Characterizing the Aging Process Using Caenorhabditis elegans and Reverse Genetics

Designed by Joslyn Mills, PhD, Brown University
Co-instructed with Louis Lapierre, PhD, Brown University
Location: Rhode Island

Context

To improve learning and immerse up to 24 students into scientific inquiry, we have designed a semester-long research project using C. elegans and RNAi libraries to identify the genetic modifiers that impact organismal aging. We aim to improve students' retention of concepts, their problem solving skills, and their capacity to evaluate science and scientific claims in everyday life. Our course aims to be more inclusive than one-on-one internships by being open to all Biology students. We aim to support their technical and conceptual learning, provide them an in-depth introduction to STEM fields, and increase their retention in STEM fields, mimicking multiple benefits of individual research internships.Finally, we want our course to have the flexibility to be modified for high school students, which would allow for earlier intervention to encourage interest in and understanding of biological research. We have a unique opportunity to present a modified version of our course as a CRE during the Summer@Brown program as a four-week course taught to pre-college students, which has been successful with minor adjustments to support high school-level learning.

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

CURE Design

Our working hypothesis is that RNAi screening will produce a number of genes that would be of interest to the aging field. The students take ownership of their project by selecting which genes they investigate more deeply, possibly determining the mechanism or pathway in which the protein is involved. Furthermore, we anticipate many targets will be identified, but only a handful can be followed up on during a single semester. Therefore, the same approach can be used for discovery over many cycles.

Students are assigned to groups to write and execute their proposals in a collaborative manner. Each group has a common goal to complete their project within the semester and present their findings as a group presentation or as a poster during the University's CURE fair. Throughout the semester, each group is required to host a "journal club," leading a discussion of a primary research article of their choosing that is related to their project, with a subsequent "lab meeting," which is a progress report of their project to that point. This journal club/lab meeting presentation is meant to be both practice for their final presentation and also an opportunity to share their work with their classmates, which might inspire new ideas or even open collaborative communication between groups if relevant.

The research theme of this course stems from the Lapierre lab at Brown University; however, the findings from the course have potential for impacts that reach into both the aging and the C. elegans ("worm") communities. Each student is required to write a final report, designed as an assessment to determine individual comprehension. When appropriate, each group also has the option to prepare a report that could be submitted to the C. elegans microPubs Biology Journal (https://www.micropublication.org/) to share with the worm community.

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

Tasks that Align Student and Research Goals

Instructional Materials

Sample Syllabus for a full semester for sophomore to senior undergraduate level: C elegans CURE Sample Syllabus.pdf (Acrobat (PDF) 114kB Aug31 21)

Assignments to be graded using a Rubric: Grant Proposal (with draft), Presentation, Final Report, Lab notebook

Additional assignments: annotated bibliographies, progress reports (participation in discussion boards and lab meetings included here),

Assessment

Assignments are designed to build upon themselves through the semester (formative) to support the submission of a large final (summative) assignment. For example, the general annotated bibliography includes four pre-selected articles for the students to read and annotate. Then they are asked to find their own relevant articles and again create an annotated bibliography. The group uses the literature to develop a hypothesis and experiments that they use to write a proposal (see below under "Group Proposal" description for the step-wise approach for this assignment.) After performing the experiments, they analyze the results and put them into figures, which are then used for their final presentations and reports.

Below are the instructions given to the students to complete the graded assignments.

