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Development and optimization of a high through-put screening methodology for rapid dynamic range improvement of FRET-based biosensors
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- Author / Creator
- Abdel Latif Ibraheem, Ahmed Abdel Mohsen
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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.
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- Subjects / Keywords
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- Graduation date
- Spring 2012
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- Type of Item
- Thesis
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- Degree
- Master of Science
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- License
- This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. 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.