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Exploring DNA Damage: What Effect Do Ultraviolet Rays Have on Yeast Colony Growth?

Difficulty
Time Required Long (2-4 weeks)
Prerequisites None
Material Availability You will need to order a UV-sensitive yeast kit online. See the Materials and Equipment list for details. Note: You might have to have the yeast delivered to a school if the company does not deliver to residential addresses.
Cost High ($100 - $150)
Safety Pouring hot agar plates should be done with caution.

Abstract

Though the Sun provides heat and light, which are essential for life on Earth, ultraviolet (UV) rays in sunlight can cause damage to DNA. In this science fair project, you will experiment with a strain of yeast that is super-sensitive to UV light. This project will demonstrate the lethal effects of UV light when DNA damage is not repaired.

Objective

Measure the lethal DNA effects of sunlight on UV-sensitive yeast.

Credits

David B. Whyte, PhD, Science Buddies

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Last edit date: 2013-09-30

Introduction

Baker's yeast (Saccharomyces cerevisiae) is used extensively by bakers and brewers around the world. This little eukaryote is also used in many laboratories as a model organism. As a eukaryote, S. cerevisiae has a nucleus, mitochondria, and other internal structures not found in bacteria. Some of the factors that make them attractive as model organisms are their small size, short generation time, common availability and well-studied genetics. As a single-celled organism, S. cerevisiae can be easily cultured on agar plates. Under favorable growing conditions, the population size can double every two hours or so. Because they grow quickly, a single cell can grow into a visible colony in 24–48 hours. Yeast cultures can be grown at room temperature, although they will grow faster if they are in an incubator at 30 degrees Celsius.

In addition to the features mentioned above, another advantage of working with yeast is that methods have been developed that allow researchers to knock out genes of interest. In a knockout strain of yeast, a particular gene has been removed from the organism's genome. Knocking out a gene is a straightforward way to learn what role the gene product is playing in the life of the organism. For this biotechnology science fair project, you will be working with a strain of yeast that is genetically engineered to be DNA-repair-deficient. In other words, DNA damage caused by the Sun is lethal to the yeast cell in which it occurs if it is not repaired. Normal yeast (and human) cells have enzymes that quickly repair DNA damage. In the DNA-repair-deficient yeast, however, some of these DNA repair enzymes have been knocked out, making the yeast super sensitive to UV light. In conditions that are easily tolerated by normal (wild type) yeast, such as exposure to light from the Sun for several minutes, many repair-deficient yeast are killed. This biotechnology science fair project is based on a kit that contains everything you will need to grow yeast colonies on agar plates, as well as the special DNA-repair-deficient strain of yeast. Though the kit contains everything you need and has detailed instructions, as well as background information, this is an advanced biotechnology science fair project, which will require creative problem solving and independent research on your part, for successful completion.

Terms and Concepts

  • Baker's yeast (Saccharomyces cerevisiae)
  • Eukaryote
  • Model organism
  • Nucleus
  • Mitochondria
  • Agar plate
  • Knockout gene
  • Genetic engineering
  • DNA-repair-deficient
  • DNA damage
  • Enzyme
  • Wild type
  • Serial dilution
  • Culture medium

Questions

  • Based on your research, where is UV light on the electromagnetic spectrum?
  • What kind of DNA damage is caused by UV light?
  • What are the main mechanisms used by wild-type yeast to repair DNA damaged by sunlight?
  • What are some human health problems that are caused by over-exposure to UV light from the Sun (don't forget to include diseases of the eye).
  • In what ways is yeast a good model organism for studying human DNA repair?
  • What are serial dilutions?

Bibliography

Materials and Equipment

  • UV-sensitive yeast culture and materials to grow yeast. The Measuring the Power of the Sun Kit is available from Carolina Biological, item # 173605B. You might have to have the yeast delivered to a school if the company does not deliver to residential addresses. The kit includes the following:
    • UV-sensitive yeast strain (mutant in several DNA repair pathways)
    • Sterile dilution tubes and sterile toothpicks
    • Yeast medium, yeast extract + dextrose (YED)
    • Petri dishes
    • Sterile distilled water
    • Pipettes
    • Glass spreading beads
    • Instructions
  • Disposable gloves (1 box). These can be purchased at a local drug store or pharmacy, or through an online supplier like Carolina Biological. If you are allergic to latex, use vinyl or polyethylene gloves.
  • Microwave oven
  • Oven mitts
  • Heat-resistant gloves
  • Permanent marker
  • Aluminum foil
  • Stopwatch or timer
  • Plastic bowl
  • Plastic wrap
  • Graph paper
  • Lab notebook

