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Development and optimization of a high through-put screening methodology for rapid dynamic range improvement of FRET-based biosensors Open Access


Other title
FRET response optimization
FRET-based biosensors
PTM biosensors
Type of item
Degree grantor
University of Alberta
Author or creator
Abdel Latif Ibraheem, Ahmed Abdel Mohsen
Supervisor and department
Campbell, Robert (Department of Chemistry)
Examining committee member and department
Cairo, Christopher (Department of Chemistry)
Hendzel, Michael (Department of Oncology)
Department of Chemistry

Date accepted
Graduation date
Master of Science
Degree level
Fluorescent protein (FP)-based biosensors based on the principle of intramolecular Förster resonance energy transfer (FRET) enable the visualization of a variety of biochemical events in living cells. The sensitivity of such biosensors is proportional to the change in ratiometric emission, and so there is a pressing need for methods to maximize the ratiometric change of existing biosensor constructs in order to increase the breadth of their utility. To accelerate the development and optimization of improved FRET-based biosensors, we have developed a method for function-based high-throughput screening of biosensor variants in colonies of Escherichia coli. We have demonstrated this technology by undertaking the optimization of a biosensor for detection of methylation of lysine 27 of histone H3 (H3K27). Application of this screening methodology led to the identification of an optimized H3K27-trimethylation biosensor with a two-fold improved emission ratio change relative to that of the initially constructed biosensor.
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.
Citation for previous publication
Ibraheem A, Yap H, Ding Y & Campbell RE (2011) A bacteria colony-based screen for optimal linker combinations in genetically encoded biosensors. BMC Biotechnol 11: 105Ibraheem A & Campbell RE (2010) Designs and applications of fluorescent protein-based biosensors. Curr Opin Chem Biol 14: 30-36

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