Figure 2: Western blot analysis of P. pastoris cultures induced with 0.1% methanol confirms expression and correct localization of Orco in the membrane of P. pastoris . An inverted image of the blot has been provided for visual clarity. Uninduced cultures do not generate a detectable signal, which suggests that the AOX1 promoter is tightly regulated methanol (data not shown). This observation is consistent with previous reports about the promoter [44].
3.2. Fluo-4-AM permeates into P. pastoris
The permeation of fluo-4-AM into the PP-Orco cells is arguably the most important step in the implementation of the odor assay and confocal microscopy confirmed that the dye permeates into approximately 21 ± 5% of Pichia cells within 45 minutes (Figure 3 ). Additionally, we observed that the proportion of cells into which the dye permeates is directly proportional to the incubation time. However, dye permeation is non-uniform. We speculate that since the cells were incubated with the dye in Hank’s buffer, fluo-4-AM diffuses into the cell as a complex with Ca2+, which lowers its diffusivity and total flux into the cells. Moreover, the non-uniformity in the distribution of fluorescent cells can also be attributed to differences in the stoichiometric ratio between the dye and Ca2+ ions in the complexes.