Part B : Yeast Experiments

Yeast Transformation

One of the most important techniques of modern molecular biology is the ability to introduce a specific gene into an organism and have that genetic information expressed by the organism. This process, called genetic transformation, played a critical role in the history of molecular biology and the discovery that DNA was the genetic material. Four scientists, Griffith in the 1920's and Avery, McCarty, and MacLeod in the 1940's, showed that the apparent "transformation" of one bacterial type into another required DNA and no other biological molecule.

Today, transformation is not limited to bacteria, and in fact, is used routinely by molecular biologists to manipulate and study the behavior of genes. This experiment uses bakers' yeast (Saccharomyces cerevisiae). As a model system, yeast has the advantage over bacteria, of being a eukaryotic organism with subcellular organization like that of plants and animals, including humans. Yeast transformation involves several steps. After completion of these steps, the yeast will express the new characteristics or phenotype of the added DNA.

Experiment: This experiment uses a yeast strain which is defective in an adenine synthesis gene, ADE1. The metabolic defect in the mutants causes them to require adenine for growth and to accumulate a red pigment which causes the colonies to appear pink or red in color. When these ade1 mutant yeast are transformed with the normal ADE1 gene, they gain the ability to grow in the absence of adenine and form normal cream colored colonies.

The DNA molecules used in this transformation system are plasmids, small, circular pieces of DNA. They behave in many ways like real chromosomes which carry genes in cells but plasmids are much more convenient. There are two types of plasmids carrying the yeast ADE1* gene available for use in this experiment. The YEpADE1 plasmid exists in multiple copies in each transformed yeast cell; however, it is unstable and can be lost from transformed yeast at relatively high frequency. The YCpADE1 plasmid exists in one copy in a haploid yeast cell, just like the normal chromosomes, and since it carries a yeast centromere, will be stably sorted into the daughter cells each time the yeast divides. Either plasmid can be used effectively to demonstrate the phenomenon of transformation.

*the cloned ADE1 gene used in these plasmids was provided by Dr. David Kaback of the University of New Jersey School of Medicine and Dentistry.

Map of Plasmid YCpADE1
Map of Plasmid YEpADE1

Time Line:

If you incubate your cultures at 30o C you will be able to follow this schedule; at room temperature you may have to double the time between steps.

The Day before: 15 min Getting ready
1st Day: 50 min Transform the cells
5th Day: 20 min Observe and analyze results

Materials:

Materials for each student or team

Common Materials

Getting Ready:

Time Line: The day before: 15 min:
Procedure:
1. Touch a sterile toothpick to the stock sample of yeast HA1L or HA1 and then make two or three streaks of the cells on a YED agar plate.
2. Incubate the plate at room temperature or in a 30o C incubator for 1-2 days.
One plate should provide enough cells for up to 4-5 students or teams.

Teacher Tips
Technical Tip: The suspension should be quite turbid. The LiAc/TE solution assists in making the yeast cells competent (ready to be transformed).
Technical Tip: The YEpADE1 or YCpADE1 plasmid DNA is mixed with a non-specific, sonicated or fragmented DNA that serves as a carrier. This carrier DNA makes the transformation more efficient and probably helps to protect the plasmid DNA from cellular nucleases which would otherwise destroy the plasmid.
Technical Tip: Be sure that the yeast are well suspended before transferring.
Technical Tip: The 40% PLT solution helps to force the plasmid DNA into the competent yeast cells.

Transforming the cells:

Time Line: 1st Day: 50 min
Procedure:
1. Prepare yeast/LiAc/TE suspension. Use the flat end of a sterile toothpick to scrape about 2-3 centimeters of a yeast streak from the agar. Suspend the cells in 0.5 mL of LiAc/TE solution in a sterile microcentrifuge tube.

2. Prepare DNA / carrier DNA tubes. To a new sterile microcentrifuge tube, add 21 uL of the plasmid DNA/carrier DNA mix. You may wish to make some control tubes. Some examples of possible control tubes are
1) carrier DNA only
2) plasmid DNA only
3) no DNA

3. Add yeast to DNA tubes. To the DNA tube(s) you prepared add 0.2 mL per tube of the yeast/LiAc/TE suspension from step 1. Discard the remainder of the yeast/LiAc/TE suspension.

4. To the DNA/yeast suspension(s), add 1.2 mL of 40% PLT solution per tube.

5. Tightly close the tube and mix well by inverting the tube several times.

6. Incubate this mixture at room temperature for approximately 20 minutes.

7. Heat shock the DNA/yeast/PLT suspension(s) for a minimum of 5 minutes (maximum of 15 minutes) in a 42oC water bath.

8. Centrifuge the suspension to pellet the yeast cells. ( Teacher Tips )
Technical Tip: The TE solution is used to replace the LiAc and PLT solutions for plating.
Technical Tip: MV medium provides only minimal nutrients, an energy source, and vitamins. MV does not supply adenine.

Plating the cells:
9. Discard the supernatant (liquid solution) from the microfuge tube(s).

10. Resuspend the pelleted yeast cells by adding 1 mL of TE solution. Tap gently, use a sterile toothpick or vortex to suspend the cells in the TE solution.

11. Plate suspension on MV agar. From each yeast suspension tube use a fresh sterile pipet to transfer 0.2 mL of yeast suspension to each of one or two MV (selective medium) plates. Use a fresh sterile spreader to spread the cells from each tube evenly over the surface of the agar. Teacher Tips
12. Incubate the plates for 3-5 days at room temperature or in a 30oC incubator.

Observe and analyze results:

Time Line: 5th Day: 20 min
1. Observe and record your results. For each experimental condition count the number of colonies record the yeast colony colors.

Questions:

1. Which cells can grow on MV (the ones with or without the ADE1 plasmid)? Explain your results in terms of the presence or absence of the plasmid.

2. Compare your plasmid DNA plate with others in the class. Did all the plates have the same number of colonies? Were the colonies all the same color? Were the YCpADE1 plates and the YEpADE1 plates the same? Write an explanation for any differences observed in your class.

3. Did any of the control plates have colonies? If they did offer a possible explanation.

( Answers )

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Last updated Wednesday, 04-Dec-2002 20:58:28 UTC