Quantification and localization of MD-2, N-Gap1, Uga4, Gap1 and Vglut1 expressed using the nitrogen catabolite repression promoter system. A) Expression control by western blot done on total cellular extract after 24 and 48 hours of culture on inductive medium. The same amount of total protein (normalized by OD measurement) was loaded on each lanes. Proteins are revealed by an antibody against GFP. Expected sizes for the GFP-tagged proteins: MD2 (47.6 kDa), N-ter Gap1 (38.7 kDa), Uga4 (91.2 kDa), Gap1 (95.2 kDa), Vglut1 (90.9 kDa). B) In vivo fluorescence of expressing cells cultivated 24 hours on inductive medium. Untransformed cells have been used as negative control (−). All fluorescence pictures were taken using the same exposure time. C) Quantification of expression by GFP fluorescence measurements. Cells cultivated for 24 and 48 hours on inductive medium were submitted to GFP fluorescence measurements. Signal from non-induced cells was used for auto-fluorescence background subtraction. Fluorescence intensity were compared to a standard curve established using purified GFP. The quantification was calculated per gram of wet weight cells, corresponding to ~25 ml of fermenter culture at OD660 40.