Green, yellow, or cyan? Introduction of color change mutations into a green thermostable fluorescent protein and characterization during an introduction to biochemistry lab course

Green fluorescent protein has long been a favorite protein for demonstrating protein purification in the biochemistry lab course. The protein's vivid green color helps demonstrate to students the concept(s) behind affinity or ion exchange chromatography. We designed a series of introduction to...

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Veröffentlicht in:Biochemistry and molecular biology education 2024-09, Vol.52 (5), p.549-558
Hauptverfasser: Anderson, Matthew R., Dargatz, Cammi J., Banerjee, Tuhina, DeVore, Natasha M.
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container_issue 5
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container_title Biochemistry and molecular biology education
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creator Anderson, Matthew R.
Dargatz, Cammi J.
Banerjee, Tuhina
DeVore, Natasha M.
description Green fluorescent protein has long been a favorite protein for demonstrating protein purification in the biochemistry lab course. The protein's vivid green color helps demonstrate to students the concept(s) behind affinity or ion exchange chromatography. We designed a series of introduction to biochemistry labs utilizing a thermostable green protein (TGP‐E) engineered to have unusually high thermostability. This protein allows students to proceed through purification and characterization without the need to keep protein samples on ice. The 5‐week lab series begins with an introduction to molecular biology techniques during weeks 1 and 2, where site‐directed mutagenesis is used introduce, a single nucleotide change that shifts the fluorescent spectra of TGP‐E to either cyan (CTP‐E) or yellow (YTP‐E). Students identify successful mutagenesis reaction by the color of a small expression sample after induction with IPTG. Next, students purify either the TGP‐E (control—typically one group volunteers), YTP‐E, or CTP‐E protein as a 1‐week lab. During the following week's lab, students run SDS‐PAGE to verify protein purity, bicinchoninic acid assay to quantify protein yield, and absorbance and fluorescence spectra to characterize their protein's fluorescent character. The final lab in the series investigates the thermostability of YTP‐E and CTP‐E compared with TGP‐E using a fluorescence plate reader. This 5‐week series of experiments provide students with experience in several key biochemistry techniques and allows the students to compare properties of mutations. At the end of the course, the students will write a research report and give a short presentation over their results.
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source MEDLINE; Wiley Journals
subjects Biochemistry
Biochemistry - education
biochemistry lab
Color
E protein
fluorescent protein
Green fluorescent protein
Green Fluorescent Proteins - chemistry
Green Fluorescent Proteins - genetics
Green Fluorescent Proteins - metabolism
Humans
Laboratories
Literary Devices
Molecular Biology
Mutagenesis
Mutagenesis, Site-Directed
Mutation
Protein purification
Protein Stability
Proteins
site‐directed mutagenesis
Students
Temperature
Thermal stability
thermostable
yellow fluorescent protein
title Green, yellow, or cyan? Introduction of color change mutations into a green thermostable fluorescent protein and characterization during an introduction to biochemistry lab course
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