The Effects of EGCG on
Microbiological Survivorship
Sean Bailey
Grade 11, CCHS
2nd Year in PJAS
This Experiment
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to determine whether or not
epigallocatechin-3-gallate has a
anti-carcinogenic effect on
Saccharomyces cerevisiae.
Previous Studies
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EGCG has been studied extensively by medical
researchers, due to its relatively low cost, abundance,
and high potential for antioxidant properties.
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A search of the PubMed database for the phrase “egcg
cancer” results in 1273 hits, indicating its widespread
research.
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Of note is a study testing EGCG’s effect on Candida
albicans, another strain of yeast, published in the
Canadian Journal of Microbiology in 2009, which was
found to have inhibitory effects on mutagenesis.
EGCG
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Epigallocatechin-3-gallate is the most
abundant catechin in green tea
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is a strong catechin and antioxidant,
and thus has the potential to inhibit
mutagenesis, preventing cancer
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other common antioxidants include
ascorbic acid, melatonin, and
anthocyanin, which is found in red
wine
Saccharomyces
cerevisiae
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commonly known as yeast
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simple to use and easy to culture
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used often as a first line of research into
anticancer drugs because it mimics the
human body’s cellular processes with a great
deal of homology
—Lysine
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The strain of yeast used in this experiment were
unable to produce lysine, a key nutrient involved in cell
growth.
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These mutants do not have enzymes that function in
the production of lysine.
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Thus, the cells require lysine in order to grow.
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They were also grown in agar that did not contain
lysine, rendering the cells unable to grow under
normal conditions.
Mutagenesis
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DNA is contained in the nucleus of eukaryotic cells.
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UV radiation is a mutagen, meaning it changes the DNA of the
cells.
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The —lysine yeast would not survive if it weren’t for
mutagenesis.
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The UV radiation mutates the yeast’s DNA, causing it in some
cases to produce lysine and grow.
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Thus, survivorship of the cells can be used as a crude measure
of mutagenesis, i.e. if cells grow, it means they mutated to
produce lysine
Ames Test
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developed by Bruce Ames in the 1970s
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cultured salmonella that did not produce histidine in
media that did not contain histidine
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exposed the salmonella to a mutagen
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growth of salmonella colonies indicated that the
mutagen caused reversion of the histidine-producing
codons in the DNA of the cell
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minus lysine test is a modified Ames test
Hypotheses
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The null hypothesis is that epigallocatechin-3-gallate will
not have a significant effect on the reversion rate of
Saccharomyces cerevisiae which have been stressed
by ultraviolet radiation.
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The alternate hypothesis is that epigallocatechin-3gallate will have a significant effect on the reversion rate
of Saccharomyces cerevisiae which have been stressed
by ultraviolet radiation.
Materials
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—lysine agar plates
sterile dilution fluid
Klett spectrophotometer
micropipettes (p1000,
p20)
sterile pipette tips
vortex
sidearm flask
spreader bar
ethanol
burner
—lysine S. cerevisiae
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UV light hood
sterile rubber gloves
test microtubes
microtube rack
Epigallocatechin-3gallate, 50mg (procured
from Santa Cruz
Biotechnology)
Procedure
1. A strain of S. cerevisiae —lys phenotype was grown for
2 days in YEPD media to a density of approximately
10^8 cells per ml
2. 4 days prior to experimentation the media was
removed from the cell pellet and replaced with 3 mL of
SDF.
3. Prior to removal, a series of washes were conducted
with SDF to remove any residual lysine nutrients
4. The pellet in SDF was resuspended, and stored in —
lys media for 2 days.
5. A stock solution of 1% EGCG was made.
Procedure (cont’d)
6. A set of dilutions were prepared from the stock solution
and sterile water in the following concentrations:
1. 0% EGCG: 0.2 mL S. cerevisiae, 0.8 mL sterile
water
2. 0.01% EGCG: 0.2 mL S. cerevisiae, 0.79 mL sterile
water, 0.01 mL 1% EGCG stock
3. 0.1% EGCG: 0.2 mL S. cerevisiae, 0.7 mL sterile
water, 0.1 mL 1% EGCG stock
7. The cells infused with the solutions for 20 minutes.
8. Eight 0.1 mL aliquots per experimental group were
spreadplated onto waiting —lys agar plates.
Procedure (cont’d)
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The plates were transported to the Mellon Institute,
Carnegie Mellon’s biology laboratory, taking great pains
to keep constant their temperature.
Each concentration of EGCG had 8 plates each exposed
to 0, 15, and 30 seconds of UV radiation.
The plates were then transported back to Central
Catholic, incubated at 37° C for four days.
The plates were removed from the incubator, colonies
were counted, and data was recorded. Each colony was
assumed to have arisen from one single cell.
The plates were then sterilized with bleach and safely
discarded.
ANOVA and Dunnett’s
Test
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An ANOVA (ANalysis Of VAriance) is a statistical test to
determine if one group differs significantly against the other.
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Each group of data is averaged and compared to each other
group, and a probability is generated (P-value).
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To determine which group had a significant effect if such an
effect is gathered, a Dunnett’s pairwise test is performed,
which compares each individual data set.
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A single factor ANOVA was performed on each timeset (0
seconds, 15 seconds, and 30 seconds) and Dunnett’s tests
were performed where significance was found.
Results - Graph and
ANOVAs
Effect of EGCG on Yeast Reversion Rate P values
0s:
0.914
0% EGCG
0.01% EGCG
0.1% EGCG
37.5
15s: 0.029
30s: 0.012
30
22.5
15
7.5
0
0 seconds
15 seconds
30 seconds
Results - Dunnett’s Test
T-crit: 2.31
T value
Significant?
15s, 0% vs 0.01%
1.81
NO
15s, 0% vs 0.1%
12.38
YES
30s, 0% vs 0.01%
0.00
NO
30s, 0% vs 0.1%
9.15
YES
Conclusions
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The null hypothesis was rejected for the 0.1%
concentration of EGCG, as the colony count varied
significantly across both times.
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The null hypothesis was accepted for the 0.01%
concentration of EGCG.
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At the higher concentration, EGCG had a significant
effect on mutagenesis, reducing the amount of
reversions.
Limitations and
Extensions
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Limitations
slight variations in the amount of cells per plate
synchronicity of plates
even exposure to UV light sources
crudeness of Ames test
temperature variations due to transport of plates in cold
weather
Extensions
test a wider variety of concentrations
use a stain assay like Trypan Blue to count cells killed
by UV radiation
test different mutagens
Sources
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Evenson, Nikki and Phyllis Braun. The effects of tea polyphenols on
Candida albicans: inhibition of biofilm formation and proteasome
inactivation. 2009, Canadian Journal of Microbiology.
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Santa Cruz Biotechnology. Material Safety Data Sheet for EGCG.
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Matuo, R., et. al. Saccharomyces cerevisiae as a model system to study
the response to anticancer agents. 2012, Cancer Chemotherapy
Pharmacology Journal.
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Stevens, S. K., et. al. The anticancer ruthenium complex KP1019 induces
DNA damage, leading to cell cycle delay and cell death in
Saccharomyces cerevisiae. 2013, Mol Pharmacology.
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Wikimedia Commons Image Library. EGCG molecule and green tea.
Thanks and Credits
I would like to take this opportunity to formally thank
Carnegie Mellon University, my sponsor Mark Krotec, and
Central Catholic High School.
Graphs were generated using matplotlib.
Statistics were done in R.