Individual assignments

Worm Olympics Quiz (15 points): 

Multiple choice and matching questions to test the general C. elegans knowledge (husbandry, developmental stages,

General Annotated Bibliography (25 pts)

Instructions: An annotated bibliography is a list of citations for papers relevant to a particular topic accompanied by a summary (annotation) of each paper. More information about annotated bibliographies. (Links to an external site.)
Task:
Read the following four journal articles
● Fire, et al. 1998: 1998 Fire...Mello RNAi in C. elegans.pdf (Acrobat (PDF) 593kB Aug31 21)
● Lopez-Otin, et al. 2013: 2013 Lopez-Otin Hallmarks of Aging.pdf (Acrobat (PDF) 3.8MB Aug31 21)
● Kern, et al. 2018: 2018 Kern Chrom I RNAi screen Proteostasis.pdf (Acrobat (PDF) 1.1MB Aug31 21)
● Mack, et al. 2018: 2018 Mack, Heimbucher, Murphy C elegans aging model.pdf (Acrobat (PDF) 780kB Aug31 21)
Write a short paragraph summarizing the main idea(s), the conclusion, and possible methods you may use in the course for each paper. You may annotate as bullet points instead of a paragraph if that format will be easier for you to refer back to.

Primer Design (10 points)

Follow the instruction in the attached file to design primers for each of your genes of interest (top 5 hits from your screen.): Designing Primers for Worm Genes (Updated).pdf (Acrobat (PDF) 1.9MB Aug31 21)

These primers will be used next week for RT-qPCR to confirm knockdown of the targeted gene in the treated worms.

Your submission must include the gene name, the forward primer, the reverse primer, the Tm, and the size of the product for each primer set (all of these are listed in your primer search results.)

For example:

Gene: lmp-1

Forward: 5'-ATGTTCAGGTCGTCTCAAGTTC-3'

Reverse: 5'-TTGTCCGAGCTCCTTAATCC-3'

Tm: 61C

Product size: 325 bp

RNAi Sequencing Analysis (10 points)

You will receive the sequence from your RNAi plasmid from GeneWiz sometime this week.  To confirm that your sequence is specifically targeting your hit gene, you will use the NCBI Blast tool found at the link below. You will analyze all 5 hits you selected last week, but pay special attention to the 1 or 2 your group discussed with us on Tuesday during the breakout sessions.

NCBI BLASTn (Links to an external site.)

Copy and paste your sequence into the "Query" box at the top of the page, and then click the "BLAST" button at the bottom of the page (you can also fill the "Organism" filter with "Caenorhabditis elegans (taxid:6239)" to more define the parameters.)

After a few moments (be patient, it can sometimes take a few minutes to get results), a new page will open with a list of genes that have a match to your sequence. Record the top 5 C. elegans genes from this list, as well as the Query Cover (you want this to be >90% for your hit), E value (0 is best), and Percent Identical (you want this to be near 100% for your hit) for each.

If you see your gene name within the first couple selections, that is good news. You can click on that result and it will show you how your sequence from GeneWiz matches up with the known gene in C. elegans (your alignment). If your gene is not listed, then your RNAi is not targeting what you thought it was targeting. If you see more than one C. elegans gene in your list with a high Percent Identical, then your RNAi could be targeting more than just the gene you are interested in.

Your submission must include:

1) The top 5 results and their values you recorded from above.

2) It would be helpful if you can include a screenshot of your alignment with your gene of interest (click your gene from the results to show the sequence alignments, plus the numbers at the top.)

3) Your evaluation if your RNAi is specific for your gene of interest

RT-qPCR Analysis (10 points)

*Due date may change depending on when we can get the data to you*

You will receive raw data (concentrations of mRNA) and the amplification curve of your RT-qPCR results of your strain on the negative control (L4440) and your hit(s) RNAi. Each set will use the primer pairs you designed for your hit (or edited primers based on feedback). You will analyze this data and determine if the results are as you would expect.

This experimental procedure, data, and analysis should all go in your lab notebook too!

Your submission will include a response to the following questions for each of your hits that was analyzed:

1) If the RNAi is working as expected, do you expect to see a decrease or an increase of targeted mRNA in your worms grown on your RNAi compared to the negative control? (You only have to answer this question once.)

