UW-Stout/Hydroxyurea

Calibration Experiment

Protocol for the calibration experiment of the stressor, Hydroxyurea, to determine the amount that best serves our purpose.

Jack Hoaby and Stephanie Strabel

Purpose:

To test the intensity of the stressor (Hydroxyurea) to see if it has an impact on growth of baker’s yeast, but that it doesn’t have too much of an impact that it destroys the cells.

Preliminary explanation:

Hydroxyurea causes nucleic acid metabolism defects that could result in defective DNA replication due to inhibition of the ribonucleotide reductase enzyme. If this stressor affects our knocked-out gene, or our classmate’s, we will know if that gene has a role in DNA replication.

Materials:

• Baker’s yeast (strain: BY 4735)
• Hydroxyurea (HU)
• Distilled H2O
• 96-well assay plate

Protocol:

1. Create a working stock of HU of 1 mL at 1 M. Should use 0.076 g of HU. Measure using the analytical balance.
2. Create multiple stock solutions: 50mM, 100mM, 150mM, and 200mM HU with distilled H2O each with a final volume of 1000 uL.
3. Vortex the yeast briefly. The starting optimal density should be 0.2.
4. Add 10 microliters of stock solution to each yeast strain (90uL) for a final volume of 100 uL.
5. Transfer the yeast solutions from the tube into separate wells on the same row of an assay plate.
6. Add 100uL of wild type yeast without HU to a well.
7. Grow yeast at 30℃ overnight. Measure every 5 min to create a graph that measures density over time.
8. Discard remaining stock solutions.
9. Store HU with Dr. Teague.

Resources:

Journal of Biology Chemistry Harvard Wiki

Proof of a good experiment:

• Controls: Step 4. Yeast without HU.
• How is it measurable? Growth is measurable by a graph that measures density over time.How is it safe: Lab equipment (gloves, eyewear, lab coat) worn at all times. HU is dangerous when exposed to skin, but it is OK to work with.
• Variable: Varying stock solutions of HU in distilled H2O. Use same volume of varying stock with same volume of yeast.

Data:

200mM HU

400mM HU

600mM HU

800mM HU

1000mM HU

0mM HU Control

Interpretation:

Each amount of Hydroxyurea used stressed out the wild-type strain an adequate amount. 800mM seemed to stress the strain the most, but due to time constraints, we guessed that 200mM would be the best option. It still turned out to be a decent option nonetheless.

Stressing the Knock-Out Genes

Protocol for stressing each gene strain with our stressor, Hydroxyurea.

Jack Hoaby and Stephanie Strabel

Purpose:

To test each strain of baker’s yeast that our class made (8 in total, 9 with the control of wild type yeast) in regards to the stressor Hydroxyurea (HU) in attempts to see the function of each knocked out gene.

Preliminary explanation:

Hydroxyurea causes nucleic acid metabolism defects that could result in defective DNA replication due to inhibition of the ribonucleotide reductase enzyme. If this stressor affects our knocked-out gene, or our classmate’s, we will know if that gene has a role in DNA replication.

Materials:

• Knock-out strains of Baker’s yeast (9 different types)
• Hydroxyurea (HU)
• Autoclaved H2O
• 96-well assay plate

Protocol:

1. Create a working stock of HU of 1 mL at 1 M. Should use 0.076 g of HU. Measure using the analytical balance.
2. Create a stock solution of 200mM HU with distilled H2O each with a final volume of 1000 uL.
3. 200 ul stock + 800 ul H2O
4. Pipette each yeast strain up and down (5 times) to re-suspend the yeast.
5. Add 10 microliters of stock solution and 90 microliters of each yeast strain to separate microfuge tubes for a final volume of 100 uL in each tube.
6. Transfer the yeast solutions from the tubes into separate wells on the same row of an assay plate (Row D, Columns 1-8).
7. Add 100uL of wild type yeast without HU to a well (Row D, Column 9). This is the control.
8. Grow yeast at 30℃ overnight. Measure every 5 min to create a graph that measures density over time.
9. Discard remaining stock solution.
10. Store HU with Dr. Teague.