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From green to blue: Site‐directed mutagenesis of the green fluorescent protein to teach protein structure–function relationships
Author(s) -
Girón María D.,
Salto Rafael
Publication year - 2011
Publication title -
biochemistry and molecular biology education
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.34
H-Index - 39
eISSN - 1539-3429
pISSN - 1470-8175
DOI - 10.1002/bmb.20467
Subject(s) - green fluorescent protein , mutagenesis , aequorea victoria , site directed mutagenesis , protein engineering , biology , plasmid , directed evolution , biochemistry , computational biology , fluorescence , transformation (genetics) , directed mutagenesis , microbiology and biotechnology , mutation , gene , enzyme , mutant , physics , quantum mechanics
Structure–function relationship studies in proteins are essential in modern Cell Biology. Laboratory exercises that allow students to familiarize themselves with basic mutagenesis techniques are essential in all Genetic Engineering courses to teach the relevance of protein structure. We have implemented a laboratory course based on the site‐directed mutagenesis of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria . The GFP is ideal because the students are able to correlate the changes introduced into the structure of the protein with the observable modification of its fluorescence properties. By using noncommercial kits, we set up a non PCR‐thermocycling reaction using mutagenic primers, followed by removal of the original plasmid template by Dpn I digestion. By introducing only one (Y66H) or two mutations (Y66H/Y145F) in the “cycle 3” variant of GFP (F99S, M153T, and V163A) or GFPuv, students are able to analyze the changes from green to blue in the fluorescence emission of the mutated proteins and to correlate these differences in fluorescence with the structural changes using three‐dimensional structure visualization software. This inexpensive laboratory course familiarizes the students with the design of mutagenic oligonucleotides, site‐directed mutagenesis, bacterial transformation, restriction analysis of the mutated plasmids, and protein characterization by SDS‐PAGE and fluorescence spectroscopy. Biochemistry and Molecular Biology Education Vol. 39, No. 4, pp. 309‐315, 2011