Vitamin C Attenuation of
Plasmid Mutagenesis
Ryan Nguyen
Grade 11
Central Catholic High School
Electromagnetic Spectrum
• Range of all types of radiation
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Radio waves
Microwaves
Infrared
Visible
Ultraviolet
X-rays
Gamma rays
UV Light Rays
• Shorter wavelengths than visible light (150nm – 300 nm)
• Greater energy than visible light
• Higher risk to life
• Naturally from the sun
• Most are absorbed by the ozone layer
Effects of UV Light
• UV light that reaches earth can cause many problems
• Humans – Heavy exposure without protection leads to skin cancer &
photokeratitis
• Used for sterilization
• Interferes with biological molecules
• Increases mutation rate of DNA
Indirect DNA Damage
• Chromophore absorbs UV
photon, exciting it
• Exciting creates singlet
oxygen (102) or a hydroxyl
radical (•OH)
• Free radicals damage DNA
by oxidation
Antioxidants
• Molecule that inhibits oxidation of other molecules
• Oxidation causes chain reactions that can cause damage to DNA
• Antioxidants terminate these chain reactions
Ascorbic Acid
• Naturally occurring organic compound
• Has antioxidant properties
• Solid form dissolves in water
• One form of Vitamin C
• Derived from glucose
pUC 18
• Extraneous non-chromosomal
plasmid DNA
• Used as vector to carry new
genes into a host cell
• Engineered to include
ampicillin resistance gene
(ampr)
• Also has Lac-Z that codes for
beta-galactosidase
Lac-Z
• Peptide product of Lac-Z complements a beta-gal
mutation
• Creates beta-galactosidase
• Breaks lactose into its monomers
• X-gal is a structural analogue of lactose
• Used to reveal change from a B-gal minus bacteria to B-gal
plus
• Cells turn blue to signify change
Transformation
• Occurs when cells
absorb extraneous DNA
to express new
characteristics
• Recombinant DNA
technology uses natural
vectors of DNA
• Plasmids often used to
transform cells
Escherichia coli
• One of most common forms of bacteria found in many environments
• Gram (-) bacilli
• Part of human flora; found in colon and digestive tract
• Reproduction time of 30 minutes
• Most are non-pathogenic
• Aerobic
DH5-Alpha E. coli
• Strand of bacteria
• Naturally not resistant to ampicillin
• Used as host for ampr plasmids for transformation
• B-gal minus
• Unable to create fully functional tetrameric Betagalactosidase enzyme
• Lac-Z restores B-gal function
Experimental Measurement and
Interpretation
• Ascorbic acid effects were assessed by analyzing the host
cell’s ability to grow in the presence of ampicillin
• Analyzing the ratio of blue to white colonies
• If colonies are blue, they are assumed to have:
• Absorbed plasmid and ampr gene functional
• Lac-Z functioning properly
• If colonies are white, they are assumed to have:
• Absorbed plasmid and ampr gene functional
• Lac-Z gene was mutated or improperly expressed
Purpose
• Primary: To see if Vitamin C can mitigate the damage
from UV radiation on DNA
• Secondary: To see if Vitamin C has to be present inside
or outside of the cell to mitigate UV damage on DNA
Hypotheses
• Null: Vitamin C does not significantly mitigate UV damage on DNA.
• Alternative: Vitamin C does significantly mitigate UV damage on DNA.
• Null: Vitamin C does not have to be present inside or outside of cells
to mitigate UV damage on DNA.
• Alternative: Vitamin C has to be present inside or outside of cells to
mitigate UV damage on DNA.
Materials
• LB (Luria Broth)
• 1% tryptone
• 0.5% yeast extract
• 1% NaCl
• Microtubes
• Micropipettes + Tips
• Incubator
• Yeast extract
• Calcium competent DH5-Alpha E.
coli
• pUC 18 plasmid DNA
• Spreader bars
• Ethanol
• Bunsen burner
• 1 M ascorbic acid stock solution
• UV hood
• 0.1 M ascorbic acid sub-stock
solution
• Gloves + safety glasses
• Matches
• SDF (Sterile Dilution Fluid)
• Turntable
• LB agar plates
• Vortex
• LB-amp agar plates
• Sidearm flasks
• LB-amp X-gal agar plates
Procedure 1 – Extracellular mitigation
1. Plasmids were diluted – 6 μL puc18 + 54 μL SDF
2. Tubes with varying concentrations were made and labeled as follows:
a) Set #1 – control of 0 seconds
b) Set #2 – 30 second exposure
c) Set #3 – 120 second exposure
3. DNA was exposed to UV light (ascorbic acid + DNA)
• Set #1 – control of 0 seconds
• Set #2 – 30 second exposure
• Set #3 – 120 second exposure
4. Cells were transformed in separate microtubes – 4 μL exposed plasmid and
Vitamin C solution + 50 μL DH5- Alpha cells
• 45 minutes was allowed for transformation in ice.
