Download S11. Using Yeast to Make Scientists-Sample

Using Yeast to Make Scientists
Goudsouzian, McLaughlin, Slee
We acknowledge that there are a few issues with this sample proposal (i.e. tense issues and the
hypothesis is more of a prediction), but this document shows the level and types of issues one
can expect when reviewing the proposals
The Effect of Energy Drinks on Actively Growing Yeast Cells
Department of Natural Science, DeSales University
Purpose:
The purpose of this experiment is to determine the effect of energy drinks on yeast cells.
Specifically, we will be observing whether or not there is a benefit to drinking an organic
energy drink as compared with a non-organic energy drink. S. cerevisiae is a good model
to study these particular variables because it is eukaryotic and is a good representation of
human cells. Our hypothesis is that the organic energy drink will allow continuous
growth of yeast cells, while the non-organic energy drink will inhibit growth.
The ingredients of energy drinks such as guarana, caffeine, and taurine, and their effects
on cell health are debated with evidence for both positive and negative effects. In a study
done on nerve cells, guarana led to cell arrest, while caffeine led to growth inhibition, but
not to a large extent (Zeidán-Chuliá 2013). However, yeast cells seemed to act contrarily
in the presence of these ingredients. In the journal Medical Microbiology in 2008, a paper
was published that displayed correlations between cell growth and caffeine treatment. S.
cerevisiae responded well to caffeine because it is believed that caffeine activated the
Ras/MAP Kinase pathway, which eventually led to an up-regulation of Rim-15,
extending lifespan (Wanke 2008). The other common ingredient, taurine, while major
companies make a point to say that their drinks are “taurine free,” is a chemical that has
shown positive effects in yeast cells. In a study done in 2000 on human intestine cells
treated with taurine, scientists found that taurine serves as an osmolyte and protects the
cells from osmotic stress (Shimizu 2000).
Materials List:
- Yeast Strain YPH499 (mating type a)
- Sterile YEPD liquid growth media
- Sterile glass test tubes
- Micropipettes and sterile tips
- Transfer pipettes
- Shaking Incubator, set at 30°C
- Waste tray for microscope slides
- Vortexer
- Spectrophotometer
- Organic Energy Drink
- Non-organic Energy Drink
- Microscope slides
- Coverslips
- Methylene Blue
- Inverted microscope
Procedure:
 YEPD tube was obtained and inoculated with “YPH499” aseptically
 Tube was incubated overnight at 30°C
 Each of 9 tubes containing 10mL were labeled and inoculated with 300µL of
cells from our overnight culture and vortexed
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9 cuvettes were labeled corresponding to the tubes
Cuvette B as filled with sterile YEPD and was used to blank the
spectrophotometer
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Test substance(s) were added to tubes 1-8 in different concentrations
o (Organic Energy Drink: tubes 1-4, Regular Energy Drink: tubes 5-8)
o Organic Energy Drink Dilutions: 0.0338mL, 0.1014mL, 0.169mL,
0.2366mL
o Regular Energy Drink Dilutions: 0.054mL, 0.162mL, 0.27mL, 0.378mL
Cuvettes were filled with YEPD + test substance and absorbance was measured
using a spectrophotometer
A small drop of methylene blue was added to microscope slide and mixed with 10
µL of each of our samples
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Cell viability of each of our samples was calculated by counting using a
microscope
Tubes were capped and placed in the 30°C shaker
Steps 7-10 were repeated at 60 minute intervals for the next six hours
Data was recorded in a table and presented in multiple graphs
Variables:
The dependent variable in this experiment will be yeast cell growth. The independent
variable will be which drink is given to the yeast cells, either organic or non-organic
energy drinks. Also a component of the independent variable will be the amount of each
that will be added to the yeast through different dilutions. The amount of caffeine would
have been a significant confounding variable. However, we calculated our dilutions based
on amount of caffeine so that caffeine concentration would be equal between the two
drinks within our samples.
Data Interpretation:
Microsoft Excel will be used to graph our data. One graph will be a line graph showing
the optical density of both organic and non-organic cultures over time. This will be our
growth curve in which time will be plotted on the x-axis and optical density on the y-axis.
We will also include a bar graph comparing the two energy drinks and the number of
dead and live cells over time. The procedure will be repeated three times to allow for
accurate statistical data.
References:
Kumagai, Akiko, et al. "The Xenopus Chk1 protein kinase mediates a caffeine-sensitive
pathway of checkpoint control in cell-free extracts." The Journal of cell biology
142.6 (1998): 1559-1569.
Kuranda, Klaudia, et al. "Investigating the caffeine effects in the yeast Saccharomyces
cerevisiae brings new insights into the connection between TOR, PKC and
Ras/cAMP signalling pathways." Molecular microbiology 61.5 (2006): 11471166.
Shimizu, M., and H. Satsu. "Physiological significance of taurine and the taurine
transporter in intestinal epithelial cells." Amino acids 19.3-4 (2000): 605-614.
Wanke, Valeria, et al. "Caffeine extends yeast lifespan by targeting TORC1." Molecular
microbiology 69.1 (2008): 277-285.
Zeidán-Chuliá, Fares, et al. "Major components of energy drinks (caffeine, taurine, and
guarana) exert cytotoxic effects on human neuronal SH-SY5Y cells by decreasing
reactive oxygen species production." Oxidative medicine and cellular longevity
2013 (2013).