Authentic Research In A Large Laboratory Course: Studies of Metabolic Channeling Properties With Malate Dehydrogenase (MDH) Isoforms In Trypanosoma brucei

Abstract

We are using a Course-Based Undergraduate Research Experience (CURE) model to provide opportunities for upper level Biochemistry and Molecular Biology (BMB) majors to gain exposure to laboratory research, and connect to the protein-centric MDH CURE community (MCC) of faculty from many institutions. Initially, students perform a four-week “boot camp” of common laboratory techniques and then work, as teams, on authentic research projects. We are interested in metabolic regulation in Trypanosoma brucei, the protozoan parasite that causes African sleeping sickness, this unicellular eukaryote is dependent on metabolic regulation for successful colonization of the Tsetse fly and subsequent infection of mammalian hosts. Growth within the fly utilizes oxidative phosphorylation of amino acids, whereas growth in the mammalian bloodstream is strictly glycolytic. Three MDH isoforms exist in T. brucei, localized to mitochondria, cytoplasm and glycosomes, respectively. We are interested in determining if any of the T. brucei MDH isoforms interact with other metabolic enzymes as appropriate to promote the proper metabolic program. Reports from other species some MDH isoforms form specific interactions with these enzymes to promote high flux of intermediates via substrate channeling. We have used student projects to explore interactions between T. brucei Malate dehydrogenase (MDH) and enzymes that act in adjacent steps in metabolic pathways of mitochondria or in cytoplasm. Students have generated clones and optimized recombinant expression of MDH, citrate synthase (CS) and fumarate hydratase (FH) isoforms. While some student teams design projects de novo, others choose to continue a project from a previous semester. Student teams have conducted expression studies, interaction assays and enzymatic analyses to characterize these enzymes. Students give presentations, a final poster and write and revise reports and a research proposal. Students also share their ideas and projects with other members of MCC, a consortium of faculty working on similar projects with other organisms. MCC CUREs have been shown to support student learning, retention and sense of ownership of research, and MCC provides a wealth of opportunities for comparative studies and to offer faculty and students access to scientific information and community beyond the students and instructors in the course, assisting development of hypotheses and sharing of reagents.