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Permanent link (DOI): https://doi.org/10.7939/R3HH6CD78

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Engineering a Chromoprotein Optimized for Photoacoustic Imaging and Biosensing Applications Open Access

Descriptions

Other title
Subject/Keyword
Photoacoustic imaging
Chromoprotein
Dark tandem dimer acceptor
FRET
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Li, Yan
Supervisor and department
Robert E. Campbell
Examining committee member and department
Zemp, Roger (Department of Electrical and Computer Engineering)
Brown, Alexander (Department of Chemistry)
Campbell, Robert E. (Department of Chemistry)
Department
Department of Chemistry
Specialization

Date accepted
2014-08-11T09:32:51Z
Graduation date
2014-11
Degree
Master of Science
Degree level
Master's
Abstract
A subset of the family of fluorescent proteins are the non-fluorescent chromoproteins which serve as important tools in biomedical research. Recently, chromoproteins have been utilized as both reporter molecules in photoacoustic imaging and acceptor chromophores in Förster resonance energy transfer (FRET)-based biosensors. Photoacoustic imaging enables imaging deep in tissue with relatively high resolution while FRET-based biosensors are the principal technology for live cell imaging of physiological events, such as enzymatic activity, protein-protein interaction and changes in small molecule concentration. However, there are few chromoproteins that have ideal characteristics for photoacoustic imaging and biosensors due to a limited ability to artificially evolve them for improved photoacoustic signals. A major challenge of directed laboratory protein evolution is establishing a simple and efficient screening method. In this thesis we describe our efforts to address this shortcoming in the area of chromoproteins evolution and application by developing a novel colony-based photoacoustic screening method. Through iterative rounds of directed evolution and subsequent screening, the best variants of chromoproteins exhibited higher photoacoustic signal and extinction coefficient and lower quantum yield. We also report the application and performance of a tandem dimer chromoprotein in FRET-based biosensors compared with monomer acceptor. The change of donor fluorescence represented the functionality of biosensor attributing to non-fluorescence of acceptor. Specifically, we demonstrated that tandem dimer-based FRET biosensors are useful for detecting activation of caspase-3 and changes in calcium ion (Ca2+) concentration in live cells.
Language
English
DOI
doi:10.7939/R3HH6CD78
Rights
This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for the purpose of private, scholarly or scientific research. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
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