Author Profile

Biochemistry II lab: Crithidia parasite metabolism

Amy Greene, Albright College
Undergraduate biochemistry student Ronald Andanje (graduating Dec 2021) helped me develop the lab protocols for this CURE.
Location: Pennsylvania

Abstract

Course Undergraduate Research Experiences (CUREs) have been shown to increase student engagement, skills, and retention in STEM. We developed a CURE using non-pathogenic Crithidia fasciculata parasites, which are insect trypanosomes related to the causative agents of Leishmaniasis, African Trypanosomiasis, and Chagas' diseases. This parasite is ideal for a CURE because it grows to high density in serum-free inexpensive media and has not been well studied in the literature, providing opportunities for students to make discoveries. Metabolically labelled 1-13C-glucose was added to the parasites, and changes in peak position was monitored over time (either in real time, or in the supernatant). The main fermentation products observed were ethanol and succinate. Student groups then designed a novel project investigating metabolism in Crithidia. Students produced novel data on metabolism in a little-studied parasite.

Student Goals

  1. Design, carry out, and interpret novel experiments
  2. Improve scientific writing and revising skills
  3. Find and read biochemical articles and explain their relevance to a research project

Research Goals

  1. Probe versatility of carbon metabolism in Crithidia fasciculata
  2. Develop a toolbox of reliable and reproducible lab assays for understanding Crithidia fasciculata parasite biochemistry

Context

Albright College is a small liberal arts college with 7 full time chemistry and biochemistry faculty. Biochemistry is a 300-level 2-semester course series. In Biochemistry I, students build skills in experimental design, PubMed research, a student-designed project based on a menu of options, science writing, and plagiarism/ethics. Then, in Biochemistry II, students in the associated laboratory carry out metabolism experiments using C-13 labelled glucose and NMR (nuclear magnetic resonance) spectroscopy then design their own experiment with the parasites.

Biochemistry I (before the CURE) enrolls 12-18 students who are a mix of biochemistry, chemistry, and biology majors. Biochemistry II, where the CURE is included, enrolls 5-10 students who are mostly or only biochemistry majors.

Biochemistry II is traditionally 15 weeks long, with about 2/3 of the lab sessions being dedicated to the CURE. Due to the pandemic, we moved to a 7.5 week schedule for the 2020/2021 year and had a mix of online and in person labs.

Target Audience: Major, Upper Division
CURE Duration: Half a term, Multiple terms

CURE Design

The semester starts with experiments I design and protocols I give the students that will help them think about sugar metabolism. Later, students design their own experiment, usually working in pairs or groups of three. Every week there is a CURE-related assignment that is a component of their proposals, data analysis, or presentations, so that everything is split into manageable pieces. For instance, drafting of research papers is divided into writing the Introduction, Methods, Figures and Legends, Results, and Discussion, each of which is due on a different week. Students finish data collection a month before the end of the semester so there is ample time for analyzing data and drafting of papers. I also teach several technique labs throughout the semester that are unrelated to the CURE. This helps meet the objectives of this core content course and enables students to learn more techniques and different types of experiments than are possible in the parasite context. At the end of the semester, every student has written and revised an individual professional-style scientific paper. They present to the class in experiment groups. Exams contain metacognitive questions about their CURE projects and I include parasite examples as often as possible in lecture or through POGIL-type group learning worksheets.

All students presented at a county-wide undergraduate research conference online in 2020 (HECBC conference). In 2021 all students attended and one research student presented at poster at the disciplinary online conference, the University of Georgia Molecular Parasitology/Vector Biology Symposium.

My work with Crithidia was inspired by Paul Ulrich at Georgia State University. Other collaborators are Megan Povelones at Villanova University and Swati Agrawal at University of Mary Washington. The four of us email or Zoom regularly to discuss research ideas and protocols, share information about online guest talks, discuss funding opportunities. In the summer of 2020, Agrawal and I organized series of Zoom parasitology talks from graduate students and faculty at University of Georgia and Clemson University that were attended by our research students.

Core Competencies: Analyzing and interpreting data, Asking questions (for science) and defining problems (for engineering), Constructing explanations (for science) and designing solutions (for engineering), Planning and carrying out investigations
Nature of Research:Basic Research, Wet Lab/Bench Research

Tasks that Align Student and Research Goals

Research Goals →
Student Goals ↓
Research Goal 1: Probe versatility of carbon metabolism in Crithidia fasciculata
Research Goal 2: Develop a toolbox of reliable and reproducible lab assays for understanding Crithidia fasciculata parasite biochemistry

