An Arabidopsis Mutant Screen CURE for a Cell and Molecular Biology Laboratory Course

Jinjie Liu, Michigan State University
Ron Cook, Michigan State University
Jon R. Stoltzfus, Michigan State University
Christoph Benning, Michigan State University
Location: Michigan

Context

The content of this CURE is part of an active research project from a faculty research laboratory. Students in this CURE sow the mutagenized Arabidopsis seeds, observe the mutants, and characterize the promising mutants, generating novel mutant lines that potentially can be further studied by the research laboratory. As the research proceeds, the research lab can update the students with new research data generated in the lab, demonstrating scientific research is a process of asking questions and using evidence to address the questions, and offering discussion opportunities about the research project. Due to the nature of this CURE, a strong partnership has formed between the research laboratory and the teaching lab, which makes this CURE sustainable and evolvable.

This CURE is designed for an introductory cell and molecular biology laboratory course, with knowledge of genetics and biochemistry integrated into the curriculum, which makes it an interdisciplinary course. Students who enroll in this course need to take math, general chemistry, and cell and molecular biology lecture course before or concurrently. The laboratory size of each section is 28 students at maximum, and the students are grouped into teams of 3 or 4 members. The instructional team for each section includes one instructor and one undergraduate learning assistant. We have implemented the course for four semesters involving about 180 undergraduates at Michigan State University. The class meets for a 3 hour laboratory period and a 50 min recitation the following day weekly during the semester. The student population is half sophomores, a quarter first-years, and the rest are juniors and seniors. This semester-long CURE starts with a knowledge and skills building module for the first four weeks (including sowing the seeds), and continues with a genotype-phenotype module and a lipid analysis module. While each of these modules serves their own purpose, the genotype-phenotype module plays a central role in this CURE.

Target Audience:Introductory
CURE Duration:A full term

CURE Design

The research theme of this CURE is to identify and characterize Arabidopsis mutants, bearing mutations that block the hormone signaling transduction or perception. This research project was built upon the functional description of a plastid lipase gene, PLIP3 (Wang et. al., 2018). The lipase PLIP3 degrades chloroplast lipids. Its over-production in Arabidopsis, a small model plant extensively used in basic plant research, releases fatty acids that are subsequently converted to the bioactive compound JA-Ile (isoleucine), an amino acid conjugate of jasmonic acid (Wang et. al., 2018). Overabundance of JA activates plant defense mechanisms against herbivores, which strongly inhibit plant growth, alter leaf morphology, and lead to increased accumulation of the pigment anthocyanin (Wang et. al., 2018). To identify new genes encoding transporters that are required for the biosynthesis of this hormone, or receptors and signaling components mediating the hormone-initiated responses, the research lab developed a suppressor mutant screen in the PLIP3-overexpressing (PLIP3-OX) plants that can be studied in a laboratory course.

Specifically, seeds of PLIP3-OX plants are randomly mutated by the research lab using the mutagen ethyl methanesulfonate (EMS). The students sow Arabidopsis seeds during the first class; these will be the plants they study in their research later during the semester. The chloroplast lipid signaling project is introduced to the students by showing a research model (can be found in the instructional materials section), and the research question is: what transporters or receptors are involved in the hormone biosynthesis, signaling transduction, or perception processes? Then the students are guided to form a hypothesis and they then predict the desired mutant phenotypes. When the plants are about 4-5 weeks, the students observe the phenotypes of the plants, select the desired mutants based on their morphology, and genotype the plant, applying the techniques and knowledge learned during the early stage of the course. This genotyping module connects concepts of genotype and phenotype, genetic information flow, and mutations and protein functions. The students also discuss with their team members how to determine the mutation sites, and what approaches can be employed to answer the questions. A lipid analysis module is included to inspire the curiosity of students given that the overall research project is about lipid signaling and transduction. Inconclusive results offer students an opportunity to think critically and discuss future experiments. As the research progress, we will replace the lipid module with a bioinformatics module targeted at the identification of the mutated gene, which can strengthen aspects of genetic information flow, protein structure and function, and the relationship between genotype and phenotype.

In this CURE, students form teams of 3 to 4 members, and they perform experiments together while each student has their own responsibilities. The students collaborate and share data within their team, and they work together to analyze the data and draw conclusions. Two classroom-wide research presentations are organized during the research stage of the course, at which time students learn from other teams. A final poster session provides a scientific communication opportunity, and a final cumulative exam or an essay based on the claim, evidence, and reasoning framework is used to individually evaluate the students' learning.

This CURE is part of an active research project from Dr. Christoph Benning's lab at Michigan State University, and Ron Cook is the graduate student who works on the research project in the Benning lab. This CURE works in a two-way approach: Mutants identified by the students can be taken back into the research lab for further biochemical analysis and study, and the research lab shares the most recent data with the students to help the students with a better understanding of scientific process.

