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.