A Multi‐year Biochemistry Lab Experiment Using a Putative Enzyme from Arabidopsis thaliana

Abstract

Until relatively recently, a typical biochemistry laboratory course format included a series of one to two‐week experiments that exposed students to the “traditional” biochemical techniques of protein purification and kinetic analyses using classic enzymes like alkaline phosphatase or alcohol dehydrogenase. However, there is ample evidence that course‐based undergraduate research (CURE) leads to increased retention and satisfaction, particularly for underrepresented groups, and as a result, there has been a rise in these types of courses for undergraduate students. In a biochemistry lab course this is often accomplished one of two ways ‐ characterizing a novel system (i.e. enzyme, substrate, reaction, or pathway) or studying a known system in a novel way (e.g. introducing a new analytical technique). Either way, it is generally difficult to maintain a level of experimental novelty year after year without redesigning some aspect of the class. This project, using the CURE model, extended over the course of three years and has the potential to extend beyond that. The experimental goal of this multi‐year project was for students to purify and characterize a putative hydroxyacid dehydrogenase from Arabidopsis thaliana, which at the start of this project was uncharacterized. To reach this goal, students in year 1 tested purification and characterization methods using a homologous enzyme from Pseudomonas denitrificans based on methods described in the primary literature. Given the results of work in year 1, the subsequent class in year 2, purified and characterized the enzyme from A. thaliana alongside students working with P. denitrificans. This was so that students had reference data to make conclusions about the quality of their own work and that published in the literature. Once the purification and characterization methods had been worked out, the next class of students in year 3, set out to more fully characterize the enzyme of A. thaliana through a side‐by‐side comparison between their native protein and a protein with a single‐amino acid mutation, which they also generated and purified. Positive outcomes of this multi‐year project are seen through increased student engagement and interest and extend beyond the class as students share their appreciation for being well prepared for summer research experiences, graduate school, and post‐college employment. Additionally, this project allows the instructor to extend their scholarly work into the classroom, maximizing time and resources to advance research productivity. Since the start of this project, the enzyme from A. thaliana was fully characterized by another research group using a different expression system. Future courses will examine the differences in enzyme function based on plasmid selection and gene length using the construct from the author's lab and that which was generated by the other research group.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.