UW-Stout/Nitrogen Starvation
Contents
Sensitivity To Starvation
Purpose:
The purpose of this lab is to test the sensitivity to the starvation of knock out strains of yeast cells. We will collect data on how our knockout yeast behaves in a different environment, in this case it would be starvation. We will test it by excluding a Nitrogen source to our synthetic complete media that we are using to grow the yeast cells on.
Materials:
- 10 plates with of Omission Media (no Nitrogen)
- 20 plates with Synthetic Defined Media (has Nitrogen)
- 10 tubes of knockout genes (the different strains of yeast)
- 1 tube of wild type yeast
- Petri dishes (42? 3 per group for experiment plus 1 control per group plus other 2 controls)
- Felt Strips
- 100 µl of each yeast cells
- Incubator (30 degrees Celsius)
Control: Every strain of yeast (even the wild type) has its own control that will grow in a normal synthetic defined medium petri dish. Then we will have a control petri dish where we starve wild type yeast cells.
Safety Note: Wear appropriate lab clothing (lab coat, goggles, gloves, etc.). There are no additional safety concerns that we need to be worried about for this lab.
Protocol:
Complete each step for each of the 10 petri plates
Day 1
1. Obtain a petri dish with synthetic defined media.
2. Transfer 100 µl of yeast cells to the petri dish.
3. Let the petri dishes incubate at 30 degrees Celsius for 2 days. Record your results.
4. Record your data and draw conclusions based off which cells die and which survive.
5. Cut out 30 pieces of 4-inch x 4-inch squares of felt
6. Pack the felt pieces in pairs of 2 and wrap them in tinfoil.
7. Put the tinfoil wrapped felt packs in the autoclave to sterilize.
Day 3
1. After 2 days of incubation, transfer the yeast cells from the synthetic complete media using a felt strip and place the cells on a petri dish with the omission media that lacks a nitrogen source.
2. Place the contaminated felt strips in diluted Lysol and let them sit for 20-30 minutes to disinfect. Let them dry out, wrap in tinfoil, and put them back in the autoclave to complete sterilization.
3. Let the petri dishes with the SD media incubate at 30 degrees Celsius for 5 days.
Day 8
1. Record the results and not if the cells die or survive.
2. Transfer the cells, again using a felt strip, into a new petri dish with SD media that has a rich nitrogen source.
3. Allow the petri dishes to incubate at 30 degrees Celsius for 2 days.
Day 10
1. Record results and draw conclusions
Additional Notes:
Label every petri dish with name of yeast strain and the media its growing in.
If the yeast cells die that means the cells are sensitive to starvation (no growth on last plate).
If the yeast cells survive that means the cells are not sensitive to starvation (growth on last plate).
How fast does the yeast recover? (record amount of growth on final plates).
Results:
All the Revival Plates have displayed full growth similar to the growth seen on the growing plates. There was no observable decrease of yeast population or evidence of any dead yeast cells. Starving the Yeast Cells for 5 days was not long enough to get conclusive results on which the strains were more sensitive to starvation. If you were to do this experiment again it is suggested that you increase the incubation time in the Nitrogen Omitted medium. Incubating the yeast strains for longer should make some differences in growth on the revival plates observable.
