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].