Student Goal 1: Design, carry out, and interpret novel experiments
  • Use information from the provided Crithidia metabolism labs and protocols to formulate a new experimental question and hypothesis
  • Select or design an appropriate protocol to address the question / test the hypothesis
  • Share and discuss your proposal with the class for feedback
  • Revise proposal and submit it as a written plan before beginning experimentation
  • At the end of the semester use metacognitive prompts to write a reflection on is learned from the CURE experimental design process

  • Write out a protocol found in the literature with revisions as needed to apply to particular experiment goals 
  • Perform appropriate and correct calculations (scale up/down, amount of reagents to weigh out, concentrations)
  • Troubleshoot and revise the protocol after each trial in the lab 
  • Discuss as a class what was learned from designing experiments that could not be learned by just following a given lab protocol


Student Goal 2: Improve scientific writing and revising skills
  • Write the methods and data analysis in the format suitable for a professional publication

  • Follow best practices in writing a methods section using advice from the MIT Com Kit
  • Revise and edit methods based on instructor feedback


Student Goal 3: Find and read biochemical articles and explain their relevance to a research project
  • Find relevant articles on Crithidia metabolism 
  • Cite relevant articles appropriately in final research paper
  • Demonstrate understanding of the data in the articles in the Results and Discussion sections of the final research paper
  • Correctly interpret primary data on metabolism from provided primary literature 
  • Compare published results to CURE results 

  • Find appropriate methods for research project in the literature and/or provided protocols
  •  Compare and discuss CURE data and data obtained from other methods in the literature in the Discussion section of the final research article
  • Reflect on how much work is involved in developing reliable protocols and generating publishable data in scientific research


Instructional Materials

The protocols and instructional materials for this CURE are evolving, especially as we move out of a different class format during the pandemic. Please email me at agreene@albright.edu with questions or for the most recent suggestions for protocols or instructional materials.

Intro PPT (PowerPoint 2007 (.pptx) 2.8MB Aug10 21) Intro PPT

Articlesciencewriting.docx (Microsoft Word 2007 (.docx) 24kB Aug10 21) Compiled science writing lessons for student designed CURE project article

CUREA.docx (Microsoft Word 2007 (.docx) 831kB Aug10 21) Crithidia online lab questions used during Biochemistry I to set up for CURE

crithidiaonlinelab.docx (Microsoft Word 2007 (.docx) 501kB Aug10 21) CURE_A Intro to using Crithidia in the lab (sterile technique, OD600, hemocytometer, ways to lyse cells)

CureB.docx (Microsoft Word 2007 (.docx) 115kB Aug10 21) CURE_B Set up 1-13C-glucose Crithidia metabolism experiments

CUREC.docx (Microsoft Word 2007 (.docx) 69kB Aug10 21) CURE_C NMR analysis of 1-13C-glucose metabolites and competition

finalarticlepresentation.docx (Microsoft Word 2007 (.docx) 14kB Aug10 21) Final Article Presentation directions

GreeneAndanjeCUREposterspring2021.pptx (PowerPoint 2007 (.pptx) 7.2MB Aug10 21) Poster presentation overview of the first 2 iterations of the Crithidia CURE

IntroductoryCURElab1.docx (Microsoft Word 2007 (.docx) 248kB Aug10 21) An earlier iteration of the intro CURE lab organized differently

OralProjectProposal.docx (Microsoft Word 2007 (.docx) 14kB Aug10 21) Instructors for oral project proposal

ProjectProposalandSciWriting.docx (Microsoft Word 2007 (.docx) 16kB Aug10 21) Proposal and materials list assignment

ResultsandDiscussion.docx (Microsoft Word 2007 (.docx) 13kB Aug10 21) Results and Discussion assignment

teachinglearning2021session2slides.pptx (PowerPoint 2007 (.pptx) 5MB Aug10 21) 2 Slides for Albright Teaching and Learning Day (online)

TLFCaseStudy.docx (Microsoft Word 2007 (.docx) 1.5MB Aug10 21) Case Study on TLF (Trypanosome Lytic Factor) for class

troubleshootingafter2weeks.docx (Microsoft Word 2007 (.docx) 13kB Aug10 21) Trouble-shooting assignment after 2 weeks of experimenting

writingandethics.docx (Microsoft Word 2007 (.docx) 56kB Aug10 21) Scientific writing and ethics (plagiarism) workshop

Assessment

The assessments for this CURE are evolving, especially as we move out of the class format employed during the pandemic. Please email me at agreene@albright.edu with questions or for the most recent suggestions for assessments.  Some grading rubrics are also included under instructional materials above.

ArticleRubric.docx (Microsoft Word 2007 (.docx) 18kB Aug10 21) Student Article Rubric

ExamQuestions.docx (Microsoft Word 2007 (.docx) 256kB Aug10 21) Some example biochemistry exam questions related to the CURE

Finalarticlechecklist.docx (Microsoft Word 2007 (.docx) 170kB Aug10 21) Final Article Checklist

Instructional Staffing

I am the only instructor of this CURE. Sometimes I have undergraduate research interns already working on parasite research who are enrolled take the class and can be resources for the other students.