2) Design a bar graph to compare your control to your RNAi worms. This will entail taking the average of each treatment and plotting these values with the x-axis labeling control or RNAi (use your gene name + i, ie: lmp-2i) and the relative mRNA level on the y-axis (no units needed in this case.) Include the standard deviation (that you calculate) for each bar if your software allows. Each gene hit will have its own graph. (Include example of the graph.)

3) Did you get the expected result? If not, what do you think could have gone wrong?

Progress Reports & Participation (2x 50 pts)

#1 Due after first month: The purpose of this assignment is to review the proposed experiments you and your group have performed until now, what your next steps will be, and to gain an individual perspective of how the process is playing out.

Please respond to the following questions in one or two paragraphs (less than 1000 words):

1. What have you (yourself, not group) accomplished since your screen? This can include experimental design or execution, literature review and contribution to your proposal, and any outside-of-class effort you have put in.
2. What is the extent of your contribution to the progress of your group?
3. Are you happy with the progress you have personally made regarding 1) foundational knowledge about your strain and genes and experimental techniques, 2) understanding of how to use certain methods to answer specific questions, and 3) performing benchtop techniques?
4. Do you believe everyone in your group is contributing equally? If not, in what ways would you like us (professors) to intervene?
5. What challenges were encountered, if any?
6. What have you done to address the challenges? What do you need help with?
7. What do you and your group plan to accomplish by the next progress report (Nov. 16)?

Your contribution to the Specific Aims peer review discussion will also be acknowledged in this assignment.

#2 Due after the second month: The purpose of this assignment is to review the proposed experiments you and your group have performed since your first progress report, what your next  and final steps will be, and to gain an individual perspective of how the process has played.

Please respond to the following questions in one or two paragraphs (less than 1000 words):

1. What have you (yourself, not group) accomplished since your last progress report? This can include experimental design or execution, literature review, contribution to your journal club, and any outside-of-class effort you have put in.
2. What is the extent of your contribution to the progress of your group?  Do you believe you could have worked harder to push your group's progress faster?
3. Do you believe everyone in your group is contributing equally? If not, in what ways would you like us (professors) to intervene?
4. What challenges were encountered since your last report, if any?
5. What have you done to address the challenges? What do you need help with?
6. What experiments remain to be done before the end of the in-person lab portion?

Your contribution to the Group Journal Club presentations will also be acknowledged in this assignment.

Lab notebook (100pts)

Your lab notebook will be a live document stored in the Google drive that you will update with each experiment. It will be due at the end of the in-lab portion of the course, but do not wait until then to complete each entry. Make it easy on yourself and record as you perform!

Find the folder with your name and create a Google Doc with your first initial and last name as the title within your Google folder. Here you will record the protocols performed, the results gathered, your interpretation of results, and next steps. Even though you will not be performing the actual experiments in the beginning, we ask that you keep track of what the scientists did in your notebook.

A sample of a lab notebook entry is included in the Google file as "A. Sample's Lab Notebook"

Your Lab Notebook entries should include the following (point breakdown):

Table of Contents: List of page numbers on the first page for important experiments/data (5)

Overall Completeness- spot checked: Can someone repeat your experiments exactly, and confirm the same outcome just by using your notebook? (15.4)

Each Entry Must Include:

Date (6)

Objective: A 1-2 sentence description of what your objective was that day. e.g. "Chunking and bleaching worms" (6)

Samples: Worm strain, RNAi, date samples were prepared, what technique was used to obtain them. Indicate if sample is DNA or protein (6)

Protocols performed: All reagents used and any alterations to the protocols/unique steps your group underwent. Someone should be able to repeat the experiment exactly as you have (15)

Measurements and observations: All concentrations, instrument readouts, and physical observations.  (12)

Data and results: Paste in any images or graphs and label them appropriately. All calculations should be included. Include figures of results that will be used in your presentation or report (15)

Summary and conclusion: 1-2 sentence summary of the overall goal of the experiment. Indicate what conclusions you can thus draw from your findings. (10.8)

Next Steps: Indication of what the next steps are for your samples (8.8)

Final report (200 pts)

Your final report is an individual assignment. This will be used to evaluate your understanding of your project and your own interpretation of the results in regard to your hypothesis.  Have fun with it! If you think something different than the rest of your group, here is where you can explain your understanding.

