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 (map) exists in multiple copies in each transformed yeast cell; however, it is unstable and can be lost from transformed yeast at a relatively high frequency. The YCpADE1 plasmid (map) 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.

Basic transformation protocol

Day One: Touch a sterile toothpick to the stock sample of yeast HA1 or HA1L and then make two or three streaks of the cells on a YED agar plate. Incubate the plate at room temperature or in a 30 C. incubator for 1-2 days. Alternatively, a liquid culture can be grown with shaking, overnight.

Day Two:

• 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 microliters 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. Discard the supernatant (liquid solution) from the microfuge tube(s).
• 9. 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.
• 10. 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.
• 11. Incubate the plates for 3-5 days at room temperature or in a 30oC incubator.

Day 5: Observe and record your results. For each experimental condition count the number of colonies and record the yeast colony colors.

Plasmid Loss: An Extension of the Transformation Exercise

Transformed cells containing the unstable YEpADE1 plasmid will maintain the white phenotype only under selective pressure (the absence of adenine in their growth medium). In these conditions, cells which do not contain the plasmid will die because they cannot make adenine. However, under non-selective conditions (the presence of adenine), cells which lack the plasmid are able to survive. The YepADE1 plasmid is unstable because it is independent of the yeast's chromosomes and is not necessarily evenly distributed during mitosis. In a population of dividing cells, some daughter cells may not receive a copy of the plasmid. Removing the selective pressure allows survival of those cells that have lost the plasmid. Colonies produced from these cells will be red or red sectored.

This exercise demonstrates that the maintenance of the white transformant phenotype is dependent on growth under selective conditions (MV). One of the white transformant colonies containing the YepADE1 plasmid is streaked onto non-selective media, YED. This medium is non-selective because it contains adenine.

Day One:

• 1. Isolate single colonies from one of the transformant colonies. Touch a sterile toothpick to one of the white transformant colonies. Pick a large white colony (avoid tiny colonies). Make a streak on a new YED plate. Then use a new sterile toothpick to make another zigzag streak across the first one on your YED plate. Continue using fresh sterile toothpicks to make 4 or 5 more zigzag streaks in this manner. The last streaks should give you some single colonies. Each colony will grow from a single transformant cell.
• 2. Follow the same procedure to streak out some of the transformant cells on an MV plate. Incubate all plates at 30 C. or room temperature until single colonies form and their color can be determined.

Recipes for solutions

Sterile Solutions: prepare working solutions from stock solutions just prior to use. Use distilled water and autoclave stock solutions and working solutions.

Working solutions: LiAc/TE solution: for 100 mL, mix 10 mL 1M LiAc stock solution + 1 mL 1M Tris-HCl stock solution + 1 mL 0.1M EDTA stock solution + 88 mL distilled water.
TE solution: for 100 mL, mix 1 mL 1M Tris-HCl stock solution + 1 mL 0.1M EDTA stock solution + 98 mL distilled water.
40% PLT solution: for 200 mL, mix 160 mL 50% PEG stock solution + 20 mL 1M LiAc stock solution + 2 mL Tris-HCl stock solution + 2 mL EDTA stock solution + 16 mL distilled water.

Stock solutions: 1M LiAc stock solution: 10.2 g LiAc.2 H2O + 80 mL distilled water; adjust pH to 7.5 with acetic acid; adjust volume to 100 mL with distilled water.
1M Tris-HCl stock solution: 12.1 g Trizma base + 80 mL distilled water; adjust pH to 8.0 with HCl; adjust volume to 100 mL with distilled water.
0.1M EDTA stock solution: 3.72 g ethylenediamine tetraacetic acid, disodium salt + 80 mL distilled water; adjust pH to 7.0 with sodium hydroxide; adjust volume to 100 mL.
50% PEG stock solution: 100 g polyethylene glycol 4000;to adjust to 200 mL with distilled water.

For additional information

For a very extensive discussion of issues involved in yeast transformation, see The Yeast Transformation Homepage maintained by Dr. Dan Gietz of the University of Manitoba. The protocol described here is based on the original transformation method of Gietz and Schiestl.

Main yeast lab page

Next lab