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, Using mathematics and computational thinking
Nature of Research: Basic Research, Wet Lab/Bench Research

Tasks that Align Student and Research Goals

Instructional Materials

This CURE course is designed in a semi-flipped style. The students study the techniques and the lab protocols and finish the pre-lab before the class. A pre-lab quiz is usually held at the beginning of the lab period to ensure the students' preparation for the class. In-lab lectures are held after the quiz to further explain experimental procedures and how the techniques can be applied to the research questions. Recitation time is used to reflect on the experiment, further introduce research project background, discuss the research project, and other course-related activities.

The CURE mainly covers three major modules: Knowledge and skill-building (Week 1-5), a genotype-phenotype module (Week 6-8), and a lipid analysis module (Week 9-10). While each of these modules serves its purpose, the genotype-phenotype module plays a central role in this CURE. This module ties the knowledge of cell and molecular biology taught in the accompanying lecture course, such as the flow of genetic information, DNA replication, mutations and mutants, genotype and phenotypes, membranes and transport, signal transduction, and cell communication, into this research project and further expands the concepts, such as forward genetics, reverse genetics, primary mutation, and secondary (suppressor) mutation, which are fundamental concepts that help students understand the genetic background of the provided plants. The background knowledge regarding the research project has been interwoven into the course materials throughout the semester to help the students connect the concepts and gain an overall understanding of the research project.

Implementing the CURE, the students started their semester by sowing Arabidopsis seeds and observing seedling development. While the plants developed, the students learned the necessary lab techniques and the background science needed for the project. Students were guided in developing a proposal and design experiments for phenotyping and genotyping to initiate the research process. When the plants were sufficiently grown, the students started the genotype-phenotype module of the course, during which they selected plants with phenotypes indicative of suppressor mutants and designed experiments to genotype them to confirm that the suppressor mutants were in the correct genetic background containing the over-expressing PLIP3 transgene. Furthermore, the students started on a lipid module designed to capture a biochemical feature of the transgenic plants, because overproduction of PLIP3 affects thylakoid membrane lipid composition in subtle ways. Towards this end, they extracted pigment and lipids from the mutant leaves and ran thin-layer chromatography to identify different polar lipid components in the plants. A classroom-wide discussion about future experiments and research interests was also included at the end of the semester.

Because this CURE is tightly linked to an active research project, we constantly update the course materials as we develop new modules or learn more as part of the science, for example, we will be replacing the lipid module with a bioinformatics module. Given the evolving nature of this CURE, it would be best if someone contacts us and asks for help and the most up-to-date materials. We are happy to provide all the course materials upon request.

Course flow chart (TIFF 6.5MB Aug6 21)
Protocol_Quick Lipid Extraction from Arabidopsis (Acrobat (PDF) 108kB Aug12 21)
Protocol_Arabidopsis seed sowing and video demonstration.pdf (Acrobat (PDF) 52kB Aug12 21)

Protocol_PCR.pdf (Acrobat (PDF) 66kB Aug12 21)

Protocol_Thin Layer Chromatography CUREnet.pdf (Acrobat (PDF) 66kB Aug12 21)

Protocol_DNA extraction (Microsoft Word 2007 (.docx) 19kB Aug6 21)

Assessment

Math worksheet (Microsoft Word 2007 (.docx) 31kB Aug6 21)
dna_report.v3.pdf (Acrobat (PDF) 269kB Sep15 21)

Instructional Staffing

• Jinjie Liu: Course instructor and researcher, curriculum design, course materials development, course implementation, course coordination, and teaching assistants training.
• Ron Cook: Graduate student collaborator who leads the research project, demonstrates techniques for video materials, provides newly generated data and discusses details of the research project.
• Linda Danhof: Lab manager in the research lab, mutagenizes the Arabidopsis seeds.
• Jon Stoltzfus: Director of Biological Sciences Program (BioSci) at MSU, initiates this CURE at BioSci, coordinates the course sections, provides the teaching platform, discusses course implementation, guides the direction of CURE.
• Christoph Benning: Principal investigator, works closely with all the personnel involving in the CURE project, guides the research project, advises course design, reviews course materials, and guides the direction of the CURE.
• Lab prep staff: Discuss with the instructor about the lab supplies and staging weekly, order lab supplies, and prepare some of the labs. A weekly prep list can be found in the instructional materials section.
• Undergraduate learning assistants: Assist in class delivering and help to grade some assignments.