You will want to start the process of writing early; we recommend about 4 weeks before it is due. As you write, you can submit individual sections to the instructors to get feedback. Use the following timeline to help you plan out your writing process.  Please note that we (the instructors) will only be giving feedback on individual sections with deadlines outlined in the syllabus and in the timeline file. We WILL NOT give feedback on a full report within the last week before it is due, so please plan accordingly.

Final report timeline: BIOL 0600 Final Report Timeline.pdf (Acrobat (PDF) 159kB Aug31 21)

The attached file might be helpful to guide your writing: Final Report Guidelines CURE 2020.pdf (Acrobat (PDF) 255kB Aug31 21)

Final Report Rubric: Final Report Rubric BIOL0600 F20.pdf (Acrobat (PDF) 157kB Aug31 21)

Group assignments

Specific Annotated Bibliography (30 pts)

For this part of your annotated bibliography, you will be working with your group to collect your own articles that are specific to your protein of interest (the tagged protein expressed in your worm strain) and your gene of interest (the hit(s) you identified in your screen.) There will be one submission for each group.

Minimum requirements: At least 2 articles each for your protein and each gene (your 2 favorites from your original list of 5), with no more than one of them being a review each. For example, you may only have a review for your protein, a review for Hit #1, and a review for Hit #2; all the other articles must be primary literature.  Your primary articles must have explicit information about your protein or gene (it cannot be a part of a general screen that doesn't suggest a biological role.)

Please specify next to the article title which protein/gene it is for and if it is a primary article or a review.

Include in your summary:

• Describe the research question or problem addressed in this paper (primary lit)
• List 3 main findings, conclusions, or main points
• State the relevance of these conclusions to your study
• State the relevance of these conclusions to the field
• Identify potential methods you would like to perform to answer your own research questions

More articles are welcomed and encouraged!  This bibliography will be useful for writing your proposals and final reports so you can remember where you read relevant information. You may continue to add more articles during the semester, but you must submit the minimum by the due date.

Written proposal (100 pts)

This assignment has several steps for feedback before submitting a final proposal.  First, the group is to fill out a logic tree (Proposal Logic Tree 2021.pdf (Acrobat (PDF) 162kB Aug31 21)) outlining the basics of their proposal. and then meet with the instructors to discuss accuracy and implementation strategies. Next, the group writes their specific aims and shares them with the rest of the class on a discussion board, where they will receive peer feedback based on . Finally, they will incorporate peer and instructor feedback to submit their proposal. Because this is a difficult assignment near the beginning of the course, I offer the opportunity for groups to resubmit an improved proposal to earn up to half the points missed if they are not satisfied with their grade on the first submission.

Instructions to students: For this assignment, you will expand on your Specific Aims to write a whole proposal. Your first page will be your specific aims that your group has edited after receiving feedback from your peers (your preliminary results will be shifted into the body of the proposal- see attached file.) The rest of the proposal includes a more extensive background, elaboration on each aim, and expected results from your proposed experiments.

Use the attached template to guide you. Please limit your proposal to 5 pages or less (not including your table of preliminary results and references.): Proposal Assignment Description CRE 2020.docx (Microsoft Word 2007 (.docx) 15kB Aug31 21)

***Differences between your SA assignment and your Proposal***

TL;DR version: Shorten your Specific Aims to one page, and put the details you took out into the body of the proposal.

