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.