BIO230 Genetics

Complete this assignment using the information from the lab video modules and the Miyawaki paper.

1. In the fluorescence microscopy video, fluorescein diacetate (FDA) was added to the trichomes and fluorescence visualised. Give two reasons why some organelles or cellular components might fluoresce when FDA is added, while others do not.

2. In fluorescence and confocal microscopes, there are usually three different lasers used. Suggest why we may need more than one laser for a given experiment.

3. A researcher has identified a new protein, protein A that they hypothesise interacts with protein B. To investigate this interaction, the researcher first wants to determine if the two proteins colocalise in the cell. List the type of microscope that would be best to study this, and indicate what other manipulations or preparations the researcher might need to do to prepare the sample.

4. Using your answer from the previous question, give one pro and one con for using this sample preparation approach.

5. When a protein of interest is tagged with a fluorescent protein like GFP, what feature of this fusion protein must be considered and tested before interpreting any experimental results? If this feature is not considered, explain why the researchers will have difficulty interpreting their results.

6. Compound light microscopes and transmission electron microscopes produce an image based on light transmission through the sample. Explain how the fluorescence microscope layout differs from that of transmission microscopes. Include the source of the light visualised in fluorescence microscopy in your answer

7. A scientist wants to study the kinetics of a specific membrane protein. They ligate the DNA coding region of the protein to the DNA coding region for GFP and introduce the DNA into cells. A day or two later, they observe the cells under the microscope and they are fluorescing green. They then shine a laser at a region of the cell repeatedly (or continuously) - that region loses its fluorescence. They monitor a separate region of the cell and find that fluorescence never decreases in that area

a) What is the full name of the technique that they performed?

b) What can they conclude from their result?

c) List another photobleaching technique and briefly indicate how it is different from the technique described here.

d) When or why might photoactivation techniques be preferred over photobleaching techniques?