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Experimental Procedure

Important Notes Before You Begin:

  • This procedure is based on the manual for the kit "Measuring the Power of the Sun" from Carolina Biological. Read the manual before starting the procedure.
  • For each phase of the procedure, the work area should be clean and free of drafts. Wipe down the surface with soapy water prior to working with yeast culture medium.
  • You will need to perform this experiment when there is some sunlight—mid-morning, noon, or mid-afternoon.
  • You will need to check with your science fair's Scientific Review Committee before starting this experiment, as the strain used is considered to contain recombinant DNA (rDNA). For more information, visit Projects Involving Potentially Hazardous Biological Agents and Scientific Review Committee.

Pouring the YED Plates

  1. Loosen the cap of the YED agar bottle.
  2. Heat the bottle in a microwave until the contents are completely melted.
    1. Stop the microwave to swirl the bottle every minute or so to keep the contents from boiling over.
  3. Pour the melted YED agar into 10 petri dishes.
    1. Cover them immediately after pouring so the dishes remain sterile.
    2. Protect your hands with heat-resistant gloves.
    3. Pour just enough agar to cover the bottom of each plate.
    4. Let the agar harden at room temperature overnight.

Streaking the Master Plate

  1. Be sure your work area is clean and free of drafts.
  2. Put on a pair of disposable gloves to keep the toothpicks sterile. Open one of the boxes of sterile toothpicks that came with the kit.
  3. Touch the tip of a toothpick on an area where you can see yeast growing in the tube.
    1. The yeast is shipped in a tube containing agar and nutrients (called the slant).
  4. Glide the toothpick in a zigzag pattern across one of the YED plates that was poured previously. See the Science Buddies page, Inoculation: How to Put the Bacteria You Desire on a Petri Dish for more information.
  5. Use a fresh toothpick (without yeast) to make another zigzag pattern through the first zigzag pattern. The idea is to get separate yeast cells that will grow into well-separated colonies.
  6. Label the plate "Master Plate" with the permanent marker. Also put the date on the plate.
  7. Wrap the plate in aluminum foil to protect it from light.
  8. Allow the yeast to grow for two days at room temperature.

Labeling the Tubes and Making a Yeast Cell Suspension

  1. Label five tubes, as follows:
    • 1.   1
    • 2.   1:10
    • 3.   1:100
    • 4.   1:1,000
    • 5.   1:10,000
  2. After the two days have passed, put on a pair of disposable gloves and use a sterile toothpick to collect a mass of yeast from the master plate. The mass of yeast should be about 1 mm in diameter.
  3. Smear the yeast inside the tube labeled "1," toward the bottom on the side of the tube.
  4. Use a 5-mL bulb pipette to add 5 mL of sterile water to the tube with the yeast.
  5. Shake the tube until the yeast are suspended.

Making Serial Dilutions

You will now create a set of serial dilutions. Use a new, different pipette for each transfer below.

  1. Use the 3.5-mL sterile pipette to add 2.25 mL of sterile water into each of the tubes labeled 1:10, 1:100, 1:1,000 and 1:10,000.
  2. Use a clean 1-mL pipette to transfer 0.250 mL of yeast from the tube labeled 1 to tube labeled 1:10.
  3. Mix thoroughly.
  4. Use a clean 1-mL pipette to transfer 0.250 mL of yeast from the tube labeled 1:10 to tube labeled 1:100.
  5. Mix thoroughly.
  6. Use a clean 1-mL pipette to transfer 0.250 mL of yeast from the tube labeled 1:100 to tube labeled 1:1,000.
  7. Mix thoroughly.
  8. Use a clean 1-mL pipette to transfer 0.250 mL of yeast from the tube labeled 1:1,000 to tube labeled 1:10,000.

Spreading the Yeast onto Agar Plates

  1. Label the bottoms of four agar plates, as follows. Labeling the bottom of the plates prevents them from getting mixed up if the lids are removed.
    • 1.   1:1,000/control
    • 2.   1:1,000/exposed
    • 3.   1:10,000/control
    • 4.   1:10,000/exposed
  2. With the lid sides down, open each plate, one at a time—just long enough to shake 4–5 glass spreading beads onto each lid.
  3. Close each plate immediately after the beads are in, and flip them back over.
  4. Use a 1-mL pipette to add 0.250 mL of yeast suspension to the appropriately labeled plate. In other words,-add yeast from the 1:1,000 to two plates and 1:10,000 to two plates.
  5. Spread the yeast cells by shaking the glass beads back and forth.
  6. Allow the plates to sit for 5–10 minutes to dry.
  7. To remove the beads, hold the plates vertically over a plastic bowl and open the plates just wide enough for the beads to drop out. The beads can safely be discarded in the trash can.