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Author Experience

Jinjie Liu, Michigan State University

A CURE project can offer students the opportunity to work on a research project and put their learning into practice. It is a great way to include more undergraduates into science research, foster critical thinking skills, and inspire their interest in scientific research. The Arabidopsis mutant screen project, serving as a crucial component of a chloroplast lipid signaling project in our research laboratory, covers core concepts and techniques of a cell and molecular biology laboratory course making it a great teaching project for a CURE course. We hope this CURE can help undergraduates to attain a deeper understanding and application of fundamental biological concepts and to gain a broader view of the underlying scientific methodology and scientific process; meanwhile, the novel data generated by students can contribute to scientific research.

Advice for Implementation

This CURE emphasizes the concepts of genetic information and genotype-phenotype, leading students to think critically about how to use techniques and knowledge they learned to address research questions. One of the challenges of the CURE project is that there is a lot of knowledge that needs to be introduced while the students have limited time. To succeed, the students not only had to learn the lab techniques and the related underlying science but also had to have a good understanding of the research project. To help with these learning goals, lectures covering these concepts are given during labs and in recitations throughout the semester, in coordination with the practical lab work. After implementing the course for four semesters, below are some of the experiences I like to share, and please do not hesitate to contact me if you have any further questions.

• Communicate with the students weekly: Let the students know what is coming up next, and what needs to be done before they come to the class. A pre-lab quiz is usually held at the beginning of the class, especially during the earlier weeks of the semester, to test the level of preparation for the class.
• Emphasize genetic information flow and explain how introducing a gene into the chromosome can change the genetic information.
• The research project is complex. When introducing the project, guiding the students to look at the big picture connecting the genotype and phenotype, and saving many of the details on the signaling pathway until the end of the semester can be helpful. Emphasizing the genetic information flow, the signaling transduction process (not the details), and the plant response by showing phenotype.
• For seeds sowing: this process occurs during the first class of the semester, and the students will need to use micropipettes to sow the seeds right after they learned how to use a micropipette. We think it is important to have the students well prepared for the class. Communicating with students and ask them to watch the Arabidopsis sowing video we made before coming to the class. We also walk the students through the protocol during class, and if time permits, we watch the video together.
• For DNA extraction: In coping with the disadvantage of not being able to use the typical tissue grinding methods in a teaching laboratory, we choose to use a kit that can break the Arabidopsis tissue by bead beating on a vortexer. The kit has many steps and has several different columns to use during the DNA extraction process, which means the students need to be very mindful about which samples to keep or discard at different steps. It is necessary to lead the class step by step and let the students know ahead of time that they need to FOLLOW the instruction step by step.
• For PCR and agarose gel: Introduce the database and guide the students to search the genes, the primers, and then check the amplicon sizes. This can help students better understand the different forms of the gene (genotype), which will show differently on the agarose gel as different sizes of bands.
• For the lipid module: As progress is made in the research laboratory, we are going to replace this module with a bioinformatics module emphasizing the concepts related to genotype and phenotype, protein structure and function.

Equipment and supply:

• Arabidopsis seeds (wild type, overexpression line, and EMS mutagenesis lines), pots, tray, soil, growth shelves, growth chamber; cold room or 4°C fridge to vernalize seeds after planting.
• Basic molecular biology equipment: micropipettors, benchtop centrifuge, vortex and adapter, water bath, PCR machine, electrophoresis tank and power supply, and refrigerator. Please refer to the "Plant CURE Equipment & Supply_weekly prep list" for more details on a weekly lab preparation basis.
• The lipid module needs some organic solvents, TLC silicon plate, and TLC tanks, with the progress made in the research laboratory, this module will be replaced by a bioinformatics module in Fall 2021, in which case concepts of the genetic information flow and genotype-phenotype will be further emphasized.

Iteration

"Failing" is part of learning, and sometimes we learn more by failing. I always tell my students that "failing" is part of the science, it is common that experiments do not go as planned, what is important is that we learn from these experiences so we can do better next time. I also mention that because we will generate novel data from this research project, it is upon us to characterize the mutants and we need to solve the problems when things did not work as planned.

At the knowledge building stage, students practice the techniques, reflect on the result or troubleshooting. For example, after DNA extraction, the students need to reflect on which factors can affect the quantity and quality of DNA during the process, and what could go wrong if there is no DNA obtained; For PCR and agarose gel electrophoresis, the students need to troubleshooting as well. These reflective processes can help the students to raise awareness and be prepared for the research project.

Structurally, there are two recitation classes following the genotyping labs are reserved for students to repeat the experiments if the experiments did not work. Students need to reflect on the process (on their own at this stage) and repeat the experiments during the reserved time.

Using CURE Data

The research results represent novel data relevant to the plant lipid and plant hormone research fields. Promising mutants are brought back to the research lab for further analysis. The students provide preliminary data for an NSF grant proposal and the CURE activities will be integral to the broader impact component of this NSF grant proposal that is going to be submitted soon.

