Yeast

Emily Rychling March 6, 2009 Biology E Per. Yeast with Different Potassium Levels Problem What affect does the mutant gene have on the cells ability to survive in environments with different levels of potassium? The job that our class is trying to complete is figuring out exactly how the gene mutation affects the organism that carries it. The class set up an experimental design to figure out the problem that Dr. Berkowitz has. ATK sands for active potassium transport. In mutated gene, it lacks the ATK-1 gene allowing it to not transport potassium into the cell. Having the ATK-1 gene is important for plants because it allows potassium flow in the cell. This happens in mutated genes. There is a blockage un-enabling KT to flow. Recent labs we did showed us that the mutant plant was dying and the wild-type was green and healthy. That is because of the lacked AKT-1 gene. Yeast, our transgenic organism, is a good to test because it is small and grows easily. We used a mutation yeast sample and wild-type yeast sample. We used both mutated yeast and wild-type to see if one worked better than the other. Our independent variable was the different concentrations of yeast, which tested where the yeast grew better. The dependent variable was the growth; stating which concentration of yeast grew better. Experimental Design Materials • Pipette • Test Tubes • Yeast concentration (Mutated and Wild-type) • 3 dishes with agar • Gloves Procedure 1. There were groups of 8. Four of them did the mutated yeast concentration, and 4 did the wild-type making the experiment flow better. 2. First, gather all materials (pipette, test tubes,10%, 1%, .1% and .01% yeast concentration, dishes with agar and gloves). 3. Take a test tube and label it “10%.” Using the pipette, add 50μl of the yeast solution and then add 450μl of distilled water. Mix it gently. 4. Then take the second test tube and label it “1%.” Add 50μl of the 10% yeast solution and put it in the new tube. Then add 450μl of distilled water and mix gently. 5. Take another tube (3rd tube) and label it “.1%.” Add 50μl of the 1% yeast solution to the third tube and then add 450μl of distilled water. Mix it gently.

6. With the fourth tube, label it “.01%” and take 50μl of the .1% yeast solution and then add 450μl of distilled water. Mix it gently. 7. All together you have 4 test tube samples with 10%, 1%, .1%, and .01% yeast concentrations. *NOTE: The different samples will help show the growth of the yeast. 8. Take your 3 agar dishes. Label the bottom of each dish with an arrow pointing to the top, so you know which is the top. *NOTE: See figure 1

Figure 1

9. Also label the bottoms with 50mM, 100mM and 150mM. NOTE: On your 50mM .. 10. With your 10% yeast tube, take a 5μl sample using the pipette and place it on the left side of the agar dish. NOTE: While you are adding your 11. Take your 1% yeast tube and take a 5μl sample using the pipette and place samples to the agar it next to the 10% yeast sample. dishes, it is very 12. Then take your .1% yeast tube and place a 5μl using the pipette sample common for different and place it next to the 1% yeast sample. DNA to come in contact and land onto 13. For the last sample, take your .01% yeast tube and place a 5μl sample the agar, be very using the pipette and place it next to the .1% yeast sample. careful and close the *NOTE: Your 50mM agar dish should look like figure 2 below. dish with the lid as much as possible!

Figure 2

14. Your are going to want to repeat steps 10-13 with the 100mM agar dish, and then repeat steps 10-13 again for the 150mM agar dish also. 15. All your class will be using the same agar dishes, so make sure you record the data where you placed your samples (They could be in different rows: See figure 3). 16. Once the class adds all their samples, place the dishes in the incubator for 24-48 hours.

After your cultures grow… 1. After the dishes are in the incubator for 24-48 hours, remove the dishes. 2. Observe your data and look at the differences between your 4 samples in each dish. Notice the growth and shape. NOTE: The bigger the spots, the better the growth and smaller shows less growth. 3. Record all your data for each dish. Data

The samples circled in red represent my group’s data. As you can tell, the first sample on the left in each dish (10% yeast) looks like an exact circle. The second sample (1% yeast) Is almost an exact circle, but in the 50mM you can tell it’s a little less growth. With the third sample (.1% yeast) it is a lot less growth and kind of looks like tiny. It is also not much of a circle anymore in any of the samples in the 50mM, 100mM and 150mM. As the concentration level goes down, so does the density level. They start losing their “circle” shape. Below is the class’ overall data in the sketches.

Conclusion In this lab, I learned a lot about potassium and the ATK-1 gene. From my problem I was originally experimenting, “What affect does the mutant gene have on the cells ability to survive in environments with different levels of potassium?,” I concluded that the mutant gene did not survive well in the potassium levels. I figured out that the mutant gene has a nonworking ATK-1 gene. The wild-type gene has a working ATK-1 Arabidopsis gene. It was harder for the potassium to get into the cells of the lower level concentration of yeast. With the 150mM, it was higher potassium level which made it easier to get into the yeast cells. The growth level in all of the mutant yeast cells was very low. The spotting showed this. Below is a diagram of what is happening with the cells.

AKT stands for active potassium transport. That is how the AKT-1 gene was a nonworking gene in the mutant yeast sample. If you take a look at the extra DNA spots on the agar dishes, you can notice that other DNA was collected on the agar. Either someone’s glove was contaminated with DNA, or another type of DNA. My group was very efficient on following directions and was as careful as possible. In future experiments I would make sure that my gloves weren’t contaminated with DNA, and I closed the dish lid as soon as possible. Even though it may have not been my group who contaminated the agar dishes, it is still important to watch out for this. It is very important because it could ruin your whole experiment, depending on what you are doing. I had fun doing this lab, and would love to further it!