Exposing the Yeast to UV Light

  1. Remove the lid from one of the plates and immediately cover it with plastic wrap, as the lid of the plates may absorb UV light.
  2. Repeat this for all of the plates.
  3. Expose the plates labeled "Exposed" to the sunlight.
  4. Wrap the plates labeled "Control" in aluminum foil to protect the yeast from light.
  5. The time for which the yeast are exposed depends on the time of day and on the season. The plates should be positioned so that the sunlight hits the yeast from directly above. Use the guide below:
    1. Summer mid-morning: 3–4 minutes
    2. Summer noon: 2–3 minutes
    3. Summer mid afternoon: 3–4 minutes
    4. Spring and fall mid-morning: 5–6 minutes
    5. Spring and fall noon: 3–4 minutes
    6. Spring and fall mid-afternoon: 4–5 minutes
    7. Winter mid-morning: 40–50 minutes
    8. Winter noon: 15–20 minutes
    9. Winter mid-afternoon: 20–30 minutes
  6. After exposing the yeast, remove the plastic wrap and replace with the lids.
  7. Wrap the plates in aluminum foil to protect them from light and let them sit at room temperature for two days.

Repeating the Procedure

  1. Repeat the entire procedure at least two more times with fresh plastic ware to show that your results are reproducible.
  2. To dispose of the yeast cultures, wrap the plates and tubes in aluminum foil and place of them in the regular trash.

Analyzing the Results

  1. Unwrap the plates and count the number of colonies on each.
    1. Each colony is formed from a single yeast cell.
    2. Ideally, one of the control plates should have about 100 colonies, as this number is small enough that you can count individual colonies, but large enough that you can get an accurate percentage of killed cells.
    3. If the colonies are too close together to count, even at the 10,000-fold dilution, repeat the dilution series and add a 100,000-fold dilution.
  2. Graph the number of colonies for each plate. Put the number of colonies on the y-axis and the treatment and dilution on the x-axis.
  3. Calculate the percentage of cells killed by UV light. See Equation 1, below. Compare colony counts from plates with the same dilution.
    1. Divide the number of colonies on the exposed plate by the number of colonies on the control plate.
    2. Subtract this number from 1.
    3. Multiply the resulting number by 100.
    4. This yields the percentage of yeast killed by the sunlight.


Equation 1:

100 × ( 1 - colonies on exposed plate/colonies on control plate) = % killed

  1. Graph the percentage of yeast killed by exposure to the sunlight.

Safety

Microorganisms such as yeast are all around us in our daily lives and the vast majority of them are not harmful. However, for maximum safety, all yeast cultures should always be treated as potential hazards. This means that proper handling, cleanup, and disposal are necessary. Below are a few important safety reminders. You can also see the Microorganisms Safety Guide for more details.

  • Keep your nose and mouth away from tubes, pipettes, or other tools that come in contact with yeast cultures, in order to avoid ingesting or inhaling any yeast.
  • Make sure to wash your hands thoroughly after handling yeast.
  • Proper Disposal of yeast Cultures
    • Yeast cultures, plates, and disposables that are used to manipulate the yeast should be soaked in a 10% bleach solution (1 part bleach to 9 parts water) for 1–2 hours.
    • Use caution when handling the bleach, as it can ruin your clothes if spilled, and any disinfectant can be harmful if splashed in your eyes.
    • After bleach treatment is completed, these items can be placed in your normal household garbage.
  • Cleaning Your Work Area
    • At the end of your experiment, use a disinfectant, such as 70% ethanol, a 10% bleach solution, or a commercial antibacterial kitchen/bath cleaning solution, to thoroughly clean any surfaces you have used.
    • Be aware of the possible hazards of disinfectants and use them carefully.

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Variations

  • Use the procedure above to make plates with diluted yeast cultures and expose the yeast to sunlight at various times of day; for example, at 10:00 AM, noon, and 2:00 PM. Use the same duration of exposure.
  • Expose the yeast for various durations at the same time of day, for example 0, 0.5, 1, 2, 4, and 8 minutes at noon.
  • Compare wild-type and DNA-repair-deficient yeast strains for UV sensitivity. Obtain wild-type S. cerevisiae from a science supply company; for example, Carolina Biological, item # 898900. Culture this strain and plate out dilutions, as described in the procedure. Compare the sensitivity of the wild-type and DNA-repair-deficient strains to ultraviolet light from the sun.

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