Reference no: EM132977101

BC2024 Principles of Molecular Cell Biology - James Cook University

Biomembranes: Active Transport in Yeast

Experimental Overview

* Additional information to the practical 1 manual is shown in green

You will be supplied with samples of Saccharomyces cerevisiae that have been glucose repressed, glucose starved (grown until glucose supplies are exhausted) and then grown in the presence of maltose, or grown in the presence of glucose. The expression of α-glucoside transporters and α-glucosidase will only be induced in the absence of glucose. You will monitor the transport of pNPαG across the membrane by measuring the presence of p-nitrophenol. p-nitrophenol is the product of pNPαG cleavage by cytoplasmic α-glucosidase. This reaction product will become bright yellow upon the addition of a strong base and has a strong absorbance at 400 nm.
There are two main objectives in this practical:
1) To demonstrate the uptake of different α-glucosides by S. cerevisiae
2) To explore the mechanism of glucose repression by S. cerevisiae

Transport Assay

1. Add pNPαG to your yeast cells to a final concentration of 5 mM (= 150 μl of 40 mM stock, final volume 1.2 ml). Mix by inversion. Incubate at 30°C [start timer immediately]

2. Remove 100 μl aliquots at specific time intervals and immediately place in a 100°C heating block for 3 min (double check the temperature is at least 95°C before using).
» Use the following time points: 0.5, 1, 2, 3, 4, 5, 7, 10, 15 min.
Note: If you miss a specific time-point then record the actual time the tube was boiled

3. Cool cells to room temperature and add 100 μl of 0.2 M NaHCO3 (pH 10).

4. Centrifuge for 2 min at ~10 000 g. Remove the supernatant to a fresh tube
Note: This step is not time sensitive. You may wait until you have all your tubes and then centrifuge all together. Make sure the centrifuge is balanced. Please speak to a demonstrator if you have never used a centrifuge before.

5. Add 100 μl to a microtitre plate and read absorbance at 415 nm to determine p-nitrophenol content. Checkpoint: Be careful not to transfer any particulates into the microtitre plate as this will interfere with your absorbance reading.

Data Analysis
Complete the following data analysis at home using Excel. If you have difficulties look back at the information from the computer workshop that was run in week 1 of this subject.
1. Create a table in Excel and input all your absorbance values. This will be the raw values you received straight from the plate reader.
a. For this you need to use blank-corrected data. If your data isn't automatically blank-corrected by the plate reader (check the absorbance of the well that contains your blank sample) then you need to create a new table of the blank-corrected values. For this table subtract the value in your blank well from the values in all of your sample wells. You should have duplicates for assays 1, 2 and 3. For your control use the data from practical 1, which had a single replicate only. An example table is shown below. This table will go in the Appendix in your report.

2. Below create another table and calculate the averages of all your duplicate experiments.

3. Calculate the standard deviations of all your duplicate experiments.

4. Draw a scatter plot comparing the two assays you did in the maltose cells (assay 1 and assay 2). Here you are comparing the transport of pNPαG to the transport of pNPαG + sucrose. Include the control in your plot. *Choose meaningful series names (that is, don't call them Assay 1, Assay 2 etc in your graph)
a. Select your averaged data (and your control) and draw a scatter plot. Do not add any lines connecting the dots.
b. Add linear trendlines. Display the R2 and equation of the line. (If your data isn't linear then you can choose a polynomial trendline instead, although this isn't the preference. Explain the need for this in your report)
c. Add errors bars. Select custom error bars and specify your calculated standard deviations for the vertical direction only (make sure you input the same values for your positive and negative error values).
d. Check axis titles, units and general formatting.
e. No chart title is required within the graph (you will have a title in your figure caption instead).

5. Draw a second scatter plot comparing pNPαG transport in the maltose cells and the glucose cells (assay 1 and assay 3).
a. Follow the points above in step 4 to create your scatter plot with trendlines and error bars. This time you do not need to include your control data.

Discussion
Interpretation of the results. Explain what your results mean and how they fit in with your aims (or objectives, as listed in the manual), expectations and with scientific consensus etc. Topics to discuss include, but are not limited to:
- Explain what your results suggest.
- Explain whether your results are as expected or unexpected.

- Compare your results to known principles and past research. Do your results fit with the literature?
- Suggest explanations of unexpected results
- Where appropriate, suggest how the experiments could be improved
Ensure you focus on the two objectives of the practical (page 4 of the practical 2 manual). This should incorporate the answers to the thought questions listed in the data analysis section of the prac manual (coloured blue, page 7), which are related to the objectives.

Attachment:- Manual_Active Transport.rar