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L-Leucine Remediation of Stress on Yeast
Colm Parrish Pittsburgh Central Catholic HS
Grade 10
Oxidative Stress • Caused by UV radiation and X-Rays • Exposure causes an increase in free
radical production • The cell may degenerate • The cell is also more susceptible to cancer
and death
Ultra Violet Radiation • Radiates from the sun, some radiation stopped
by the ozone layer • Have short wavelengths, which are more
powerful than that of visible light • UVC 254nm radiation used in this experiment
Effects of UV Radiation • Leads to sunburn, skin cancer, and sunstroke,
all due to exposure to epithelial cells • Also have been linked to causing cataracts in
the eyes • FDA recommends sunscreen, hats, and
sunglasses to protect the body from radiation • Can cause dimers in the DNA, which can lead to
replication errors and mutations
Stress Proteins • Free Radicals can disrupt the shape and
function of many molecules of life such as lipids, carbohydrates,
proteins, and sometimes even nucleic acids
• Stress proteins are involved in restoring the structure and function of critical cell proteins
that have been damaged by stress !
Can amino acid supplementation increase the response of stress proteins?
L-Leucine • An essential amino acid that is part of the
Branched Chain Amino Acid group • Cannot be produced by the body, must be
consumed through food or supplements • An activator of proteins that causes
muscle growth through protein production
Saccharomyces cerevisiae!!• More commonly know as Baker�s or Brewer�s
yeast • Most studied Eukaryotic cell model • Easily grown and cultured • Similar to epithelial cells in cell cycle and
biochemistry
Problem!!
Ultra violet radiation is harmful and damaging to cells
Purpose To discover if L-Leucine can remediate the oxidative stress caused by ultra violet radiation
Hypotheses • Null: The L-Leucine will not significantly
remediate the oxidative stress of ultraviolet radiation on Saccharomyces cerevisiae
• Alternative: The L-Leucine will significantly remediate the oxidative stress of ultraviolet radiation on Saccharomyces cerevisiae. The L-Leucine will significantly increase the survivorship of UV stressed yeast.
Materials!• 72 YEPD agar plates(1% yeast extract, 2% peptone, 2% dextrose, 1.5% agar) • Sterile dilution fluid [SDF] (10mM KH2PO4, 10mM K2HPO4, 1mM MgSO4, .1mM CaCl2, 100mM NaCl)
• Klett spectrophotometer • Sterile pipette tips and Micropipettes • Vortex • Sidearm flask • Spreader bar • Ethanol • Micro burner • Saccharomyces cerevisiae (yeast) • UV Hood • Rubber Gloves • Test tubes • Test Tube Rack • SDF Test Tubes • Microtubules • L-Leucine • Incubator • YEPD media
Procedures!1. Saccharomyces cerevisiae was grown overnight in
sterile dilution YEPD media 2. A sample of the overnight culture was added to fresh
media in a sterile sidearm flask 3. The culture was incubated at 30 degrees Celsius
until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 107 cells/mL
4. The culture was diluted in sterile dilution fluid to a concentration of approximately 105 cells/mL
5. The L-Leucine was diluted with sterile dilution fluid to the chosen concentrations to a total of 9.9 mL
!
Test Tube Concentrations!
13"
Control" 0.1% 1%
Yeast! 0.1mL 0.1mL 0.1mL
SDF! 9.9mL 9.8mL 8.9mL
L-Leucine (10% Stock)
0mL 0.1mL 1mL
Total! 10mL 10mL
10mL
6. 0.1 mL of cell culture was then added to the test tubes, yielding a final volume of 10 mL and a cell density of approximately 103 cells/mL 7. The test tubes were then vortexed and 0.1 mL of the solution was pipetted onto the YEPD plates then spread plated 8. The plates were then exposed to UV radiation for 0, 20, 30, and 40 seconds 9. The plates were incubated at 30 degrees Celsius for 48 hours 10. The resulting colonies were counted. Each colony is assumed to have arisen from one cell
Procedures Con’t!
15"
L-Leucine Remediation Effects Concentration of L-Leucine!
Exposure Time
Num
ber
of
Colo
nie
s P-Value 2.54E-6 "
P-Value 5.65E-7 "
P-Value 1.87E-9
P-Value 1.6E-11 "
Interaction 1.1E-7 "
• Did the L-Leucine have an effect by itself? P-Value 2.54E-6 Significant
• Did the UV reduce the survivorship of the cells? P-Value 5.65E-7 Significant
• Did 0.1% L-Leucine affect survivorship after exposure?