Long version:
Your specific aims is the first page of your proposal. This is the first thing a grant reviewer sees and might be the deciding factor if they want to put a lot of effort into reading the rest of your proposal. This means you have to get your points across succinctly but still get the reader interested: What is known in the field, what isn't known yet, why is it important to know, and how are you going to figure it out. A short background about your protein, gene (hit), and your novel idea of how they affect each other (your hypothesis) is needed to set up your aims and subaims. The aim descriptions themselves shouldn't be too detailed either; just a quick sentence or two describing what experiments will be done to answer the aim.

The details are in the rest of the proposal. This is where you extend the background about your protein and gene (there might be some repetition from your Specific Aims page, but only if it is to elaborate on the point). You still want to make sure what you include is relevant to what you are proposing (for instance, you don't need to say the gene has been found to have a role in leukemia when you aren't proposing a cancer or blood based project.) You can also elaborate on the rationale behind your hypothesis here. Further, you will be more specific about your methods and approaches (you plan to do qPCR and Western blotting? Then describe specifically what targets you will look at for each experiment and why.) You are also expected to include predicted results, and you need to provide rationale for those as well, and also why you may get a different result.

All of these points show the reader that you have thought through your proposed experiments and determined them to be worth doing (and to get grant money to pay for it!) So keep these point in mind as you are writing each section of your proposal.

Rubric for final proposal: Proposal_Rubric BIOL0600.pdf (Acrobat (PDF) 155kB Aug31 21)

Final presentation (200 pts)

This was done in an online, asynchronous format; however, a poster presentation is another option.

***If we are unable to do live presentations via Zoom, your group presentations will be a recorded video that you will upload through this Assignment window.  We will add your video to the Google Drive (or you can directly upload it) so everyone can access and watch it.  Each presentation will have an associated Google Doc where the audience can post questions and comments, and the presenters will respond the following day.

You can use Zoom to record your presentation unless you have a different preferred recording app. Everyone will be expected to participate, both in design and presentation. At the beginning of your video, please have everyone's video on so you can introduce yourselves before moving into the presentation, and you can turn them off once you start.  If there are any technical issues, we will address them as they occur, so please start on this early.

Use the attached file to guide your presentation design. You may choose to do a PowerPoint (or similar slide show) presentation or a poster.

Presentation Guidelines: CURE Presentations Guidelines F2020.pdf (Acrobat (PDF) 342kB Aug31 21)

Point breakdown: Title Slide (20), Intro/background slides (35), Hypothesis slide (10), Results slides (60), Discussion slides (25), Presentation (35),  Questions & Responses (15)

Instructional Staffing

We have been successful teaching this course with 2 co-instructors and an Undergraduate Teaching Assistant (UTA) for each group of 3 or 4 students (3 UTAs for a class of 12)

UTAs are assigned to their own group that they will assist over the semester, with flexibility to assist other groups when needed. UTAs are trained to handle C. elegans, make and seed plates for husbandry and experiments, and assist with other experiments as needed.

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Head shot of Joslyn Mills

Provenance: Joslyn Mills, Brown University
Reuse: This item is offered under a Creative Commons Attribution-NonCommercial-ShareAlike license http://creativecommons.org/licenses/by-nc-sa/3.0/ You may reuse this item for non-commercial purposes as long as you provide attribution and offer any derivative works under a similar license.

Author Experience

Joslyn Mills, Brown University

Hands-on learning is essential for students to fully grasp complicated concepts, but a single exposure to a technique or idea during a semester is usually not enough to achieve long-term retention. Through the iteration of a number of techniques, students become proficient at the bench while also understanding the theoretical concepts behind the technique. Furthermore, the availability of research laboratories at Brown University that can accommodate a student to meet their research requirement for their degree is lacking, and these opportunities have become increasingly competitive for students. For this reason, students with prior experience in high school are typically favored, thereby limiting inclusivity of undergraduate research and diversity of undergraduate researchers. I have designed my CURE to help students gain hands-on laboratory and project design experience needed to become more competitive to join a research lab for their eventual thesis work, particularly for those students that have not had the advantage of working in a lab before. Therefore, the long-term goal of this course is to help many students from a variety of backgrounds meet their research requirement in support of the academic programs and associated labs and prepare them for future exciting STEM careers.

