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.
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 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.
Hypothesis
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
3.
4.
5.
6.
7.
8.
and exposed to the following durations of UV light: 0 seconds, 5 seconds, 15
seconds, 30 seconds, and 60 seconds of ultraviolet light.
After ultraviolet light exposure, 100 µl of competent cells (DH5-Alpha E. coli)
were pipetted into each microtube.
The microtubes were placed in a microrack and immersed in ice for 40
minutes to allow transformation to occur.
After ice incubation , the microtubes incubated for 3 minutes in an incubator
set to 37°C.
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 LBAmp agar (- control).
The plates were incubated overnight at 37° C.
The number of surviving colonies were counted. Each colony was assumed to
have arisen from one transformed cell.
UV Radiation Effects on Plasmid DNA/Gene
Expression
180
160
Amount of Colonies
140
120
100
80
60
40
20
0
0 seconds
5 seconds
15 seconds
UV Light Exposure
30 seconds
60 seconds
UV Radiation Effects on Plasmid DNA/Gene
Expression
Positive Control=lawn
P=0.002743
Negative Control=0
140
Amount of Colonies
120
100
80
60
40
20
0
o seconds
5 seconds
15 seconds
UV Light Exposure
30 seconds
60 seconds
Dunnett Test Results
t-critical= 3.48
UV Exposure
T-value
Interpretation
5 seconds
1.99
Not Significant
15 seconds
3.48
Significant
30 seconds
4.39
Significant
60 seconds
4.04
Significant
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, 2005.
Clark, David. Molecular Biology Simple and Fun. New York: Warner Books, 2007.
“C.T. Chung.” One Step Prep of Compotent Escherida Coli. 20 Oct. 2006
<http://www.pnas.org/>
Krotec, Mark. "Transformation and its Importance." Personal interview. Sept. 2008.
"NCBI." Cloning and Transformation. 20 Oct. 2006
<http://www.ncbl.nih.gov/>.