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Permanent link (DOI): https://doi.org/10.7939/R3057D01P

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Theses and Dissertations

Generation of a fluorescent protein-derived biosensor based on alternate frame folding Open Access

Descriptions

Other title
Subject/Keyword
Tandem alternate frame switching
alternate frame folding
avGFP
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Saini, Ritesh K.
Supervisor and department
Campbell, Robert E. (Chemistry)
Examining committee member and department
Gibbs-Davis, Julianne M. (Chemsitry)
Loppnow, Glen R. (Chemistry)
Department
Department of Chemistry
Specialization

Date accepted
2012-09-26T15:55:46Z
Graduation date
2012-09
Degree
Master of Science
Degree level
Master's
Abstract
To develop a single fluorescent protein (FP)-based scaffold that could potentially serve as a universal platform for biosensor creation, we have introduced the tandem alternate frame switching strategy. We genetically inserted a non-identical copy of 7th β-strand in Aequorea victoria greenFP(avGFP) to create protein conformational switch. By creating a series of variants, we demonstrated that equilibrium between conformations can be manipulated by substitution, deletion and insertion of residues. The chromophore absorption spectra unique to each conformation allowed the determination of bound equilibrium through independent component analysis. To verify the potential of this strategy, we strategically created calcium-ion (Ca2+) and cyclic adenosine-monophosphate (cAMP) biosensors and evaluated the change in the fluorescence spectra corresponding to binding of the analyte. We performed preliminary study to explore switch generation in cpmCherry. We anticipate that this rational strategy for construction of genetically-encoded indicators could be applicable to a variety of FPs and sensing motifs.
Language
English
DOI
doi:10.7939/R3057D01P
Rights
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
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File author: Ritesh
Page count: 143
File language: en-US
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