By: Alec DiVito Central Catholic High School

Problem Does X-ray radiation damage plasmid DNA and alter gene expression? Does microwave radiation damage plasmid DNA and alter gene expression?

X-Ray Radiation X-Ray Radiation is light that has shorter wavelengths than ultraviolet. It ranges from 10nm to 0.01nm. Greater energy than UV and visible light yields a greater risk to life. They are given off during medical imaging techniques. This is where most people are exposed.

Measurement of the Strength of X-Rays A unit known as the kVp is used to measure the strength of X-rays. kVp is short for Kilovolt Peak. Instead of a constant strength and varying time intervals, the DNA was exposed to different strengths with the same amounts of time.

Effects of X-Ray Radiation Exposure in medical imaging is usually not long enough to cause any problems. However, prolonged exposure can lead to third degree burns. Thought to interfere with many important biological molecules including DNA. Increased mutation rate of DNA.

Microwave Radiation Microwave Radiation is light that has shorter wavelengths than radio waves and longer wavelengths than infrared waves. It ranges from one meter to one millimeter. Some, but not all, studies suggest that long-term exposure can have a mutagenic effect. During WWII, it was observed that individuals in the radiation path of radar installations experienced clicks and buzzing sounds in response to the microwaves radiation. Research by NASA in the 1970s has shown this to be caused by thermal expansion in parts of the inner ear.

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. LAC Z AMP r

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 affects of X-ray or microwave radiation on DNA structure and function. Specifically, to investigate the affect of X-ray or microwave radiation on DNA plasmid transformation and gene expression.

Experimental Measurement and Interpretation The effects of x-rays and microwaves on DNA transformation/gene expression was assessed by analyzing the host cell’s ability to grow in the presence of ampicillin and observing the ratio of blue to white colonies. 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 presence of ampicillin. If the cells are blue, those cells are assumed to have: 1. Absorbed the plasmid. 2. Expressed the Lac-Z gene. Allowing the cells to cut the X-Gal molecule resulting in a vivid blue color.

Hypothesis X-rays and microwaves will damage plasmid DNA and reduce transformation and gene expression efficiency. Null Hypothesis The X-rays and microwaves will not significantly affect cell transformation/gene expression.

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

Procedure 1. Two µl of plasmid DNA (pGEM 7) were transferred into eight microtubes. 2. For microwave radiation, two microtubes of DNA were exposed to the following durations of X- ray radiation: 0, 5, 10, and 20 seconds. 3. After microwave radiation, 100 µl of competent cells (DH5-alpha E. coli) were added into each microtube. 4. The cells were allowed to transform for 40 minutes immersed in ice. 5. After ice incubation, the microtubes were incubated in for three minutes in an incubator set to 37° C. 6. After heat shock, 120 µl of LB were added into each microtube µl aliquots of cell suspension were spread onto LB-amp X-gal agar plates, plating two plates per tube. Non transformed cells were plated onto LB agar (+ control) and LB-amp agar (- control) plates. 8. For X-ray radiation, four µl of plasmid DNA were transferred into eight microtubes. 9. Two microtubes were exposed to the following strengths of X-rays: 0, 50, 60, and 70 kvp. 10. After X-ray radiation, 200 µl of competent cells were added into each microtube. 11. The cells transformed. 12. After ice incubation, the microtubes were heat shocked. 13. After heat shock, 240 µl of LB were added into each microtube. 14. Cells were plated, plating four plates per tube. 15. Plates were incubated overnight at 37° C.

Ratio of Blue to White Colonies

Microwave Radiation Effects on Plasmid DNA/Gene Expression

Microwave Radiation Effects on Plasmid DNA/Gene Expression (Averages) P-value = Positive Control = lawn Negative Control = 0

Dunnett Test Results Microwave Exposure T-ValueInterpretation 5 seconds3Insignificant 10 seconds3.08Insignificant 20 seconds6.88Significant T-Critical = 3.29