Aug 18, 2022
Growth Curve Stress Test
This protocol is a draft, published without a DOI.
- Brian Teague1
- 1University of Wisconsin - Stout
- Yeast ORFans CURE
Protocol Citation: Brian Teague 2022. Growth Curve Stress Test. protocols.io https://protocols.io/view/growth-curve-stress-test-cffjtjkn
License: This is an open access protocol 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
Protocol status: Working
We use this protocol and it's working
Created: August 17, 2022
Last Modified: August 18, 2022
Protocol Integer ID: 68811
Keywords: yeast, phenotype, growth curve, yeast growth study, growth curve stress test, single yeast, stressor
Abstract
This is one (fairly quantitative and reproducible) way to run a yeast growth study to analyze the effect of a stressor on yeast. It is not the only way! It is also less of a "protocol" and more of a "template" -- it measures the growth of a single yeast strain at one concentration of "chemical X." Makes sure to adapt it to your own needs!
Image Attribution
Chalik1, CC BY 3.0 , via Wikimedia Commons
Guidelines
Remember, if you see a difference between the knockout and the wild-type under stress, it could be that the stress is slowing down the knockout strain. However, there's another possible explanation: it could just be that the knockout strain is growing more slowly under all conditions. Hence the no-stress controls.
Materials
Equipment
- Vortexer
- Microplate reader (with incubation and shaking)
- Sterile workspace (such as a biosafety cabinet or laminar flow hood)
Materials
- Corning COSTAR 96-well clear flat-bottom assay plate
- Wild-type yeast in double-strength synthetic complete media, OD600 ~ 0.1-0.2
- Knockout yeast in double-strength synthetic complete media, OD600 ~ 0.1-0.2
- A solution of Chemical X in water
Troubleshooting
Safety warnings
Yeast and yeast media are not hazardous.
However, you may be using a hazardous "chemical X." Make sure to use appropriate PPE and engineering controls and dispose of it appropriately.
The yeast we are using are genetically modified. Make sure to inactivate them (by bleach or autoclaving, for example) before disposing of them.
Assay setup
Note
If possible, perform steps 1 and 2 in a sterile environment such as a biosafety cabinet or laminar flow hood.
Vortex the yeast culture briefly to resuspend the yeast cells.
- Set up four wells according to the following table:
| A | B | C | D | E | |
| Well 1 | Well 2 | Well 3 | Well 4 | ||
| Yeast culture (wild-type) | 50 µl | 50 µl | -- | -- | |
| Yeast culture (knockout) | -- | -- | 50 ul | 50 ul | |
| Chemical X, 100 mM | 10 µl | -- | 10 µl | -- | |
| Water | 40 µl | 50 µl | 40 µl | 50 µl |
Set up the plate reader as follows:
- Temperature: 30°C
- Mode: Kinetic
- Wavelength: 600 nm
- Interval: 5 minutes
- Total run time: 24 hours
- Shake before read: 30 seconds
Transfer the assay plate to the reader and read for 24 hours.
Data analysis
Plot each growth curve with the Y axis scale set to "logarithmic."
Find the part of the curve that is linear.
Expected result
In this case, I might choose from 250 minutes to 350 minutes.
Find the OD600 reading at these two times.
Expected result
At 250 minutes, my OD600 is 0.2086; at 350 minutes, my OD600 is 0.3583.
Compute the doubling time using a bit of math:
Where t1 and t2 are the start and end times you found in step 2, and q1 and q2 are the OD600 at those times.
Expected result
Thus, for my BY4735 strain growing in YPD, the doubling time is 128 minutes.