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Team:Illinois/Bimolecular Fluorescence Biosensor
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Core Team Members
Luke Edelman, Adam Zoellner, Meghan McCleary, Katrina Keller
Project Abstract
We hope to design a soluble molecular biosensor that, when it comes in contact with an 'activating' ligand such as a virus, bacterium, or specific antibody, generates a fluorescent response using bimolecular complementation. Traditionally unimolecular constructs such as Green Fluorescent Protein (GFP) can be split into two heterologous protein fragments, which can then bind and reinitiate fluorescence upon close spatial proximity. GFP fragments are often fused to endogenous intracellular proteins to study protein-protein interactions: complementation between these GFP fragments is achieved only when they are tethered to proteins which interact (bind) strongly.
We seek to harness this powerful molecular tool for an inverse task: instead of studying putative interactions between known proteins, we are designing fusion constructs to detect the presence of proteins known a priori to interact, for deployment as a one-step diagnostic assay. For example, virus envelopes are often composed of large multiprotein complexes; one GFP fragment could be fused to an antibody against on envelope protein, and the complementary fragment fused to an antibody against an adjacent protein. In solution, only the presence of this specific multiprotein complex would generate GFP complementation and the resulting fluorescence, providing a robust one-step method for the detection of biological pathogens. Similarly, many secreted bacterial enterotoxins form multiprotein complexes, such as cholera toxin. Additionally, many immunoglobulin biomolecules can form aggregates in the circulation.
Specific Plans, Supplies, and Protocols
We are currently considering possible biological targets for use as an initial proof-of-concept. Specifically, this project requires a known protein-protein interaction: for example, a ligand-receptor pair, or an antibody-epitope pair, for each of two unique sites on a target pathogenic protein.
One possibility we are currently considering is an antibody to the cholera toxin B subunit (CTB) which forms a homopentameric complex in solution and can be purchased commercially. In this case, we would fuse both GFP fragments to an antibody against CTB. Fluorescence would occur when a CTB complex exists to tether two complementary GFP fragments through the CTB-antibody interaction.
A second possible
We must then acquire genes for our desired proteins- both the GFP fragments, and
Literature References
Bimolecular Fluorescence Complementation Review
Yeast BiFC Plasmid Construction
