Week 3 Tetrahymena Experiment

1. Week 3Tetrahymena Experiment Student researcher images

2. Overexpression: An example in muscle A model for the role of myostatin during muscle growth and differentiation. Myotubes Myoblasts Precursor cells Differentiation MyoD Proliferation Myf5 Myogenin Myostatin MyoD Image by Lina Dahlberg, adapted from Langley, et al., Journal of Biological Chemistry, 2002

3. Overexpression: An example A model for the role of myostatin during muscle growth and differentiation. Myoblasts Precursor cells Differentiation MyoD Proliferation Myf5 Myogenin Myostatin MyoD ? Image by Lina Dahlberg, adapted from Langley, et al., Journal of Biological Chemistry, 2002

4. Under-expression in Myostatinresult in overexpression of MyoD and increased muscle mass • Examples from selective breeding include the Belgian Blue breed of cattle and the Bully Whippet breed of dogs

5. Overexpressing Cdt1 • We are using cells that have additional copies of DNA to promote overexpression of our gene of interest: CDT1 MTT1 promoter in presence of heavy metals, like Cadmium, is activated MTT1 Promoter MTT1 DNA engineered to contain Cdt1 downstream of MTT1 promoter MTT1 Promoter Cdt1 When Cadmium is present the gene is turned on. When Cadmium is absent the gene is turned off. Image by Lina Dahlberg

6. Our conditions for this lab: • WT +CdCl2 • O/E Cdt1 – CdCl2 • O/E Cdt1 + CdCl2 As [CdCl2] increases so does gene expression! Shang, et al., 2002., PNAS  3734–3739, doi: 10.1073/pnas.052016199 What do you expect levels of CDT1 expression to be in WT? In O/E CDT1 without CdCl2?

7. OE of CDT1 in Tetrahymena Image by Lina Dahlberg, adapted from MBoC, 6th ed. Alberts, et al.

8. Hypothesis Building • First, come up with a specific research question you wish to answer… • A hypothesis is a statement, not a question. It is a prediction of what will happen • Keep the variables in mind (what variables are we interested in?) • Your hypothesis should be an “educated prediction” rooted in scientific understanding • Important: make sure your hypothesis is specifically “Testable”

9. Example from a Grad TA’s experience: • Question: Do logs put on ground reduce the evaporation demand on plants? • We hypothesizethat logs significantly decrease the evaporative demand nearby compared to far away from logs • Rationale: logs block wind and provide shade. Decreasing wind and reducing solar radiation are known to decrease evaporative demand (Rosenberg et al. 1983).

10. Overview of our Question • How does overexpression of CDT1 affect the phenotype of Tetrahymena? • Narrow it down into testable questions: • How will overexpression of CDT1 affect the number of viable cells/ml compared to WT? • How will overexpression of CDT1 affect cell length, cell width, and nuclear diameter compared to WT?

11. In Pairs… • Choose TWO conditions to test your hypothesis • Why did you choose them? • Research questions: • How will your chosen conditions affect the number of viable cells/ml? • How will your chosen conditions affect cell length, cell width, and nuclear diameter? • Generate a hypotheses for each question • Generate a detailed protocol to test the hypotheses using the tools we learned last week • You cannot reuse measurements taken last week • After 10 minutes share your protocol with the other lab group, comment, critique, and come up with a plan to optimize lab time and equipment • Turn in at end of class "If overexpression does this _____, then _____[this]_____ will happen." “We can test [this]by _____________________”

12. Class Data from last week:(Wednesday) Woah! WT WT Fixed Cell measurements WT Fixed cell counts (individual data points) (average of averages)

13. End Goal- fill out data entry, and create awesome figures with stellar captions Living Cells Fixed Cells Figure 1: Ratio of nuclear diameter to cell length… Figure 2: Measurements of Tetrahymena cells…. Tables are found in the lab manual

14. Dispersing Concentration of Cells • Cells sink fast! Fixed cells even faster! • Cells will clump! Make sure they un-clump • Look into the light to make sure cells looks evenly dispersed • May take a bit to get them mixed well • IMMEDIATELY take sample from tube • Let’s practice flicking the microfuge tubes

15. Hazards • Cadmium chloride is an acute and chronic toxin as well as a human carcinogen. • Harmful if inhaled, swallowed or in contact with the skin. Can also be fatal if inhaled or ingested.  Exposure can cause damage to the respiratory tract, kidneys, and liver.  • Students will be working with a dilute solution (1-2 ppm, 1ug/ml), which essentially eliminates the high hazard that are associated with powder. • None the less, students are required to follow basic safety precautions when handling this solution, including wearing disposable nitrile gloves and washing hands when gloves are removed be familiar with emergency spill procedures. • Cadmium is very toxic to aquatic organisms and long-term effects in aquatic environments, do not empty into drains. • Collect as hazardous waste all disposable items such as slides, pipet tips, Kimwipes, gloves, etc., anything that is being used for the lab. Items that are not disposable, the hemocytometer and hemocytometer cover slip, wash as instructed and collect all items used in the cleaning as hazardous waste.

16. Clean Up • Same procedure as last week…. • GLOVES: Do NOT touch laptops, microscopes, personal items, etc… with gloves on! • Clean hemacytometerand special coverslip (do not throw away) • All other waste goes into hazardous waste bin (slides, coverslips, pipet tips, Kimwipes, gloves, etc)

17. Before you leave • Please fill out the excel spreadsheet on the computer upfront. Enter raw data (not averages). • Exit question (please leave answers at the front of the room): • Did you enjoy the structure of the past two labs? What did you enjoy/learn? How would you change or improve it?