Figure 2.

The activity of different variants of the KAR2 upstream region in P. pastoris X33. The promoter activity of the different variants of the KAR2 upstream region was measured as a green fluorescence of sfGFP (channel FL1) with flow cytometry, as the gene of sfGFP was inserted downstream of the KAR2 upstream region. A variant with no promoter in front of the sfGFP gene (x) was used as a negative control. P. pastoris strains with the different variants of the KAR2 upstream region (FL, −190 bp, −190 bp mut., −77 bp) controlling sfGFP expression, and the negative control strain (x-sfGFP) were cultured overnight in shake flasks with YPD, then split into three parallels and further incubated for another 2 h. One parallel was cultured under normal conditions without experimental stress (black bars), the second parallel was cultured in the presence of 3 mM DTT (dark gray bars) and the third parallel was cultured at an increased temperature of 39°C (light gray bars). The displayed values are median FL1 values of all events belonging to a gate defined in a FSC-SSC dot plot (distinguishing P. pastoris cells from the background, data not shown). The error bar is showing a standard deviation of three measurements.