Hey there,

I'm now doing an experiment where I want to follow the expression of a gene during a batch culture of yeast (cerevisiae). I first started to fuse my gene promoter of interest with lacZ, and using B-gal assay as a readout of gene expression (normalized to protein content). After this, I wanted to optimize the technique, by switching the reporter gene from lacZ to GFP, and recording a full growth curve in microplate reader. My OD curves look as in normal batch culture, and I'm able to detect GFP increase. However, it is very discrepant from the results using the lacZ: from the lacZ data, the gene starts to be transcribed at the middle of the growth curve, while by GFP, starts to increase from time wero, than decreases and stabilizes. We know from previous data that the correct trend should be the one of lacZ, so I'm having a problem with GFP. I normalized GFP according to the optical density, and subtracted the background of the same strain not expressing GFP. So, is it a problem of normalization? The signal of the strain without GFP shows an increase proportional to the cell density - which is normal, considering the cells autofluorescence. Is it degradation? Some mathematical operation I'm missing? Hope I'm not the first one facing this problem xD Thanks in advance