C. ELEGANS AS A MODEL ORGANISM
Caenorhabditis elegans (C. elegans) is a small, free-living, nematode worm. C. elegans are organisms that most resemble human genes and biomolecular functions. Over time C. elegans has become a model organism in academia for studying gene manipulation and human reactions to disease. It is the first multicellular organism for which scientists have been able to sequence its whole genome and is as widely used as drosophila melanogaster, or more commonly known as the fruit fly.
PHASE ZERO: Prototyping
Phase Zero of the C. elegans BioCrowd Kit includes the design of the required items in the kit as well as troubleshooting any issues that might occur during production, package transport, initial experimentation, and data extraction.
Phase Zero Goals: Time length of set up, experimentation, and data collection. Issues and constructive criticism that arose with setup, experimentation and disposal, and data extraction.
C. Elegans Experimentation
Experiments will be carried out with the wild-type strain (N2) during the prototyping phase. Given that mutants will not be used, experiments will not include genotype-phenotype classification. The student using the kit will carry out three experiments.
- The first experiment consists of obtaining the kinematics of worms using video recording. Then, common worm behaviors can be identified.
- The second experiment consists of using sodium chloride solutions of different concentrations to perform a chemotaxis assay. The bottom of the NGM plates should be marked into 4 equal quadrants. The student will label diagonal quadrants and place droplets of each solution. After some time (typically 1 hour in chemotaxis assays), note where the majority of the worm population is located.
- The third experiment consists of observing how the worms move within the agar (e.g. burrowed) instead of the agar-air interface. The student should puncture multiple holes in the agar and see if worms burrow into the agar. Once a worm is burrowed, the student can record the kinematics of the worms using video recording.
C. Elegans BioCrowd Kit Setup
The C. elegans BioCrowd Kit includes a manual that instructs students throughout the lab, C. elegans agar plates with E. coli, plain agar plates, a phone microscope, a Na+ solution with pipettes, and a needle for puncturing the plain agar plates. The phone microscope has multiple parts that must be assembled together before experimentation.
- Set up the LED light and battery on the floor of the microscope
- Choose the magnification lens and insert the slide
- Apply the calibration sticker accurately to both sides of the microscope for the height adjustment
- Insert the acrylic plate holder into the plate clips and screw the wing nuts onto the clips through the height adjustment slots. Make sure both sides are level by using the calibration sticker.
- Place the C. Elegan agar plate on the acrylic and begin imaging. Adjust the height accordingly, and record the height and magnification at which imaging is most clear.
C. Elegans Data Analysis
Worms will be tracked using Tierpsy Tracker and WormLab 2.0, both of which are open-source trackers. Tierpsy Tracker requires python, making it available without a license to external software. Worm midlines will be extracted and post-processed externally as necessary.
PHASE ONE: Phenotype Experimentation
Once students receive the C. elegans BioCrowd Kit, they will be able to begin the Research Workshop. The day of the workshop consists of set up, experimentation, collecting image data, and worm kinematics extraction. Students will first assemble a miniature lab set up according to the directions given so that imaging can be done hours at a time.
Students will then conduct a series of three experiments on each strain of C. elegans given.
Experiment 1: Worm Locomotion on NGM and Morphological Defects
- Steps: Each participant will be given three different strains of worms with distinct locomotion patterns: N2 (wild type/control), unc-54(uncoordinated omega shape), and rol-9 (circular motion). The goal for the participants will be to do genotype-phenotype classification of the worm strains. The participants, while uninformed of the strain identity, will record the kinematics of each of the worms and by observing the differences in postures and/or behaviors will then identify each worm. A brief description of each mutant will be provided to aid the participants. This will help students become accustomed to worm imaging and identification.
- Extracted data: Participants should extract kinematics via video recordings using their smartphones. Identification of each strain during the workshop will be done qualitatively. Post-processing of the videos will be done to study the behaviors of each of the strains.
Experiment 2: Chemotaxis Assay and Behavioral Affinity
- Steps: Each participant will be given Che-5, a worm strain with sodium affinity. The goal for the participants will be to do another genotype-phenotype classification of the worm strains. Participants will transfer the C. elegans strains onto the plain agar plates by “chunking”. The participants will record the before and after kinematics of each of the worms after adding the different sodium solution concentrations to the worm environment.
- Extracted data: Participants should extract kinematics via video recordings using their smartphones. Overall observations on changes in locomotion and behavior will be done qualitatively. Post-processing of the videos will be done to study the behaviors of each of the strains.
Experiment 3: Burrowing
While this worm has been extensively studied in smooth agar, less is known about the worm’s biomechanics in burrows. Studying how the worms move in burrows can provide insight on how the worms navigate confined environments similar to the ones they traverse in nature (e.g. rotten fruit).
- Steps: Each participant will puncture multiple holes in previously provided NGM plates and see if the worms burrow into the agar. Once a worm is burrowed, the participant can record the kinematics of the worms using video recording. If desired, participants can explore the locomotion of previously provided mutants, and compare them to the wild-type strain. If the participants are unable to get worms to burrow, plates will be provided with worms within the NGM.
- Extracted data: Participants should extract kinematics via video recordings using their smartphones. Overall observations on changes in locomotion and behavior will be done qualitatively. Post-processing of the videos will be done to study the burial locomotion. Notable features to look for include 3D vs 2D locomotion and variations between mutant strain patterns.