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By: Alec DiVitoCentral Catholic High School
ProblemDoes X-ray radiation damage plasmid DNA
and alter gene expression?Does microwave radiation damage plasmid
DNA and alter gene expression?
X-Ray RadiationX-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-RaysA 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 RadiationExposure 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 RadiationMicrowave 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 DNAA 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
TransformationCells 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.
PurposeTo 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.
HypothesisX-rays and microwaves will damage plasmid
DNA and reduce transformation and gene expression efficiency.
Null HypothesisThe 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
Procedure1. 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.7. 100 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
Ratio of Blue to White Colonies
0
100
200
300
400
500
600
700
20 s. 10 s. 5s. 0s.
Microwave Exposure
Am
ou
nt
of
Co
lon
ies
Blue Colonies
White Colonies
Total
Microwave Radiation Effects on Plasmid DNA/Gene Expression
Microwave Radiation Effects on Plasmid DNA/Gene Expression
0
20
40
60
80
100
120
140
160
180
20 s. 10 s. 5 s. 0s.
Microwave Exposure
Am
ou
nt
of
Co
lon
ies
Microwave Radiation Effects on Plasmid DNA/Gene Expression (Averages)
Microwave Radiation Effects on Plasmid DNA/Gene Expression
0
20
40
60
80
100
120
140
160
20 s. 10 s. 5 s. 0 s.
Microwave Exposure
Am
ou
nt
of
Co
lon
ies
P-value = 0.000175Positive Control = lawn
Negative Control = 0
Dunnett Test Results
Microwave Exposure
T-Value Interpretation
5 seconds 3 Insignificant
10 seconds 3.08 Insignificant
20 seconds 6.88 Significant
T-Critical = 3.29
