Reporter genes - Reporter proteins - Fluorescence reporters
3 important questions on Reporter genes - Reporter proteins - Fluorescence reporters
What two things can fluorescence visualize?
1. Promoter activity (fuse in gene)
2. Protein localization (fuse after protein gene)
2. Protein localization (fuse after protein gene)
Name 4 problematic situations with protein localization using GFP
1. The GFP may block the signal peptide (SP), as a result the localization cannot happen normally
2. Expression levels are too high --> cell gets saturated (too strong promoter)
3. Change in diffusion/transport of protein by GFP
4. Blocking the catalytic center by GFP
2. Expression levels are too high --> cell gets saturated (too strong promoter)
3. Change in diffusion/transport of protein by GFP
4. Blocking the catalytic center by GFP
5 step process of GFP construct setup
1. Find gene of interest
2. Get the mutant
3. Create construct
4. Transform mutant
5. Complemented = no disruption of gene function by GFP.
2. Get the mutant
3. Create construct
4. Transform mutant
5. Complemented = no disruption of gene function by GFP.
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