Someone has just given you the cDNA for GFP and you would like to design a construct that would result in GFP becoming resident within the ER. What would you do to this cDNA that would result in this final location. Explain.

To design a construct that would result in GFP (Green Fluorescent Protein) becoming resident within the Endoplasmic Reticulum (ER), you would need to add an ER targeting signal sequence to the cDNA for GFP. This ER targeting signal sequence, also known as an ER retention signal or ER retrieval signal, helps direct proteins to the ER and prevent them from being secreted out of the cell.

Here's how you can modify the cDNA for GFP to achieve ER localization:

1. Obtain the cDNA sequence for GFP: This is usually provided in a plasmid or a vector format by the person who gave it to you. The cDNA contains the coding sequence for the GFP protein.

2. Identify an ER targeting signal sequence: The most commonly used ER targeting signal sequence is the KDEL motif. This sequence targets proteins to the ER and is recognized by the KDEL receptor, which ensures their retention within the ER.

3. Determine the position for adding the ER targeting signal sequence: The ER targeting signal sequence is usually added at the C-terminus of the protein. This way, it does not interfere with the folding and function of GFP.

4. Design primers and perform PCR amplification: Design forward and reverse primers specific to the cDNA sequence of GFP. The reverse primer should also include the ER targeting signal sequence (e.g., KDEL motif) at its 5' end. Perform PCR amplification using these primers to introduce the ER targeting signal sequence.

5. Verify the PCR product: Analyze the PCR product, containing the modified cDNA for GFP with the ER targeting signal sequence, by gel electrophoresis or any other suitable method. Confirm the expected size of the PCR product.

6. Clone the modified cDNA into an appropriate expression vector: Use restriction enzymes to cut and clone the modified GFP cDNA into a suitable expression vector that has a strong promoter and an ER retention signal. This vector should also provide antibiotic resistance or a selectable marker for efficient selection of transfected cells.

7. Transfect cells with the construct: Use a suitable transfection method to introduce the GFP-ER construct into target cells. This can be done via transfection reagents, electroporation, or viral transduction, depending on the specific cell type.

8. Analyze expression and localization: After transfection, confirm the expression of modified GFP by visualizing green fluorescence using fluorescence microscopy or flow cytometry. To verify ER localization, co-stain the transfected cells with an ER-specific dye or antibody that binds to an ER marker protein.

By following these steps, you can modify the cDNA for GFP to contain an ER targeting signal, resulting in the localization of GFP within the ER.