From gene mutation to protein characterization

Fluorescent proteins are commonly used in cell biology to assess where proteins are within a cell as a function of time and provide insight into intracellular protein function. However, the usefulness of a fluorescent protein depends directly on the quantum yield. The quantum yield relates the efficiency at which a fluorescent molecule converts absorbed photons into emitted photons and it is necessary to know for assessing what fluorescent protein is the most appropriate for a particular application. In this work, we have designed an upper-level, biochemistry laboratory experiment where students measure the fluorescence quantum yields of fluorescent proteins relative to a standard organic dye. Four fluorescent protein variants, enhanced cyan fluorescent protein (ECFP), enhanced green fluorescent protein (EGFP), mCitrine, and mCherry, were used, however the methods described are useful for the characterization of any fluorescent protein or could be expanded to fluorescent quantum yield measurements of organic dye molecules. The laboratory is designed as a guided inquiry project and takes two, 4 hr laboratory periods. During the first day students design the experiment by selecting the excitation wavelength, choosing the standard, and determining the concentration needed for the quantum yield experiment that takes place in the second laboratory period. Overall, this laboratory provides students with a guided inquiry learning experience and introduces concepts of fluorescence biophysics into a biochemistry laboratory curriculum. V C 2014 by The International Union of Biochemistry and Molecular Biology, 43(1):52-59, 2015.

Section: Introductionmentioning

confidence: 99%

Fluorescence quantum yield measurements of fluorescent proteins: A laboratory experiment for a biochemistry or molecular biophysics laboratory course

Wall

Dillon

Knowles

2014

“…The inspiration for this project came from Liskin Swint‐Kruse, who currently has an NIH‐funded project that has already used 10 different homologous proteins to assess the outcomes for >1000 mutations at nonconserved positions and are now focused on pyruvate kinase, aldolase, and an organic anion transmembrane transporter as model systems to test the hypothesis “that rheostat positions can be detected by a particular pattern of evolutionary change” . David Moffet has created a “gene to protein” biochemistry lab sequence where students create mutant versions of a fluorescent protein (EGFP) to alter the fluorescent properties . Farham and Dube have already created a 13‐week biochemistry lab course were students design and implement their own experiments to study a biomedically relevant enzyme, Heliobactor pylori (Hp) urease .…”

Section: Laboratory Pitfalls and Potential Modificationsmentioning

confidence: 99%

A course‐based undergraduate research experience investigating the consequences of nonconserved mutations in lactate dehydrogenase

Ayella

Beck

2018

There is a growing movement to involve undergraduate students in authentic research experiences. A variety of studies have indicated the strength of this approach in developing scientific aptitude, confidence, critical thinking skills, and increasing the likelihood to become career scientists. Course-based undergraduate research experiences (CUREs) foster opportunities for students to carry out authentic research at both primarily undergraduate and large research institutions. Here, we describe a novel CURE-based biochemistry laboratory course to explore the consequences of mutagenesis of non-conserved amino acid sites in the structure and function of the lactate dehydrogenase enzyme and demonstrate how collaborations between institutions can facilitate real research experiences at any type of institution. © 2018 by The International Union of Biochemistry and Molecular Biology, 46(3):285-296, 2018.

“…The project in BIOC 312 combines elements of two published student laboratory projects . Each group begins the project with a culture of a standard cloning strain of E. coli carrying a plasmid for expression of a different translation factor.…”

Section: Project Overviewmentioning

confidence: 99%

“…While they proceed with the experimental procedures on the wild‐type protein, students undertake an in silico investigation of their protein similar to that described by Moffet . They explore the research literature with a focus on the 3D structure of their protein and learn how to use structure visualization and analysis software.…”

Section: Project Overviewmentioning

confidence: 99%

“…Based on their investigations, they choose a specific mutation, generate a model 3D structure for the mutant translation factor, and design primers to generate the mutant by site‐directed mutagenesis of their wild‐type plasmid. The student‐generated wild‐type plasmid and mutagenesis primers serve as the starting materials for the expression and purification of the mutant protein as described .…”

Section: Project Overviewmentioning

confidence: 99%

See 1 more Smart Citation

Protein analysis using real‐time PCR instrumentation: Incorporation in an integrated, inquiry‐based project

Southard

2013

Instrumentation for real-time PCR is used primarily for amplification and quantitation of nucleic acids. The capability to measure fluorescence while controlling temperature in multiple samples can also be applied to the analysis of proteins. Conformational stability and changes in stability due to ligand binding are easily assessed. Protein structure studies possible with a real-time PCR instrument address core topics in biochemistry and have valuable highthroughput applications in the fields of drug discovery and protein engineering. Protein analysis using real-time PCR instrumentation has been incorporated in an undergraduate laboratory project based on previously described projects. Students express, purify, and characterize a protein.Based on literature research and analysis using bioinformatics tools, they select a specific mutation to investigate. They then attempt to express, purify, and characterize their mutated protein. Thermal denaturation using a real-time PCR instrument is the primary tool used to compare the wild-type and mutated proteins. Alternative means for incorporation of protein analysis by real-time PCR instrumentation into laboratory experiences and additional modes of analysis are also described. V C 2013 by The International