Incorporation of 3-fluorotyrosine into green fluorescent protein

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Description: Green Fluorescent Protein (GFP) is a 27 kDa protein isolated from the jellyfish Aequorea victoria . It functions as a secondary emitter of green fluorescence via radiationless energy transfer from the light-emitting protein aequorin. GFP forms an intrinsic fluorophore through cyclization and oxidation of an internal Ser-Tyr-Gly tripeptide. It has been used in various biological applications, however there is still a need for new and improved GFPs. DNA was amplified using PCR (polymerase chain reaction), inserted into an expression plasmid using a topoisomerase-based cloning system, transformed into E. coli 3-fluorotyrosine incorporated, induced with isopropyl-β-D-thiogalactoside (IPTG), then purified and analyzed for interest. The goal of this research project was to not only incorporate 3-fluorotyrosine into Green Fluorescent Protein, but also investigate the unique changes in expression, fluorescence, pH dependence, and the possible applications of this new F-GFP.
Language: English
Format: Degree Work