Advice for Implementation

The most successful execution of this CURE course has used a flipped classroom approach, splitting the semester into weekly or biweekly modules that include lecture videos students are required to watch before coming to class. This allows the optimal use of time at the bench to perform experiments.

I use a six module setup

• Week 1/Module 1:  Introduction to Caenorhabditis elegans and RNA interference
  • Lecture videos include Model Organism intro, Why C. elegans?, Worm Assays and Experimental Methods, and Information the strains we will use in the course
  • Assignments include the Worm Olympics Quiz, General Annotated Bibliography, and a Student Introduction Discussion Board
  • The instructors lead a journal club of two of the papers from their general annotated bibliography in a live classroom session
• Week 2/Module 2: The Reverse Genetics Screen
  • Lecture videos include Intro to RNAi screens and the Central Dogma refresher
  • Assignments include Screen Results (begin using their online lab notebook), Strain Ranking Discussion (to help form groups based on strain), Primer design, and Group Specific Annotated Bibliography.
  • Groups begin completing their logic tree to prepare for meeting with the instructors
  • The instructors lead a journal club of the other two papers from their general annotated bibliography in a live classroom session
• Week 3/Module 3: The Research Direction
  • Lecture videos include Confirmatory Experiments (reproducibility and robustness, confirm sequence of RNAi and knockdown of expected gene) and "Technique Toolbox #1" (describes worm assays the students might consider for their proposal.)
  • Assignments include RNAi sequencing analysis (after they have mini-prepped their bacteria that express the RNAi) and Specific Aims Discussion Board with peer feedback
• Week 4-5/Module 4: The Proposal
  • Lecture Videos include "Technique Toolbox #2" (RT-qPCR and Western blot)
  • Assignments include RT-qPCR analysis and Group Proposal
• Week 6-8/Module 5: Back to the Bench
  • Mainly benchwork and analysis of incoming results over these weeks
  • Assignments include Progress Report #1 and Results figure design
• Week 9-13/Module 6: Data Presentations
  • Finishing benchwork
  • Suggest students start working on their final reports, and have them submit sections for feedback over a course of a few weeks (see Timeline under Final Report description in Assessments)
  • Each group hosts a journal club of a relevant paper of their choosing with a subsequent lab meeting to present their project and progress
  • Assignments include Progress Report #2, Group Presentation, and Final Report

Iteration

This CURE has been executed in traditional (fully in person), hybrid (some in person aspects with some online work), and fully online and asynchronous formats. The main difference with the online course is that someone (usually the instructor) performs the proposed experiments in the lab and then shares the results with the students for their analysis, since the students are not on site. To accommodate the missing hands-on experience, the course is supplemented with technique videos or "lab look-ins" to give the students a sense of what the experiments look like as they are being done.

Using CURE Data

This course content is original and based on research done in Dr. Louis Lapierre's lab at Brown University. To develop this idea into a genuine CURE course, my instruction came from the Sheridan Teaching Center at Brown University (supported by HHMI), the CUREnet Institute at CCRI, and the listed resources below. Brown University's Sheridan Center has collected CURE-specific data from two iterations of this course, along with other CUREs developed at Brown University, and may be available upon request.

Resources

Cooper 2017 Backwards Design.pdf (Acrobat (PDF) 603kB Sep1 21)

CUREs_Learning Outcomes Draft.pdf (Acrobat (PDF) 155kB Sep1 21)

goal_conflicts and solutions.pdf (Acrobat (PDF) 4.2MB Sep1 21)

researchandstudentgoals_conflicts and solutions.pdf (Acrobat (PDF) 5.2MB Sep1 21)1_introandwhycures_ccri.pdf.pdf (Acrobat (PDF) 11.2MB Sep1 21)