Apr 13, 2020
Yeast growth profile analysis
- 1Southern Illinois University-Edwardsville
- Labyrieth
- Yeast Protocols, Tools, and Tips
Document Citation: Monica Rieth 2020. Yeast growth profile analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.33jgqkn
License: This is an open access document distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Created: June 11, 2019
Last Modified: April 13, 2020
Document Integer ID: 24395
Keywords: yeast, growth, profile
Abstract
This method outlines a procedure for analyzing the growth curve of Baker's yeast, Saccharomyces cerevisiae, for the assessment of growth medias, the addition of metabolites to media, and the incorporation of heterologous genes to our yeast strain.
PROCEDURE
Day 1:
1. Streak a YPD-agar plate from a glycerol stock.
2. Incubate for 48-72 hours at 25-30 °C or until colonies are large enough to pick (1-2 mm diam.)
Day 2:
3. Check plates. There likely won’t be any colonies that are large enough to pick
Day 3:
4. Colonies should be large enough to pick (1-2 mm diameter)
5. Propagate colony (ies) in 5 mL YPD media
6. Allow colonies to grow overnight at 30 °C shaking at 240-250 rpm
Day 4:
7. Propagate triplicates in 5 mL fresh YPD using 50 μL of the overnight growth added directly to the 5 mL of fresh media
8. Begin collecting data for the growth curve. This marks the t=0 time point.
9. To begin collecting time points, 50 μL of culture can be added to 750 μL sterile media (or ddH2O) using a polystyrene semi-microcuvette. Measure the absorbance at 600 nm
NOTE: Yeast grow more slowly than bacteria and time points can safely be collected every 2 hours. Additionally, after the fresh media is inoculated and time point 0 is collected, it can take up to 4 hours for any appreciable absorbance to be reached.
10. Time points data can be collected up to and including 48 hours. After 12 hours data was collected every 4 hours up to 24 hours and every 12 hours thereafter.
11. After an OD of 1.0-1.2 is achieved, yeast can be gently harvested at RT and fresh YPG added to induce protein expression
- Fix the cap of the culture tube so it is sealed (2nd stop)
- Using the Sartorius benchtop centrifuge (or other), spin at 500 x g for 3-5 minutes or until the supernatant is clear
- Gently remove the supernatant with a micropipette (save if interested in metabolite studies at - 20 °C in sterile 15-mL
conical tubes)
- Replace with 5 mL of fresh YPG media (warmed to 30 °C) and place back in the incubator at 30 °C. Continue collecting
time point data
12. Pellets can be retained and saved at the end of the study for additional analysis
Yeast Growth Curve Analysis_unlinked.pdf