P-Value 1.87E-9 Significant • Did 1% L-Leucine affect survivorship after
exposure P-Value 1.6E-11 Significant
• Did effect of UV exposure depend upon the concentration of L-Leucine (Variable Interaction)
Interaction P-Value 1.1E-7 Significant
16"
ANOVA!• Short for Analysis of Variance • Statistical test to find variance between and within
groups • If the P-Value is smaller than the Alpha Value (0.05), the
analysis is significant, an example from the experiment is shown below! ANOVA" " " " " " "
Source of Variation" SS" df" MS" F" P-value" F crit"
Between Groups" 49114.4" 1" 49114.4" 76.97876" 1.64E-07" 4.493998"
Within Groups" 10208.41" 16" 638.0254" " " " " " " " " " "Total" 59322.81" 17" " " " "
16"
ANOVA Results!P- Value" Significant/ Insignificant"
No UV and L-Leucine 2.54E-6 Significant
0.1% L-Leucine and UV 1.87E-9 Significant
1% L-Leucine and UV 1.6E-11 Significant
No L-Leucine and UV 5.65E-7 Significant 20 Seconds UV and L-
Leucine 0.00048 Significant 30 Seconds UV and L-
Leucine 0.12138 Insignificant 40 Seconds UV and L-
Leucine 0.2517 Insignificant Synergy!
(Two-Factor Anova) 1.1E-7 Significant
Dunnett�s Test!• Follow up to an ANOVA • Statistical test to find the source of variance • If the T-value is larger then the T-Crit, the effect is
significant !
19"
Dunnett�s Test! T-Crit = 2.86"
T-Value" Significant/ Insignificant"
No UV, 0.1% L-Leucine 3.95841695 Significant
No UV, 1% L-Leucine 8.276689986 Significant
20 Sec UV, 0.1% L-Leucine 0.457784958 Insignificant
20 Sec UV, 1% L-Leucine 4.425254594 Significant
19"
Conclusion!Alternative Hypothesis: The L-Leucine will significantly remediate the stress of Ultra Violet radiation on Saccharomyces cerevisiae
- Rejected, unsupported by data Null Hypothesis: The L-Leucine will not significantly remediate the stress of Ultra Violet radiation on Saccharomyces cerevisiae
-Accepted, supported by data -Data suggests that L-Leucine concentrations
decreased the survivorship of the UV stressed yeast
Limitations!• Only 6 replicates • Only 4 exposure time • Only 1 wavelength of UV radiation
• (UVC 240nm) • Synchronizing the exact times of plating • Cannot analyze the health of cells or growth rate of cells
that recovered from stress
22"
Extensions!• More replicates and concentrations • More exposure times • Utilize more wavelengths of UV radiation specifically UVA
and UVB • Analyze the growth rate of yeast exposed to UV
radiation and L-Leucine • Perform a Trypan Blue Exclusion Assay to separate the
living and dead cells!
23"
21"
Bibliography!• http://www.who.int/uv/health/uv_health2/en/index2.html • http://ntp-server.niehs.nih.gov/index.cfm?objectid=BD4CD88D-
F1F6-975E-792094AC1CE4B062 • www.FDA.com • http://examine.com/supplements/Leucine/ • http://www.arpansa.gov.au/images/basics/uvabc.gif • http://www.sciencebuddies.org/Files/2642/5/MicroBio_img021.gif • http://www.microbiologyonline.org.uk/themed/sgm/img/slideshows/
3.1.4_fungi_2.png • http://www.lifesciencesfoundation.org/content/media/
2011/06/29/1995_Saccharomyces_cerevisiae-large.jpg
Analysis of Variance (One-Way)
Summary !! !! !! !! !! !!Groups Sample size Sum Mean Variance
0% Leucine 6! 774.! 129.! 54.8!0.1% Leucine 6! 969.! 161.5! 113.9!1% Leucine 6! 1,188.! 198.! 456.8!!! !!
ANOVA !! !! !! !! !! !!Source of Variation SS df MS F p-level F crit
Between!Groups! 14,299.! 2! 7,149.5! 34.29017! 0.! 5.13545!Within!Groups! 3,127.5! 15! 208.5!
Total 17,426.5! 17!!! !! !! !!
Analysis of Variance (One-Way)
Summary
Groups Sample size Sum Mean Variance 156 5 813. 162.6 133.3
92 5 733. 146.6 532.8 43 5 272. 54.4 1,334.8
5 5 90. 18. 176.5
ANOVA Source of Variation SS df MS F p-level F crit
Between Groups 74,045.2 3 24,681.73333 45.34166 0. 4.36094 Within Groups 8,709.6 16 544.35
Total 82,754.8 19
Analysis of Variance (One-Way)
Summary Groups Sample size Sum Mean Variance
No UV 6 1,188. 198. 456.8 20 UV 6 297. 49.5 1,753.9 30 UV 6 96. 16. 35.2 40 UV 6 25. 4.16667 34.56667
ANOVA Source of Variation SS df MS F p-level F crit
Between Groups 144,097.5 3 48,032.5 84.2503 1.60647E-11 4.1134 Within Groups 11,402.33333 20 570.11667
Total 155,499.83333 23
Analysis of Variance (One-Way)
Summary !! !! !! !! !! !!Groups Sample size Sum Mean Variance
0% Leucine 6! 822.! 137.! 801.6!0.1% Leucine 6! 825.! 137.5! 923.1!1% Leucine 6! 297.! 49.5! 1,753.9!!! !!
