Series 1 Lab 3 Variable Testing in Tetrahymena
Adapted from Bozzone, M.D., and D.A. Martin 2000. An experimenal system to study phagocytosis. Pages 405-415, in Tested studies for laboratory teaching, Volume 21 (S.J. Karcher, Editor). Proceedings of the 21st Workshop/Conference of the Association for Biology Laboratory Education (ABLE).
In lab this week, you will have the opportunity to test the hypothesis you formulated in Lab 2. Each group of two students will focus on one inhibitor, and no more than two student groups can work on any one inhibitor.
I. POTENTIAL INHIBITORS OF VACUOLE FORMATION
You will have the opportunity to test either a cytoskeleton inhibitor or a phosphatase inhibitor. The cytoskeleton, which consists of intermediate filaments, microtubules, and actin filaments, plays an important role in the structure and mobility of cells and cell components. You can test to see if actin and/or microtubules play a role in food vacuole formation in Tetrahymena.
Because protein function is dependent on protein shape and charge, many biochemical pathways add a phosphate group to a protein as an effective way to regulate protein function. The addition of a phosphate group is termed phosphorylation, and an enzyme that adds a phosphate group to an amino acid within a protein is called a protein kinase. In contrast, protein phosphatases are enzymes that dephosphorylate (remove a phosphate group from) certain amino acids within proteins. Depending on the protein, phosphorylation can either activate or inhibit protein function, and it plays an important role in many cell signaling events that occur in the cell. Today in lab, you may choose to test whether protein phosphatases are important for food vacuole formation in Tetrahymena.
All of the reagents that you used in Lab 2 will be available again in Lab 3. In addition, you will be able to use one of the following inhibitors. CAUTION: Wear gloves and exercise caution when handling the inhibitors because they are toxic.
A. Cytochalasin B is an inhibitor of actin.
B. Colchicine is an inhibitor of microtubules.
A. Phenylarsine oxide (PAO) is an inhibitor of tyrosine phosphatases.
B. Cantharadin is an inhibitor of serine/threonine phosphatases.
1. Place used micropipette tips and microcentrifuge tubes in autoclave bags taped above your bench.
2. Clean the oil immersion objective lens (100x) of your microscope using only lens tissue (NOT Kimwipes®). Check the 40x objective lens as well. Sometimes it gets inadvertently contaminated with immersion oil.
3. Rotate the 4x objective lens into place.
4. The binocular head must be rotated into the storage position, to protect the ocular lenses from damage. Loosen the setscrew on the right, rotate the head 180°, then retighten the screw. Turn off the microscope light. Return the microscope to the cabinet under your bench with its plastic cover on.
5. Put all used microscope slides and cover slips in the glass disposal box.
You have been given a stock glucose solution with a concentration of 100mg/mL. To perform the glucose assay in lab today, you must dilute the 100mg/mL stock solution of glucose to concentrations of 80mg/mL, 60mg/mL, 40mg/mL, and 20mg/mL. You need to prepare 1mL of each dilution. Explain how you would make the 4 dilutions from the glucose stock solution.
Preparation for Science Writing Workshop in Lab 5
1. In lab 5, you and your partner will have the opportunity to give a 5 minute oral presentation of your data. Please prepare a PowerPoint presentation that includes a figure and figure legend summarizing your lab 3 data. Although figure legends are not typically included in PowerPoint presentations, you will include one this time so we can discuss effective figure design.
2. Read the journal article assigned by your instructor. Pay careful attention to the format of the different sections of the paper. You and your partner may be assigned figure or other part of the paper to present informally to the class as part of the science writing workshop discussion we will have in lab 5. Don’t worry if you have trouble understanding every detail in the paper. The main purpose of this exercise is to help you understand the style of scientific writing and how data is presented efffectively in figures.