From green to blue: Site‐directed mutagenesis of the green fluorescent protein to teach protein structure–function relationships

Abstract

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 DpnI 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.

Immersing undergraduate students in the research experience

“…Given the importance of molecular cloning within biological research, there have been numerous studies published describing the integration of molecular cloning skills into undergraduate practical classes [20][21][22]. Over a number of inquiry-driven weeks, these studies involved students investigating one specific gene or protein using a broad range of experimental techniques [20][21][22].…”

“…Over a number of inquiry-driven weeks, these studies involved students investigating one specific gene or protein using a broad range of experimental techniques [20][21][22]. In contrast, the URE within this project focuses more upon the experimental skills used in the initial stages of molecular cloning, and gives students the opportunity to repeat this process in a subsequent module for the characterization of a different set of candidate genes.…”

“…In addition to the commonly used student self-assessment questionnaires [17,[20][21][22] that gauged student learning outcomes in experimental, analytical, and reporting skills, student comprehension of theoretical content was also evaluated via cloning mastery quizzes after each module; professional scientific reporting skills were examined via weekly laboratory notebook assessment as well as extended project reports. Furthermore, the qualitative data obtained from in-depth focus group interviews corroborated the quantitative findings on student attitudes toward scientific research and the laboratory environment.…”

Small laccase from streptomyces coelicolor—an ideal model protein/enzyme for undergraduate laboratory experience

“…It is difficult to find one model protein with enzymatic activity that satisfies all the criteria above. Green fluorescent protein or similar fluorescent proteins are commonly used in many undergraduate laboratories . However, these proteins do not have enzymatic activity and therefore enzymatic experiments and characterization are not possible with them.…”

An adaptable investigative graduate laboratory course for teaching protein purification

“…The use of green fluorescent protein (GFP) fusion proteins in laboratory classes is ideal for teacher‐scholars. Educators have effectively employed GFP as a tool to teach the fundamental principles of protein purification in vitro , and it has been used to involve students in structure‐function studies through site‐directed mutagenesis and spectrophotometry . However, these approaches do not allow the instructor the flexibility to incorporate their own research interests into the course material.…”