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
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.
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.
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 14:58:28 CST