Vitamin C Attenuation of Plasmid

MutagenesisRyan Nguyen

Grade 11Central Catholic High School

Electromagnetic Spectrum

• Range of all types of radiation• 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 Beta-

galactosidase 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

• UV hood

• Gloves + safety glasses

• SDF (Sterile Dilution Fluid)

• LB agar plates

• LB-amp agar plates

• LB-amp X-gal agar plates

• Calcium competent DH5-Alpha E. coli

• pUC 18 plasmid DNA

• Spreader bars

• Ethanol

• Bunsen burner

• 1 M ascorbic acid stock solution

• 0.1 M ascorbic acid sub-stock solution

• Matches

• Turntable

• Vortex

• Sidearm flasks

Procedure 1 – Extracellular mitigation 1. Plasmids were diluted – 6 μL puc18 + 54 μL SDF2. Tubes with varying concentrations were made and labeled as follows:

a) Set #1 – control of 0 secondsb) Set #2 – 30 second exposurec) 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 SDF2. Tubes with varying concentrations (minus ascorbic acid) were made and labeled as follows:

a) Set #1 – control of 0 secondsb) Set #2 – 30 second exposurec) 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 concentrations5. 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  0 M 0.1 M 0.001M

Stock Solution [1M] 0 μL 1 μL 0 μL

Sub Stock Solution [0.01M]

0 μL 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

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 TestT- 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 TestT- 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