Figure captions
Figure 1: Design of the odor biosensor. (A) The complete odor transduction pathway in mosquitoes involves odor binding proteins (OBPs), olfactory receptors (ORs) and the olfactory receptor co-receptor (Orco). (B) Previous studies have shown that Orco can function as a cationic channel and individually transduce some olfactory signals. As a consequence, the refactored olfactory transduction pathway was constructed in P. pastoris by solely employing Orco.
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].
Figure 3: Confocal microscopy and permeation statistics. (A)The confocal micrograph confirms permeation of fluo-4-AM into P. pastoris . The cells were imaged using an Olympus FV-1000 laser-scanning confocal microscope under 60x magnification and at excitation and emission wavelengths of 488 and 505 nm, respectively. (B) The permeation is non-uniform. However, 45 minutes incubation is sufficient to stain about 21% of the population and generate a detectable signal.
Figure 4: Functional testing of the biosensor in Hank’s buffer containing 1 mM Ca2+ confirms dose-dependent activation of Orco by VUAA1. The response is analogous to membrane fluctuations observed following activation of a TRP cationic channel. The p-values for the difference in normalized fluorescence between PP-Orco and GS115 strains were lower than 0.01 for the 0.5 mM, 1 mM and 2 mM runs and 0.001 when the concentration of VUAA1 was 1.5 mM.
Figure 5: Functional testing of the biosensor in Hank’s buffer containing 5 mM Ca2+ reveals that the signal can be amplified by increasing the concentration of calcium ions in the extracellular medium. The p-value for the difference in normalized fluorescence between PP-Orco and the control is less than 0.01 when the VUAA1 concentration is 0.125 mM. The corresponding p-values for the remaining VUAA1 concentrations are below 0.001.
Figure 6: EC50 for the interaction between VUAA1 and Orco is estimated to be 0.85 mM and 0.41 mM for extracellular calcium concentrations of 1 mM and 5 mM, respectively.
Figure 7: DMSO does not interact with Orco and can be used to solubilize hydrophobic odorants in the assay buffer.
Figure 8: Citronellal or a minor constituent of citronella oil interferes with Orco. The ligand is either a negative allosteric modulator of Orco or an antagonist that is weakly competitive with VUAA1. The dilutions correspond to volumetric ratios of citronella oil and DMSO in an injection of 1 µL into the assay buffer.
Figure 9: The Pichia biosensor does not respond to oct-1-en-3-ol , which is corroborates previous reports that Orco is not activated by the odorant. The dilutions correspond to volumetric ratios of oct-1-en-3-ol and Hank’s buffer containing 5 mM Ca2+ in an injection of 1 µL into the assay buffer. ‘None’ refers to no dilution.