• Heat shocked for 5 min. in incubator.
5. Cells plated – Add 210 μL LB to cells and plasmid
• 50 μL of mixture was added to a plate.
• Five plates per group total of 45 plates.
6. Incubated for 48 hours
Procedure 2 – Intracellular mitigation
1. Plasmids were diluted – 6 μL puc18 + 54 μL SDF
2. Tubes with varying concentrations (minus ascorbic acid) were made and labeled as
follows:
a) Set #1 – control of 0 seconds
b) Set #2 – 30 second exposure
c) Set #3 – 120 second exposure
3. DNA was exposed to UV light (DNA only)
• Set #1 – control of 0 seconds
• Set #2 – 30 second exposure
• Set #3 – 120 second exposure
4. Treated Plasmid DNA was exposed to Vitamin C to make the concentrations
5. Cells were transformed in separate microtubes – 4 μL exposed plasmid and Vitamin C
solution + 50 μL DH5- Alpha cells
• 45 minutes was allowed for transformation in ice.
• Heat shocked for 5 min. in incubator.
6. Cells plated – Add 210 μL LB to cells and plasmid
• 50 μL of mixture was added to a plate.
• Five plates per group total of 45 plates.
7. Incubated for 48 hours
Concentrations
0M
0.1 M
0.001M
0 μL
1 μL
0 μL
Sub Stock Solution 0 μL
[0.01M]
0 μL
1 μL
Plasmid solution
5 μL
5 μL
5 μL
SDF
5 μL
4 μL
4 μL
Total Volume
10 μL
10 μL
10 μL
Stock Solution
[1M]
P-value: 1.14E-11
Interaction P-value: 0.015619
P-value: 3.29E-20
Interaction P-value: 0.012511
Interaction P-value: 0.020321
0.001 M Dunnett’s Test
T- critical: 5.143
UV Exposures
T-value
Significance
Extracellular
30 seconds
N/A
N/A
120 seconds
1.5578
Not significant
Intracellular
30 seconds
4.6464
Not significant
120 seconds
5.1779
Significant
0.1 M Dunnett’s Test
T- critical: 5.143
UV Exposures
T-value
Significance
Extracellular
30 seconds
12.4821
Significant
120 seconds
6.3291
Significant
Intracellular
30 seconds
11.7619
Significant
120 seconds
10.5617
Significant
Conclusions
• First Null Hypothesis: Rejected
• Vitamin C (ascorbic acid) does significantly mitigate damage done by
UV radiation.
• Second Null Hypothesis: Rejected
• Vitamin C (ascorbic acid) has to be present inside cells (intracellular)
to mitigate damage done by UV radiation.
• It is not known in this experiment whether Vitamin C has to be
present outside cells (extracellular) to mitigate damage done by UV
radiation.
• Dunnett’s tests showed that ascorbic acid was significantly more
effective at higher concentrations with greater doses of radiation.
Limitations
• Cells were not Alpha
complement
• Slight lag in synchronization
of plating
• Only two concentrations
were used
• Only one method was used
• Small sample size
Extensions
• Properly identify that cells
are Alpha complement
• More trials
• Sequence the plasmid to see
if genes were truly mutated
• Utilize different plasmids
• Investigate genes of other
plasmids
• Utilize various types of
radiation and antioxidants
References
• Betsey, Tom. Microbiology Demystified. New York: Wagner, 2005. Print.
• Chung, C. T. "PNAS." PNAS. Web. 16 Dec. 2014.
• Clark, David. Molecular Biology Simple and Fun. New York: Warner, 2007. Print.
• "Cloning and Transformation." Web. 16 Dec. 2014.
• Ferguson, L.R., ed. "Mutation Research." Fundamental and Molecular
Mechanisms of Mutagenesis 12.1 (2007): 1+. Print.
• "Nutrigenomics." ScienceDirect - Home. Ed. L.R. Ferguson. Elsevier. Web. 16 Dec.
2014. <http://www.ScienceDirect.com>.
• "X-Rays." NASA Science. Ed. Ruth Netting. National Aeronautics and Space
Administration. Web. 04 Jan. 2010.
<http://science.hq.nasa.gov/kids/imagers/ems/xrays.html>.
UV Hood Specifications
• Model: Labconco Biosafety Cabinet
• Uses 254 nm UV lightbuilbs
• Generates 20-40 microwatts per cm2
• At the work surface it generates 0.7-0.9 microwatts per cm2