Initial Publication Date: May 24, 2019

Molecular Parasitology

Paul Ulrich, Georgia State University
Mariya Campbell, Georgia State University
Michael Sanderson, Georgia State University
Location: Georgia

Abstract

Of the approximately 1000 different proteins that populate eukaryotic mitochondria, ~50% have no known function. Molecular Parasitology is a course-based intensively engages undergraduates by investigating roles of uncharacterized, mitochondrial proteins in trypanosomatid parasites. Students utilize basic bioinformatics (subcellular localization, conserved domain prediction, BLAST, secondary structure) to predict protein function followed by construction and transfection of GFP-fusion constructs into cell lines to validate their predictions. The CURE is designed to encourage independent problem-solving, science identity, and support career success.

Student Goals

Students utilize computational analysis and resources to answer biological questions.
Students will independently perform experiments, interpret results, and progress on a research project.
Students will collaborate effectively in team contexts.

Research Goals

Identify the full mitochondrial proteome of the trypanosomatid Crithidia fasciculata from genomic data.
Characterize hypothetical, conserved proteins of the trypanosomatid mitochondrion.

Context

Molecular Parasitology is a single semester course at the level of junior and senior students. The course meets for two, three hour sessions per week. Maximum class size is 18 students. This is a time intensive course (10-15 hours per week outside of schedule lab periods), but flexibility in laboratory access (24/7 access) permits students of diverse backgrounds, employment, and family responsibilities to pursue intensive research experience. The prerequisite skills and knowledge for the course include basic chemistry laboratory exposure (e.g. pH and solution preparation) and a some understanding of molecular and cell biology. Prior experience with PCR, DNA extraction, blotting, and electrophoresis is not necessary.

Target Audience:Major, Upper Division
CURE Duration:A full term

CURE Design

The bean-shaped mitochondrion and focus on the TCA cycle and ETS common to all biology and biochemistry textbooks gives a false impression that this organelle is largely understood. However, ~50% of the 1000 proteins in the mitochondrion have no known or empirically-derived function. Molecular Parasitology engages students in answering questions of protein function in a non-pathogenic protozoans closely related to human pathogens in Leishmania and Trypanosoma. The course is designed to cultivate ownership in students by either assigning by letting them select one of ~25 proteins currently under investigation, by focused training sections (e.g. scientific writing, methods discussions, group troubleshooting of results, and a mixture of group and individuals presentations and writing.

Core Competencies: Analyzing and interpreting data, Asking questions (for science) and defining problems (for engineering), Planning and carrying out investigations, Using mathematics and computational thinking
Nature of Research: Basic Research, Informatics/Computational Research

Tasks that Align Student and Research Goals

Research Goals →
Student Goals ↓

Research Goal 1: Identify the full mitochondrial proteome of the trypanosomatid Crithidia fasciculata from genomic data.

Research Goal 2: Characterize hypothetical, conserved proteins of the trypanosomatid mitochondrion.

Student Goal 1: Students utilize computational analysis and resources to answer biological questions.

Students will predict subcellular localizations, secondary structure, and function on amino acid sequences derived from publicly-available genome data (TriTrypDB.org). Students will use literature searches via Google Scholar, build an EndNote library relevant to their specific protein targets, and present findings in three minute, oral research pitches.

Students will design primers for PCR for subcloning and/or CRISPR and and build an argument for mitochondrial protein function that relies on conserved domain analysis, presence or absence of essential motifs, and BLAST.

Student Goal 2: Students will independently perform experiments, interpret results, and progress on a research project.

Students will amplify genes for their selected targets via PCR, subclone into a GFP-expression vector, transfect Crithidia fasciculata, confirm expression via western blot, and validate subcellular localization with fluorescence microscopy. Progress will be monitored via laboratory note checks, weekly results updates, and biweekly reflective, "learning log" assignments.

Students will knock-out genes or tag the endogenous locus of the target genes using CRISPR. Mitochondrial localization of tagged proteins will be confirmed by fluorescence microscopy, and growth phenotypes of knock-outs will be assessed. Progress will be monitored via laboratory note checks, weekly results updates, and biweekly reflective, "learning log" assignments.

Student Goal 3: Students will collaborate effectively in team contexts.

Though students self-select a protein of choice, they will be part of a working group that completes routine lab tasks together (e.g. solution prep assignment, pipette tip racking, sterilizing waste), presents results as a single unit (e.g. final presentation), and journal club discussions relevant to their projects.

Instructional Materials

Syllabus - Molecular Parasitology - Spring 2019 (Microsoft Word 2007 (.docx) 67kB May24 19)
Second semester, peer leadership syllabus (Microsoft Word 2007 (.docx) 54kB May24 19)
Bioinformatics assignment (Microsoft Word 2007 (.docx) 75kB May24 19)

Assessment

Sample Assignment and Rubric (Microsoft Word 2007 (.docx) 14kB May24 19)

Instructional Staffing

Offering this CURE requires, at minimum, a faculty instructor and one graduate teaching assistant (GTA). Because this CURE provides novice researchers a high degree of independence in their laboratory work, it is critical that the instructor and the GTA attend to skill development of CURE students, sufficient student training, monitor and respond to progress or troubleshooting, and manage consumables. Students taking the course for a second semester continue to develop their research project but take on peer leadership and lab management roles, and they are an invaluable and dedicated resource for both the laboratory staff and the first semester students. Helpful but not essential to the process are hourly, graduate student graders who can assist with detailed commenting on student written papers.

GTA roles and responsibilities. GTA's should be encouraged to take ownership in the CURE and work alongside the instructor to lead group discussions, facilitate journal club, monitor attendance, and contribute to activity and assessment design. The GTA is responsible for supporting the instructor by assisting with grading of various assessments of lab notebooks, learning logs, and journal club activities.

Hourly grader roles and responsibilities. Hourly graders are utilized in Molecular Parasitology for extensive commenting and editing of student papers using rubrics designed by the instructor.