Mutating GFP

Graduate Fellow Alyssa Wu-Zhang & Teacher Partner Jesse Wade Robinson

This lesson explores the concept of how a protein’s function is dictated by its structure and how a protein’s structure and consequently its function can be manipulated by mutating the DNA coding for that protein.  Students learn this concept through the mutation of green fluorescent protein to fluorescent proteins of other colors by using the technique of site-directed mutagenesis to make targeted point mutations in the DNA for GFP, resulting in changes in particular amino acid residues known to be critical for GFP’s fluorescence color.

GRADE LEVEL: Upper level high school or undergraduate biology classes 

DURATION: Three or possibly four 50-minute periods

Day 1:
Introduction to protein structure/function, manipulating protein structure by mutating
DNA.
Optional exercise (for long or extra period): design primers for mutating GFP to BFP/YFP.
Set up site-directed mutagenesis PCR reactions.
Reactions thermocycle overnight (takes ~3 h).

Day 2:
Transform mutated plasmids into bacteria.

Day 3:
Observe results and draw conclusions.

LEARNING OBJECTIVES:

• Students will understand that mutations in DNA, whether occurring spontaneously in nature or through manipulation of DNA in the lab, cause changes in protein amino acid sequence that lead to changes in protein structure and hence function/phenotype.
• Students will understand that DNA is manipulated and purposely mutated in the lab in order to study how changes in protein structure can affect their function.
• Students will be able to explain the purpose and steps of site-directed mutagenesis, a technique used in the lab to generate targeted amino acid mutations through a special PCR reaction using mutagenic primers followed by transformation of the mutated DNA into bacteria.
• Students will know that site-directed mutagenesis was used to generate mutants of GFP and RFP that fluoresce in a spectrum of different colors.