Author Experience

Amy Greene, Albright College

I want every student in my class to engage in the process of biochemical research. My Ph.D. background is in parasite biochemistry and I want my teaching and research to be inter-connected so that students can build senior thesis projects from their CURE projects.  In addition to CUREs, I have had students design their own projects for 2-4 lab periods in general chemistry, organic chemistry, analytical chemistry, and biochemistry courses.


Advice for Implementation

The workload for this course is intense because students design their own projects and write a full scientific style article with 6-8 literature sources. Some students are unhappy about the workload, while others are so proud of their final products that it is worth it. I do my best to respect students' time by allowing them to complete their experimental work during class. Students are never required to work outside of weekly 4-hour lab periods. If students need to work outside of that time for their planned experiment (e.g., 24-hour assays, etc.), they can then miss one of the regular CURE lab periods because they put in the time outside of class. I learned the hard way to finish collecting data almost a month before the end of the semester so students have time to complete a full draft of their articles well before finals week. Then, when finals roll around they only have revisions left to complete.

Equipment/Supplies: 400MHz NMR, labeled 1-13C-glucose, and Crithidia fasciculata parasites donated to me by Megan Povelones (Villanova University). Note that these parasites as BLS1 cells which grow in serum-free media at 27°C and don't require CO2.

Iteration

Students have three or four 4-hour lab sessions to complete their CURE experiments. There are also three to four weeks during which they learn methods we will use with the parasites, as well as dry labs on finding research articles, science writing, and final presentations. Students are encouraged to design an experiment that can be completed within one lab period so there is greater opportunity for iteration. They are rarely able to collect useful data the first week, but they learn how to organize their time and their work such that they can collect reliable data by the final week. There is an assignment where they have to think about what to revise/change after the first week or two of experimenting. Students usually wish they had more time on the projects because they only just got to the point of getting some data when we are done with the CURE, but this is variable between groups based on how complex their projects are, how prepared they are for each lab, and whether there is a clear result from initial data collection.

Using CURE Data

So far, I have only collected the final papers into a folder and saved the data on the instrument computers, partly because my classes have been small. It would be helpful to collect and organize data in a more central location and standardized format or webpage. We made a draft page but have not used it. Collaborators Paul Ulrich (Georgia State University), Megan Povelones (Villanova University), Swati Agrawal (University of Mary Washington), and I share data and protocols via email. I also share examples from the previous CURE with students the next year to get them excited about the process.

My students and I have presented posters and oral presentations at local and online conferences, with authors being the students who are presenting or who have been responsible for most of the data shown. I encourage students to revise their work for publication in an undergraduate journal. If data contribute to a professional publication, students who contributed an entire figure or whole panel of data and/or where involved heavily in writing and method development would be authors. Students who were part of a group to help do experiments leading to the data, students who contributed only to a few data points, and students only involved in method development would be acknowledged without authorship.

Resources

1. de los Santos, C., Buldain, G., Frydman, B., Cannata, J. J., & Cazzulo, J. J. (1985). Carbon‐13 nuclear magnetic resonance analysis of [1‐13C] glucose metabolism in Crithidia fasciculata: Evidence of CO2 fixation by phosphoenolpyruvate carboxykinase. European Journal of Biochemistry, 149(2), 421-429. The experiments I did are based on this paper and students compare our data with published data.
2. Giles, B. J., Matsche, Z., Egeland, R. D., Reed, R. A., Morioka, S. S., & Taber, R. L. (1999). An in vivo 13C NMR analysis of the anaerobic yeast metabolism of 1-13C-glucose. Journal of chemical education, 76(11), 1564. This is the experiment I read first and decided to try in Crithidia. This reference is helpful for faculty to learn more about the pedagogical background. It is not necessary for students to read.

People to contact:
3. Megan Povelones at Villanova University is interested in mitochondrial biogenesis in Crithidia and studying adherent cells. Old site with protocols: https://sites.psu.edu/bwtryps/crithidia-protocols/ This is her new webpage at Villanova:https://www1.villanova.edu/villanova/artsci/biology/facstaff/biodetail.html?mail=megan.povelones@villanova.edu&xsl=bio_long 
4. Paul Ulrich at Georgia State University has been leading Crithidia CUREs for years. Here is his page: https://cas.gsu.edu/profile/paul-ulrich/
5. Swati Agrawal at University of Mary Washington also works with Crithidia and is a collaborator: https://cas.umw.edu/biology/swati-agrawal-ph-d/