1. Vitamin C Attenuation of Plasmid Mutagenesis Ryan Nguyen Grade 11 Central Catholic High School

2. Electromagnetic Spectrum Range of all types of radiation Radio waves Microwaves Infrared Visible Ultraviolet X-rays Gamma rays

3. 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

4. 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

5. Indirect DNA Damage Chromophore absorbs UV photon, exciting it Exciting creates singlet oxygen ( 1 0 2 ) or a hydroxyl radical (OH) Free radicals damage DNA by oxidation

6. Antioxidants Molecule that inhibits oxidation of other molecules Oxidation causes chain reactions that can cause damage to DNA Antioxidants terminate these chain reactions

7. Ascorbic Acid Naturally occurring organic compound Has antioxidant properties Solid form dissolves in water One form of Vitamin C Derived from glucose

8. pUC 18 Extraneous non-chromosomal plasmid DNA Used as vector to carry new genes into a host cell Engineered to include ampicillin resistance gene (amp r ) Also has Lac-Z that codes for beta-galactosidase

9. 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

10. 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

11. 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

12. DH5-Alpha E. coli Strand of bacteria Naturally not resistant to ampicillin Used as host for amp r plasmids for transformation B-gal minus Unable to create fully functional tetrameric Beta- galactosidase enzyme Lac-Z restores B-gal function

13. 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 amp r gene functional Lac-Z functioning properly If colonies are white, they are assumed to have: Absorbed plasmid and amp r gene functional Lac-Z gene was mutated or improperly expressed

14. 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

15. 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.

16. 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

17. 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

18. 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

19. Concentrations 0 M0.1 M0.001M Stock Solution [1M] 0 μL1 μL0 μL Sub Stock Solution [0.01M] 0 μL 1 μL Plasmid solution5 μL SDF5 μL4 μL Total Volume10 μL

20. P-value: 1.14E-11

23. Interaction P-value: 0.015619 P-value: 3.29E-20

24. Interaction P-value: 0.012511

25. Interaction P-value: 0.020321

26. 0.001 M Dunnett’s Test T- critical: 5.143 UV ExposuresT-valueSignificance Extracellular 30 secondsN/A 120 seconds1.5578Not significant Intracellular 30 seconds4.6464Not significant 120 seconds5.1779Significant

27. 0.1 M Dunnett’s Test T- critical: 5.143 UV ExposuresT-valueSignificance Extracellular 30 seconds12.4821Significant 120 seconds6.3291Significant Intracellular 30 seconds11.7619Significant 120 seconds10.5617Significant

28. 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.

29. 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

30. 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.. "X-Rays." NASA Science. Ed. Ruth Netting. National Aeronautics and Space Administration. Web. 04 Jan. 2010..

31. UV Hood Specifications Model: Labconco Biosafety Cabinet Uses 254 nm UV lightbuilbs Generates 20-40 microwatts per cm 2 At the work surface it generates 0.7-0.9 microwatts per cm 2