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Genetically encoded fluorescent biosensors for detection of ligands and protein-protein interactions
- Author / Creator
- Wu, Sheng Yi
Fluorescence imaging enables direct visualization of cellular activities with remarkable resolutions supported by advancing technologies. The fluorescent markers for biomolecules complement the hardware and software for imaging a specific target in detail. Genetically encoded fluorescent proteins enable imaging in live cells with a spatiotemporal resolution and can be targeted to a specific subcellular region of a cell or a specific cell type of a tissue. While most biomolecules can be visualized with a fused fluorescent protein, more sophisticated biosensors are required to report a specific small molecule or ion (e.g., Ca2+ biosensors), protein-protein interactions (e.g., Split GFP), or enzymatic activity (e.g., kinase biosensor). These biosensors are valuable tools that have revolutionized the way scientists investigate biological questions.
Well aligned with the expertise of the Campbell lab in genetically encoded indicator development, my thesis focuses on genetically encoded biosensors to detect ligands including potassium ion (K+), insulin, and the brain derived neurotropic factor (BDNF); and protein-protein interactions with dimerization-dependent fluorescent proteins (ddFPs) and FRET pairs of DipdTFP (Discosoma peripheral dTFP) and dTomato. The proposals of biosensors are based on two criteria: the potential impact and feasibility. All the projects listed above can potentially make a difference in biological research. Potassium ion is one of the most abundant biological ions that has many implications in human; abnormal level of K+ concentration has been reported in various diseased conditions. Insulin is a major biomolecule for blood glucose regulation and is associated with diabetes, a common and uncurable metabolic condition. BDNF is a nerve growth factor that has implications in neurological diseases. Protein-protein interactions are commonly investigated in biology. The feasibility of biosensor development largely relies on the availability of binding proteins: potassium binding protein (Kbp), human insulin receptor (hIR), insulin degrading enzyme (IDE), and BDNF pre-peptide. The ddFPs and FRET based DipdTFP/dTomato pair uses engineered interface interaction to detect protein-protein interactions.
The main projects in this thesis are on the K+ biosensor development. KIRIN1 and GINKO1 were developed for intracellular K+ detection as described in Chapter 2. GINKO1 was further optimized to GINKO2a and GINKO2b in Chapter 3, with greatly improved sensitivity and specificity. In Chapter 4, I describe the effort towards tackling the major obstacle of the K+ biosensor project, developing a functional extracellular displayed version.
The ddFP optimization work in Chapter 5 was a continuous effort to further improve the previous development ddFPs in the Campbell lab. The optimization led to some improvements in ddRFP brightness but not sufficiently brighter to show a difference in the mammalian system. The work on ddGFP did not yield variants much brighter.
In Chapter 6, a DipdTFP/dTomato FRET pair was engineered by replacing the exterior residues of dTFP with that of dTomato. The surface residue engineering successfully allows heterodimer to form, but with a high affinity. More work is required to weaken this interaction for proper protein-protein interaction detection.
Finally, the appendix chapter describes my attempt on prototyping insulin and BDNF biosensors. The designed prototype indicators were constructed and tested with the corresponding ligands, further investigations and optimizations is required to for the development of functional biosensors.
- Graduation date
- Spring 2021
- Type of Item
- Doctor of Philosophy
- 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.