Resources

References for instructors:
1. Wang, K., Guo, Q., Froehlich, J. E., Hersh, H. L., Zienkiewicz, A., Howe, G. A., & Benning, C. (2018). Two Abscisic Acid-Responsive Plastid Lipase Genes Involved in Jasmonic Acid Biosynthesis in Arabidopsis thaliana. Plant Cell, 30(5), 1006-1022. doi:10.1105/tpc.18.00250.
2. Liu J, Cook R, Danhof L, Lopatto D, Stoltzfus JR, Benning C (2021). Connecting research and teaching using an Arabidopsis suppressor mutant screen in an Introductory Course-based Undergraduate Research Experience. Biochemistry and Molecular Biology Education (under revision).
3. Wang, Z., & Benning, C. (2011). Arabidopsis thaliana polar glycerolipid profiling by thin layer chromatography (TLC) coupled with gas-liquid chromatography (GLC). J Vis Exp, e2518, doi:10.3791/2518. 
4. Chloroplast lipid synthesis and lipid trafficking through ER-plastid membrane contact sites. Biochem Soc Trans, 40(2), 457-463. doi:10.1042/BST20110752. 
5. Avanci, N. C., Luche, D. D., Goldman, G. H., & Goldman, M. H. (2010). Jasmonates are phytohormones with multiple functions, including plant defense and reproduction. Genet Mol Res, 9(1), 484-505. doi:10.4238/vol9-1gmr754.
6. Benning, C. (2009). Mechanisms of lipid transport involved in organelle biogenesis in plant cells. Annu Rev Cell Dev Biol, 25, 71-91. doi:10.1146/annurev.cellbio.042308.113414
7. Luz Rivero, R. S., Nicholas Holomuzki, Deborah Crist, Erich Grotewold, and Jelena Brkljacic. (2014). Handling Arabidopsis Plants: Growth, Preservation of Seeds, Transformation, and Genetic Crosses. Methods in Molecular Biology. doi:10.1007/978-1-62703-580-4
8. Morris, J., Hart, D., A., K., Lue, R., Michael, M., Berry, A., Biewener, A., Farrell, B., Holbrook, N.M., Heitz, J., Hens, M., Merrill, J., Phillis, R., Pires, D., Lozovsky, E., and Liu, J. (2019). Biology: How Life Works. ISSN: 9781319017637. Macmillanlearning.com.
9. Nguyen, C. T., Martinoia, E., & Farmer, E. E. (2017). Emerging Jasmonate Transporters. Mol Plant, 10(5), 659-661. doi:10.1016/j.molp.2017.03.007. 
10. Qu, L., Qin, G. (2014). Generation and Identification of Arabidopsis EMS Mutants. Methods in Molecular Biology, 225-229. doi:10.1007/978-1-62703-580-4. 
11. Wasternack, C., & Hause, B. (2013). Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth and development. An update to the 2007 review in Annals of Botany. Ann Bot, 111(6), 1021-1058. doi:10.1093/aob/mct067.

References for students:
1. Wang, K., Guo, Q., Froehlich, J. E., Hersh, H. L., Zienkiewicz, A., Howe, G. A., & Benning, C. (2018). Two Abscisic Acid-Responsive Plastid Lipase Genes Involved in Jasmonic Acid Biosynthesis in Arabidopsis thaliana. Plant Cell, 30(5), 1006-1022. doi:10.1105/tpc.18.00250.
2. Liu J, Cook R, Danhof L, Lopatto D, Stoltzfus JR, Benning C (2021). Connecting research and teaching using an Arabidopsis suppressor mutant screen in an Introductory Course-based Undergraduate Research Experience. Biochemistry and Molecular Biology Education (under revision).
3. Morris, J., Hart, D., A., K., Lue, R., Michael, M., Berry, A., Biewener, A., Farrell, B., Holbrook, N.M., Heitz, J., Hens, M., Merrill, J., Phillis, R., Pires, D., Lozovsky, E., and Liu, J. (2019). Biology: How Life Works. ISSN: 9781319017637. Macmillanlearning.com.
4. Wang, Z., & Benning, C. (2011). Arabidopsis thaliana polar glycerolipid profiling by thin layer chromatography (TLC) coupled with gas-liquid chromatography (GLC). J Vis Exp, e2518, doi:10.3791/2518. 
5. Wachsman, G., Modliszewski, J. L., Valdes, M., & Benfey, P. N. (2017). A SIMPLE Pipeline for Mapping Point Mutations. Plant Physiol, 174(3), 1307-1313. doi:10.1104/pp.17.00415.