I did some comparison of GFP expression for comparing different constitutive promoters in E. coli in my previous lab. I used flow cytometry to rank the promoters but I came across this paper which uses other methods to rank constitutive promoters (http://www.pnas.org/content/102/36/12678.full Tuning genetic control through promoter engineering).
-- For an absolute quantification of the number of molecules, the only thing I remember was from a conference where people were using fluorescent microscopes to count the number of fluorescent molecules per cell for investigating promoter stochasticity. For flow cytometrry, I have been told that ~200 GFP molecules are needed for having a signal, below you cannot really differentiate from noise. My data with really weak promoters were in fact around the noise threshold.
On Friday, May 15, 2015 at 6:09:31 PM UTC+1, Linden wrote:
On Friday, May 15, 2015 at 6:09:31 PM UTC+1, Linden wrote:
Hey guys,Thanks Nathan and Sebastian, I will compare the promoters. Just to clear it out, there is no way to know the amount of GFP inside the cell using only the spectrophotometer results? Lambert-beer equation doesn't apply in this case?
On Thursday, May 14, 2015 at 8:02:45 PM UTC-3, Linden wrote:Hi guys!I would like to rank in strength a few promoters I have. The promoters are cloned upstream GFP, so I used a spectrophotometer (485 nm excitation 525 nm emission) to measure the GFP in E. coli, just like the paper "Measuring the activity of BioBrick promoters using an in vivo reference standard". I did everything, and now I have a some numbers, but I don't know how to turn it in something useful. Anyone know which formula should I use to get a estimate of the GFP or mRNA concentration inside the cell?Thanks a lot!
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