Photoreactivation, the process in which
the enzyme photolyase uses the energy from
sunlight to split pyrimidine dimers, is easy to
demonstrate. When you irradiate cells with
UV-C from a germicidal lamp most of the
damage to DNA is in the form of pyrimidine
dimers. Adjacent pyrimidine bases
(Thymine and Cytosine) in the DNA become
joined by covalent bonds formed between
their aromatic rings. If the cells are kept in
the dark, the excision, error-prone or
recombination repair systems will fix many
of the dimers. However, in this experiment
you will discover that another DNA repair
system, photoreactivation, can repair
additional dimers. Photoreactivation
requires exposure to a relatively intense
source of visible or UV-A light, so little if
any repair occurs under normal laboratory
lighting conditions. You can achieve a striking difference in survival of irradiated
cells by keeping some in subdued light and
exposing others to direct sunlight or bright
artificial light.
Experiment:
In this experiment you will spread known
numbers of yeast cells in Petri plates and
then expose the cells to various amounts and
combinations of UV-C and visible light.
You will use three types of conditions;
1)
unirradiated,
2) exposed to UV-C and
3)
exposed to both UV-C and visible light.
From your colony counts you will
calculate the amount of photoreactivation
from the difference between survival levels
for the photoreactivated samples and the
controls. Table 1 summarizes the results of
an experiment that demonstrates how UV-C
damage can be repaired by the visible
wavelengths of sunlight.
Time Line:
Day before:10 min Getting Ready
45 min Discussion of the
strategy and
objectives
Day 1: 50 min Dilution, Plating and
Irradiation of Cells
Day 3 or 4: 50 min Counting Colonies
and Analyzing
Results
Materials:
For each student or team:
Common Materials:
UV irradiation chamber with germicidal lamp Optional Materials:
1. Make a clean sterile work space by
wiping the table or bench with an
alcohol wipe. Because most
contamination is airborne select a
place free from drafts.
2. Open the yeast storage vial.
3. Using the broad end of a sterile
toothpick, pick up a small amount of
yeast from the agar slant in the vial.
4. Replace the lid. Tighten. ( Store in a
refrigerator to keep the cells viable for
up to nine months.)
5. Open the YED Petri dish just enough
so that you can reach into it with the toothpick full of cells.
6. Gently make several streaks of the
culture on the surface of the agar.
(Remember that you need not be able
to see the streaks to have enough to
grow into a visible culture overnight.)
7. Close the lid and incubate the culture
overnight at 30oC, or 2 days at room
temperature. (Most microbial cultures
should be incubated with the agar side
up to prevent condensation from
dropping on the colonies.)
(Teacher Tips)
Technical Tip:
The concentration factor is the ratio of
the number of cells plated on the
irradiated plate to the number of cells
plated on the unirradiated control plate,
based on the dilution series.
Time Line:
Day 1: 50 min
Procedure:
1. Use your survival data for strains HA2
and HB2 or the sample survival data in
Figure 2 to design your experiment.
Estimate the time of exposure and number of cells
you will need to plate so that 1/10th of the cells
survive and they produce 50 to 100 colonies per
plate.
2. Prepare a dilution series of strain HA2
or HB2 and spread the appropriate
dilutions on your plates using the
dilution and plating procedure
described in Figure 1.
3. Irradiate the samples.
Place your unirradiated control plate in the dark.
Remove the lids from two other plates, place the
plates in the irradiation box and expose them for
the chosen exposure time.
Then expose one of these irradiated plates to
visible light through a glass cover (keep the other
irradiated plate in the dark).
4. Incubate the plates until the colonies
are large enough to count.
At 30øC that will take about two days and at room
temperature it will take three or four days.
5. Count the colonies and tabulate your
data:
Make a table of dose, concentration factor, colonies/plate for each duplicate plate, and mean colonies/plate.
Calculate the surviving fraction for each treatment.
Teacher Tip
Figure 1 Serial Dilution and plating strategy for a survival
curve.
Name of Investigators
Date
Time
Ultraviolet Radiation Source
Yeast Strain
Growth medium
Incubation temperature
Diagram of Dilution and Plating Procedure:
Return to Contents
Treatment
Count
Plate 1
Count
Plate 2
Count
Plate 3
Mean
Count
Conc.
Factor
Surviving
Fraction
0 sec UV, kept in dark
__________ sec UV,
kept in dark
__________ sec UV plus
2 min in sunlight
Last updated Wednesday, 04-Dec-2002 15:00:06 CST