By: Alec DiVito St. Elizabeth Elementary

Problem Does UV light damage plasmid DNA and alter gene expression?

UV Light Rays Ultraviolet (UV) rays are light rays that have shorter wavelengths than visible light. They range from 400nm to 10nm. Greater energy than visible light yields greater risk to life. They are naturally given off by the sun, but most are absorbed by the ozone layer.

Effects of UV Light The UV light waves that reach Earth can lead to many problems in vast numbers of species. Humans - Heavy exposure to UV light without protection may lead to skin cancer Used for sterilization Thought to interfere with many important biological molecules including DNA Increased mutation rate of DNA

pGEM 7 Plasmid DNA A plasmid is extraneous DNA that is not chromosomal. Often employed as a vector in biotechnology to carry new genes into a host cell. pGEM 7 is a much utilized plasmid containing a resistance gene to ampicillin. Contains 2977 base pairs. AMP r LAC Z

Transformation Cells that absorb extraneous DNA and express a new characteristic are referred to as transformed. Plasmids commonly used to transform cells. Recombinant DNA technology makes use of naturally occurring vectors of DNA.

Escherichia Coli Escherichia coli (E. coli) are a large and diverse group of bacteria commonly found in the intestine of most mammals. It has been the most studied type of bacteria in biological research. The majority of E. coli are harmless, but some can cause harmful, and even fatal, illnesses. E. coli are also commonly used as markers for water contamination. The E. coli in drinking water is not always harmful itself but can indicate that the water is contaminated with other bacteria and pollutants. E. coli is one of the most commonly used host cells for transformation (recombinant DNA) experiments.

Purpose To assess the effects of UV light on DNA structure and function. Specifically, to investigate the affect of UV light on DNA plasmid transformation and gene expression.

Experimental Measurement and Interpretation The effects of UV light on DNA transformation/gene expression was assessed by analyzing the host cells’ ability to grow in the presence of ampicilin. If cells grew, those cells are assumed to have: 1. Absorbed the plasmid. 2. Expressed the ampicillin resistance gene. Allowing the cells to survive in the ampicillin drug.

Hypothesi s UV light will damage plasmid DNA and reduce transformation and gene expression efficiency. Null Hypothesis The UV light will not significantly affect cell transformation/gene expression.

Materials LB (Luria Broth) LB agar plates LB-amp agar plates Competent DH5-alpha E. coli pGEM 7 plasmid DNA Spreader bar Ethanol Burner Microtubes Micropipets + Tips Incubator UV hood Gloves + Safety glasses

Procedures 1. 4 µl of plasmid DNA (pGEM 7) were transferred into 10 microtubes. 2. The microtubes of plasmid DNA were transferred to a sterile UV-culture hood and exposed to the following durations of UV light: 0 seconds, 5 seconds, 15 seconds, 30 seconds, and 60 seconds of ultraviolet light. 3. After ultraviolet light exposure, 100 µl of competent cells (DH5-Alpha E. coli) were pipetted into each microtube. 4. The microtubes were placed in a microrack and immersed in ice for 40 minutes to allow transformation to occur. 5. After ice incubation, the microtubes incubated for 3 minutes in an incubator set to 37°C. 6. After heat shock, 30 µl aliquots of cell suspension were spread onto LB-Amp agar plates. Non-transformed cells were plated on LB agar (+ control) and LB- Amp agar (- control). 7. The plates were incubated overnight at 37° C. 8. The number of surviving colonies were counted. Each colony was assumed to have arisen from one transformed cell.

Positive Control=lawn Negative Control=0

Dunnett Test Results t-critical= 3.48 UV ExposureT-valueInterpretation 5 seconds1.99Not Significant 15 seconds3.48Significant 30 seconds4.39Significant 60 seconds4.04Significant

Conclusion The null hypothesis was rejected for the following time periods: 15, 30, and 60 seconds. Exposure to UV light did appear to significantly affect the ampicillin resistance gene expression. Two possibilities seem likely: 1.the plasmid DNA was in some way altered resulting in less efficient transformation. 2. The ampicillin resistance gene was altered (mutated) preventing the host cell from acquiring ampicillin resistance.

Limitations/Extensions Increase the sample size. Utilize different plasmids. Investigate other plasmid genes and their expression (Lac Z). Synchronize the exact times of plating. Utilize various wavelengths of UV light.

Bibliography Betsey, Tom. Microbiology Demystified. New York: Wagner, Clark, David. Molecular Biology Simple and Fun. New York: Warner Books, “C.T. Chung.” One Step Prep of Compotent Escherida Coli. 20 Oct Krotec, Mark. "Transformation and its Importance." Personal interview. Sept "NCBI." Cloning and Transformation. 20 Oct