ANOVA !! !! !! !! !! !!Source of Variation SS df MS F p-level F crit
Between!Groups! 30,801.! 2! 15,400.5! 13.28164! 0.00048! 5.13545!Within!Groups! 17,393.! 15!
1,159.53333!
Total 48,194.! 17!!! !! !! !!
Analysis of Variance (One-Way)
Summary !! !! !! !! !! !!Groups Sample size Sum Mean Variance
0% Leucine 6! 321.! 53.5! 2,250.3!0.1% Leucine 6! 315.! 52.5! 1,089.5!1% Leucine 6! 96.! 16.! 35.2!!! !!
ANOVA !! !! !! !! !! !!Source of Variation SS df MS F p-level F crit
Between!Groups! 5,479.! 2! 2,739.5! 2.43511! 0.12138! 5.13545!Within!Groups! 16,875.! 15! 1,125.!
Total 22,354.! 17!!! !! !! !!
Analysis of Variance (One-Way)
Summary !! !! !! !! !! !!Groups Sample size Sum Mean Variance
0% Leucine 6! 87.! 14.5! 280.7!0.1% Leucine 6! 95.! 15.83333! 169.36667!1% Leucine 6! 25.! 4.16667! 34.56667!!! !!
ANOVA !! !! !! !! !! !!Source of Variation SS df MS F p-level F crit
Between!Groups! 489.33333! 2! 244.66667! 1.51455! 0.2517! 5.13545!Within!Groups!
2,423.16667! 15! 161.54444!
Total 2,912.5! 17!!! !! !! !!
Anova:!TwoAFactor!With!ReplicaFon!
SUMMARY' Count' Sum' Average' Variance'Row!1! 3! 478! 159.3333333! 908.3333333!Row!2! 3! 427! 142.3333333! 457.3333333!Row!3! 3! 493! 164.3333333! 1406.333333!Row!4! 3! 503! 167.6666667! 2025.333333!Row!5! 3! 516! 172! 2061!Row!6! 3! 514! 171.3333333! 933.3333333!Row!7! 3! 269! 89.66666667! 4974.333333!Row!8! 3! 322! 107.3333333! 5942.333333!Row!9! 3! 352! 117.3333333! 9464.333333!Row!10! 3! 352! 117.3333333! 1416.333333!Row!11! 3! 327! 109! 1389!Row!12! 3! 322! 107.3333333! 142.3333333!Row!13! 3! 182! 60.66666667! 2886.333333!Row!14! 3! 180! 60! 1776!Row!15! 3! 161! 53.66666667! 1564.333333!Row!16! 3! 105! 35! 1813!Row!17! 3! 51! 17! 1!Row!18! 3! 53! 17.66666667! 40.33333333!Row!19! 3! 50! 16.66666667! 564.3333333!Row!20! 3! 35! 11.66666667! 134.3333333!Row!21! 3! 40! 13.33333333! 204.3333333!Row!22! 3! 35! 11.66666667! 408.3333333!Row!23! 3! 26! 8.666666667! 8.333333333!Row!24! 3! 21! 7! 49!
Column!1! 24! 2004! 83.5! 3499.913043!Column!2! 24! 2204! 91.83333333! 4219.623188!Column!3! 24! 1606! 66.91666667! 6760.862319!
ANOVA!Source'of'Varia7on' SS' df' MS' F' P;value' F'crit'
Rows! 259631.5! 23! 11288.32609! 7.072725457! 1.07EA08! 1.766805319!Columns! 7722.333333! 2! 3861.166667! 2.41922244! 0.100232419! 3.199581706!Error! 73417.66667! 46! 1596.036232!
Total! 340771.5! 71!!! !! !! !!
UV Hood Specs • 254 nm • Average of 0.03 mWt/cm2 at12in from exposure
(0.01-0.03) • 0.07-0.09mW/cm2
![Accessories for the Laser Scattering Particle Size ......[Measurement range] 0.01–1000 μm, suits organic solvents Cell volumes: 5 mL, 10 mL, 15 mL Paste Cell The Paste Cell can](https://img.pdfslide.net/doc/110x75/5f30a22d4122bd0397092e6d/accessories-for-the-laser-scattering-particle-size-measurement-range-001a1000.jpg)
