified:04 Oct 2019 00:02

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