Stress exposure of yeast

SOP

Six (6) questions in this handout

Plates need to be available and left at room temperature overnight.

Your group will have three Petri dishes containing about 25 mL of Make YPD growth medium filter sterilized with agar (24 g/liter). The media also contains ampicillin, a broad-spectrum antibiotic.

Question 1. I added 1 mL of a 50mg/mL stock solution to 499 mL media. What was the concentration of ampicillin (μg/ml) in the plates?

I set up 5 mL media containing 0.03 g/mL yeast (Red Star, dry yeast weight) at 11:30AM on Wednesday 19 October. Six tubes will be available, the following putative mutagens were added

Question 2. What are the molecular weights of colchicine, cycloheximide, and β-estradiol?

Question 3. What was the molarity of each mutagen in the tubes?

Question 4. Why is ampicillin included in the plates?

Procedure

Day 1

Your group will be assigned one of putative mutagens or a control sample. Wednesday’s group will work with cells setup with media on Wednesday (see above), and therefore exposure of yeast cells to the agents will have only been since 2PM on Wednesday, the time the agents were added to the culture tubes. Thursday’s group will work with the same cells, 24 hours later.

Plates with poured media will be provided for you. Label your plates (bottom) with the following information:

Date:
Group ID:
Mutagen source:
UVC: Yes/No

Question 5. Why label the bottom of the plate, why not the top?

Streak yeast from the mutagen sample with a sterile toothpick onto two plates as demonstrated by your instructor. Reserve one plate for UVC exposure (Dr Dohm will do this, using one of our Biosafety Level 2 hoods (equipped with germicidal lamp). UVC exposure time will be five minutes.

Plates will be kept in Henry Lab 2 in the hood at room temperature. Place plates upside down to minimize contamination and condensation buildup on the agar.

Question 6. What might the UVC exposure do to the cells? What if exposure time was increased from 5 to 15 minutes, what is likely to happen to the yeast cells?

The future

24 hours after, check plates for growth. Image capture plates with the Gel Doc or by smart phone camera

See Mutagenesis – a tool to make new genetic variants — we will use PCR and fingerprinting technology to screen the cells for mutations.

References

Joosten HFP, van Acker FAA, van den Dobbelsteen DJ, Horbach GJMJ, Krajnc EI. (2004) Genotoxicity of hormonal steroids. Toxicology Letters 151:113-134. (link to pdf)

Kaàufer NF, Fried HM, Schwindinger WF, Jasin M, Warner JR (1983) Cycloheximide resistance in yeast: the gene and its protein. Nucleic Acids Research 11:3123-3135 (link to pdf)

Landry CR, Townsend JP, Hartl DL, Cavalier D. 2006 Ecological and evolutionary genomics of Saccharomyces cerevisiae. Molecular Ecology 15:575-591. (link to pdf)

Rieder CL, Palazzo RE. 1992. Colcemid and the mitotic cycle. Journal of Cell Science 102:387-392 (link to pdf)