Fsample and Fblank are the fluorescence
recordings for the sample and control wells, respectively, at a given
time point, whereas Fosample and
Foblank are the fluorescence
recordings for the sample and control wells at the initial time point.
Hereinafter, fluorescence refers to the normalized fluorescence, F. We
analyzed three technical and two biological replicates for each assay
condition, and statistical significance was assessed by performing a
one-way ANOVA paired test with a Tukey post hoc test on the highest
value of the normalized fluorescence for each sample.
3. Results
3.1. P. pastoris successfully
expresses Orco
We tested two concentrations of methanol in the BMMY medium, 0.01% and
0.1%. Although Orco is expressed by the cells at both induction levels,
expression is markedly higher at 0.1% methanol. Additionally, we did
not observe any expression after 24 hours of culturing, which suggests
that expression and localization of Orco within the membranes ofP. pastoris cells occurs between 24 and 48 hours. As a result, we
used 0.1% methanol in BMMY medium for all subsequent experiments.
Western blotting confirms that P. pastoris expresses and
localizes Orco to its cellular membrane (Figure 2 ). The
unmodified form of Orco has a molecular weight of 54 kDa whereas its
glycosylated form weighs approximately 56.5 kDa. The blot image confirms
the presence of a protein within the appropriate size window in the
PP-Orco samples. Although we did not mutagenize the protein at its sole
glycosylation site (N167) to determine if the engineered cells are
post-translationally modifying the protein at the correct location,
functional studies presented subsequently confirm that fact. Crucially,
no bands were observed in the whole cell fraction of P. pastorisGS115. Moreover, the band intensities for the two PP-Orco fractions
suggest that a majority of the heterologous expressed proteins correctly
localize to the membrane. Incidentally, we had previously attempted to
construct a version of the odor biosensor in Escherichia coli . We
observed that the prokaryotic host was unable to localize Orco to its
periplasmic membrane and instead formed inclusion bodies despite
co-expression of chaperones such as DnaK and AAA proteinases such as
FtsH [43].