Pick A Color, Any Color: An Inquiry‐Based Laboratory Course Based On Fluorescent Protein Mutagenesis, Expression, Purification And Characterization

A major problem in science education is the disconnect between a typical laboratory class and the experience of doing research. In most laboratory classes, students are given background reading and a protocol to follow, follow the procedure, and then analyze and report their results in a lab report. They then move on to another experiment, typically unrelated to the previous one. Contrast this to the typical research experience in which students work towards a goal, using whatever techniques are needed, often troubleshooting experiments that fail or have outcomes different from those expected. Undergraduate researchers become intellectually and emotionally invested in their projects, and as a result can get more out of a research experience than a lab class. Unfortunately, the resources to give every undergraduate experience in a research lab are unlikely to become available. A potential solution is the growth of project‐based laboratory classes, which convert laboratory classes into more research‐like experiences where students pursue one project throughout the semester. I implemented such a class for second‐semester junior biochemistry majors. Students were given the DNA sequence for a plasmid encoding a fusion protein derived from my research lab. Students first used bioinformatic tools to determine what these domains were, then, after doing some research, designed mutations to change the fluorescent properties of the Green Fluorescent Protein (GFP) portion (to create a blue, cyan or yellow variant). Students designed oligonucleotide primers for their chosen mutations, and then went on to use molecular biology techniques to make the mutations and determine if the mutations had been successfully made. In some cases this required troubleshooting and repetition of experiments (with variations), something that enhanced the “research‐like” aspect of the project. After successfully making their mutant plasmid, students over‐expressed the protein in bacteria, and purified the resulting fusion protein, visually determining if the mutation had the desired effect. More biochemical characterization (SDS‐PAGE, western blotting, absorbance, fluorescence) followed, including experiments to determine how the mutation changed both the fluorescence properties and the folding properties of the GFP domain. Finally, students were able to use their protein in a pull‐down assay to test a literature hypothesis. The semester culminated in a poster presentation designed to integrate the entire research experience, and ensure that students connected the dots between the weeks of the lab, rather than taking each lab as an isolated event. In the initial iteration of this class, students reported higher levels of learning than in the previous semester's more traditional biochemistry lab.