I planning to build a promoter trap GFP transposon library and FACS sorting to identify strong promoters under various growth conditions. Do you think it would be better to use a stable GFP variant to get more signal strength? Or would a high turnover GFP variant give you more sensitivity since strong GFP signal would require more expression?
I would definitely go with a stable GFP.
I assume you are talking about a PEST-GFP, that gets proteolitically cleaved in the proteasome, as an unstable GFP.
With the PEST-GFP, the degradation is quick and you get very little signal even in the high expressors. A stable GFP variant should give you a high signal to noise ratio and you will be sort out finer fractions to determine promoter strength on a greater gradation.
Unstable fluroescent proteins are useful to observe outputs that are changing in time. For example, if you want to measure how promoter output changes when the growth condition is varied, an unstable reporter might be useful to only see recently expressed fluorescent protein. If you're worried about the signal being too high or toxicity at high expression levels, maybe it's better to reduce the translation rate or increase the transcript degradation rate than to use an unstable GFP.
If you are just looking at a single output value per sample after some time of growth, then go ahead and use stable. The unstable variants are useful when you want to look at change, i.e. does expression go down when you change some condition.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology