Part B: Yeast Experiments

Plasmid Loss

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 (Manney and Manney, 1992). Experiment: In this experiment you will demonstrate that the maintenance of the white transformant phenotype is dependent on growth under selective conditions (MV). You will take one of the white transformant colonies containing the YepADE1 plasmid and streak it onto non-selective media, YED. This medium is non-selective because it contains adenine. Time Line:

If you incubate your cultures at 30xC, you will be able to follow this schedule. At room temperature, you may have to double the incubation time.

1st Day : 10 min Streak Transformants
3rd Day: 15 min Count Red and Sectored Colonies
Materials:

Transformed yeast cells containing YEpADE1 plasmid Transformed yeast cells containing YCpADE1 plasmid
YED agar plate MV agar plate
Sterile toothpicks

Map of Plasmid YCpADE1
Map of Plasmid YEpADE1

Time Line: 1st Day 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 (See Figure 1).

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. (See Figure 1) Time Line: 3rd Day 3. Count the total number of single colonies on each plate. Count the number of white colonies on each plate. Use these numbers to compute the percent of transformants in each plate. 4. Which condition maintained the highest percentage of transformants?
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