Download X-Ray Radiation Effects on Plasmid Gene Expression

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
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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:
Absorbed the plasmid.
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:
Absorbed the plasmid.
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
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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
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Two µl of plasmid DNA (pGEM 7) were transferred into eight microtubes.
For microwave radiation, two microtubes of DNA were exposed to the following durations of Xray radiation: 0, 5, 10, and 20 seconds.
After microwave radiation, 100 µl of competent cells (DH5-alpha E. coli) were added into each
microtube.
The cells were allowed to transform for 40 minutes immersed in ice.
After ice incubation, the microtubes were incubated in for three minutes in an incubator set to
37° C.
After heat shock, 120 µl of LB were added into each microtube.
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.
For X-ray radiation, four µl of plasmid DNA were transferred into eight microtubes.
Two microtubes were exposed to the following strengths of X-rays: 0, 50, 60, and 70 kvp.
After X-ray radiation, 200 µl of competent cells were added into each microtube.
The cells transformed.
After ice incubation, the microtubes were heat shocked.
After heat shock, 240 µl of LB were added into each microtube.
Cells were plated , plating four plates per tube.
Plates were incubated overnight at 37° C.
Ratio of Blue to White Colonies
Ratio of Blue to White Colonies
700
Amount of Colonies
600
500
Blue Colonies
400
White Colonies
300
Total
200
100
0
20 s.
10 s.
5s.
Microwave Exposure
0s.
Microwave Radiation Effects on Plasmid
DNA/Gene Expression
Microwave Radiation Effects on Plasmid DNA/Gene Expression
180
Amount of Colonies
160
140
120
100
80
60
40
20
0
20 s.
10 s.
5 s.
Microwave Exposure
0s.
Microwave Radiation Effects on Plasmid
DNA/Gene Expression (Averages)
Positive Control = lawn
Negative Control = 0
P-value = 0.000175
Microwave Radiation Effects on Plasmid DNA/Gene Expression
160
Amount of Colonies
140
120
100
80
60
40
20
0
20 s.
10 s.
5 s.
Microwave Exposure
0 s.
Dunnett Test Results
T-Critical = 3.29
Microwave
Exposure
T-Value
Interpretation
5 seconds
3
Insignificant
10 seconds
3.08
Insignificant
20 seconds
6.88
Significant