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Genetically engineered sensors of cell signaling

Citation

Siegel, Micah Seth (2000) Genetically engineered sensors of cell signaling. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/92c5-r851. https://resolver.caltech.edu/CaltechTHESIS:04082013-160433360

Abstract

Measuring electrical activity in large numbers of cells with high spatial and temporal resolution is a fundamental problem for the study of neural development and information processing. To address this problem, we have constructed FlaSh: a novel, genetically-encoded probe that can be used to measure trans-membrane voltage in single cells. We fused a modified green fluorescent protein (GFP) into a voltage-sensitive potassium channel so that voltage dependent rearrangements in the potassium channel induce changes in the fluorescence of GFP. A voltage sensor encoded into DNA has the advantage that it may be introduced into an organism non-invasively and targeted to specific developmental stages, brain regions, cell types, and sub-cellular compartments.

We also describe modifications to FlaSh that shift its color, kinetics, and dynamic range. We used multiple green fluorescent proteins to produce variants of the FlaSh sensor that generate ratiometric signal output via fluorescence resonance energy transfer (FRET). Finally, we describe initial work toward FlaSh variants that are sensitive to G-protein coupled receptor (GPCR) activation. These sensors can be used to design functional assays for receptor activation in living cells.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Computation and Neural Systems
Degree Grantor:California Institute of Technology
Division:Biology
Major Option:Computation and Neural Systems
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Mead, Carver
Thesis Committee:
  • Fraser, Scott E.
  • Laurent, Gilles J.
  • Lester, Henry A.
  • Mead, Carver
Defense Date:16 December 1999
Record Number:CaltechTHESIS:04082013-160433360
Persistent URL:https://resolver.caltech.edu/CaltechTHESIS:04082013-160433360
DOI:10.7907/92c5-r851
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:7588
Collection:CaltechTHESIS
Deposited By: Benjamin Perez
Deposited On:09 Apr 2013 14:26
Last Modified:16 Apr 2021 23:13

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