Aug 18, 2022

Public workspaceGrowth Curve Stress Test (Instructor Protocol)

This protocol is a draft, published without a DOI.
  • 1University of Wisconsin - Stout
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Protocol Citation: Brian Teague 2022. Growth Curve Stress Test (Instructor Protocol). protocols.io https://protocols.io/view/growth-curve-stress-test-instructor-protocol-cffitjke
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: 68810
Keywords: saccharomyces, growth, phenotype
Abstract
This is the instructor protocol for
Protocol
Growth Curve Stress Test
NAME
Growth Curve Stress Test
CREATED BY
Brian Teague

Image Attribution
Chalik1, CC BY 3.0 , via Wikimedia Commons
Materials
Equipment
  • Shaking incubator (or preferably a roller drum in an incubator)
  • Spectrophotmeter for measuring OD600

Materials
  • ReagentYeast Nitrogen Base (YNB)Sunrise ScienceCatalog #1500-100
  • ReagentCSM-Leu-Ura PowderSunrise ScienceCatalog #1038-010
  • ReagentAmmonium sulfate Sigma AldrichCatalog #A4418
  • ReagentUracil Yeast Culture GradeSunrise ScienceCatalog #1906-010
  • ReagentL-Leucine Yeast Culture GradeSunrise ScienceCatalog #1980-010
  • Reagentα-D-GlucoseSigma AldrichCatalog #158968
  • Test tubes for growing yeast cultures
Protocol materials
ReagentAmmonium sulfate Merck MilliporeSigma (Sigma-Aldrich)Catalog #A4418
Materials, Step 1
ReagentUracil Yeast Culture GradeSunrise ScienceCatalog #1906-010
Materials, Step 1
ReagentL-Leucine Yeast Culture GradeSunrise ScienceCatalog #1980-010
Materials, Step 1
Reagentα-D-GlucoseMerck MilliporeSigma (Sigma-Aldrich)Catalog #158968
Materials, Step 3
ReagentYeast Nitrogen Base (YNB)Sunrise ScienceCatalog #1500-100
Materials, Step 1
ReagentCSM-Leu-Ura PowderSunrise ScienceCatalog #1038-010
Materials, Step 1
Safety warnings
Yeast and yeast media are not hazardous.
The yeast we are using are genetically modified. Make sure to inactivate them (by bleach or autoclaving, for example) before disposing of them, following your institution's guidelines.
Prepare 2x synthetic media
Prepare 2x synthetic media
30m
30m
Put Amount200 mL of deionized water in a 250 ml bottle. Then add:
  • Amount0.855 g ReagentYeast Nitrogen Base (YNB)Sunrise ScienceCatalog #1500-100
  • Amount0.335 g ReagentCSM-Leu-Ura PowderSunrise ScienceCatalog #1038-010
  • Amount2.5 g ReagentAmmonium sulfate Sigma AldrichCatalog #A4418
  • Amount5 mL 100X uracil solution (Concentration2 mg/mL ReagentUracil Yeast Culture GradeSunrise ScienceCatalog #1906-010 )
  • Amount5 mL 100X leucine solution (Concentration12 mg/mL ReagentL-Leucine Yeast Culture GradeSunrise ScienceCatalog #1980-010 )

Autoclave Temperature121 °C for Duration00:30:00

30m
Cool, then add Amount50 mL Concentration40 Mass / % volume glucoReagentα-D-GlucoseSigma AldrichCatalog #158968 using aseptic technique.

Prepare the yeast cultures
Prepare the yeast cultures
The afternoon or evening before lab, prepare a test tube with 5 ml 2xSDC media for each yeast strain you will be testing.
Pick a colony from each strain's plate and resuspend it in the media.
Grow DurationOvernight at Temperature30 °C , either on a roller drum (preferred) or on an orbital shaker at Shaker180 rpm .

The next morning, several hours before class, measure the OD600 of each culture.
Prepare another test tube with 5 ml of 2xSDC media for each yeast strain. Subculture the overnights to an OD600 of 0.1. Place the tubes back in the incubator or shaker.
Optimally, grow the cultures for at least 3 hours before lab.

Note
The experiments will be more reproducible if the cells always start the experiment in logarithmic growth phase!

Instructor Tips & Common Student Errors
Instructor Tips & Common Student Errors
Instructor Tips
  • This assumes that, once you or your students have identified yeast colonies with successful knockouts, you have struck them out on plates
  • Log-phase growth is the most reproducible growth mode for yeast. This is why the experiment should start in log phase and why doubling time should be computed in log phase.
  • Growing the cells in double-strength media means that they can be diluted 1:1 and still be in "complete" media for the stress experiment.
  • Yeast cells are heavy and settle rapidly toward the bottom of a test-tube. Make sure you vortex them before measuring OD600; vortex again before subculturing; and vortex again before putting them in an assay plate.
  • Computing doubling time is not optional! Because we're looking at an exponential phenomenon, a small difference in starting concentration is rapidly amplified and can "look" on a graph like a strain is growing slower.
  • I thought everyone graduated highschool with a basic ability to use a spreadsheet? I am often surprised by the number of students that have trouble with "make a graph of this data."
Common student errors
  • Not computing doubling time to compare growth, but just drawing conclusions from eyeballing a graph.
  • Not vortexing the yeast cells before putting them in the assay plate.
  • General data manipulation and interpretation difficulty