2. Materials and methods
2.1. Molecular cloning and transformation ofP.
pastoris
We used the P. pastoris GS115 as the chassis for all experiments
in this study. P. pastoris exhibits a significantly shorter
doubling time, is easier to work with, and its toolkit for molecular
cloning [33–36] is much more developed than competing platforms
such as Xenopus oocytes [30], Drosophila empty neurons
[30], HEK 293 [37] and Sf9 cells [38]. Moreover, P.
pastoris has been used previously to express heterologous membrane
proteins [39–42]. The amino acid sequence of Orco was retrieved
from the UniProt database and reverse-translated to acquire its cDNA
sequence. The latter was then codon-optimized for expression in P.
pastoris and synthesized through a commercial service (GenScript). ThePichia cells were transformed using the pPICZA plasmid. The
coding sequences on the plasmid are under the transcriptional control of
the AOX1 promoter, which is inducible in a titratable manner with
methanol. The plasmid also bears a zeocin (phleomycin D1) selection
marker and its multicloning site (MCS) is configured to allow the
inclusion of C-terminal c-myc and polyhistidine (6xHis) tags. TheP. pastoris GS115 strain and the pPICZA plasmid were generously
donated to us by Prof. Steven Hallam (Department of Microbiology &
Immunology, The University of British Columbia).
All sub-cloning was performed in E. coli DH5α and commenced with
digestion of the pPICZA plasmid and PCR product of Orco cDNA using EcoRI
and NotI and their subsequent ligation using T4 DNA ligase to generate
the pOrco plasmid. We subsequently linearized the pOrco plasmid using
the SacI enzyme. All restriction endonucleases used in the study were of
the high-fidelity form, and all enzymes were purchased from New England
Biolabs. The Pichia cells were then made electrocompetent and
transformed with the previously linearized pOrco plasmid via
electroporation [33]. The transformed cells were plated onto YPD
plates (1% yeast extract, 2% peptone, 2% dextrose and 2% agar)
containing 100 µg/mL of zeocin and incubated in a darkened incubator at
30°C for 2-3 days to yield colonies that are sufficiently large to be
observable with the naked eye. Transformation of P. pastorisGS115 was confirmed through colony PCR and the resultant clone is
labelled as PP-Orco.
2.2. Culturing conditions for expression of
Orco
We arbitrarily selected three colonies for further testing. The colonies
were transferred to 5 mL of YPD medium (1% yeast extract, 2% peptone
and 2% dextrose) and cultured overnight at 30oC under
constant agitation at 200 rpm. Unless otherwise noted, all liquid
culturing media described hereinafter also contained 100 µg/mL of
zeocin. We then inoculated 0.1 mL of these cultures in triplicate in 30
mL of BMGY medium (1% yeast extract, 2% peptone, 1% glycerol, 400
μg/L biotin, 0.1 M potassium phosphate and pH 6.0) and propagated the
cultures in 250 mL baffled flasks for 24 hours. Next, we centrifuged the
cultures at 2,000 rpm for 3 minutes and resuspended the cell pellets in
30 mL of BMMY media (1% yeast extract, 2% peptone, 0.1% methanol, 400
μg/L biotin, 0.1 M potassium phosphate and pH 6.0). The approximate
average optical densities of the cultures at this point was 1. The BMMY
cultures were propagated at 30oC for 48 hours under
constant agitation at 200 rpm. Methanol in the culture medium was topped
up to 0.01% every 18 hours. At the end of culturing, we centrifuged the
cultures at 3,000 rpm and 4oC and subsequently
harvested the cells and stored them at -80oC prior to
Western blotting. We also tested BMMY medium containing 0.01% methanol
to evaluate the impact of the concentration of the inducer on expression
of Orco.
2.3. Processing of harvested Pichiacells for Western
blotting
The previously frozen cell pellets were resuspended in 10 mL of breaking
buffer (50 mM potassium phosphate, 100 mM NaCl, 1 mM EDTA, 5% glycerol
and 100 mM PMSF) and centrifuged for 5 minutes at 3,500 rpm and
4oC. The supernatant was collected and centrifuged for
another 15 minutes at 14,000 rpm and 4oC. The
supernatant that emerges from the second centrifugation was centrifuged
once more for 1 hour at 100,000 rcf and 4oC. The
resulting pellet, which contains the membrane fraction, was resuspended
in a membrane buffer solution (50 mM Tris, 120 mM NaCl, 2 mM EDTA, 10%
glycerol and pH 8) and used directly for Western blotting. We also
diluted 25 μL of the supernatant produced in the second centrifugation
step and diluted it with 25 μL of 2x Laemmli buffer for analysis using
Western blotting. This sample represents the whole cell fraction.
2.4. Western blotting
The membrane and whole cell fractions of the PP-Orco cultures were
resolved using protein gel electrophoresis on pre-cast Mini-PROTEAN TGX
gels. Electrophoresis was performed for 1 hour at 90 V. The gels were
then transferred to an Immun-Blot PVDF membrane in a blotting cell that
was filled with ice-cold transfer buffer (25 mM Tris, 1.92 M glycine,
20% methanol and pH 8.5). The transfer took place over 1 hour at 100 V
and the membrane was then blocked for 1 hour under constant, gentle
shaking at room temperature using a mixture of TBST solution (137 mM
NaCl, 19 mM Tri-Base and 1% Tween-20) and 5% skim milk. The blocked
membrane was subsequently washed with the TBST solution and incubated
overnight with the primary antibody for the c-myc tag at
4oC under constant shaking, which was followed by a
shorter incubation of 1 hour with a suspension of the HRP conjugate of
the goat anti-mouse IgG secondary antibody in 5% skim milk at room
temperature. The membranes were imaged on a Clarity ECL substrate in a
ChemiDoc MP Imager.
2.5. Assessment of dye permeation intoPichia cells
We used confocal microscopy to assess the permeation of fluo-4-AM into
the Pichia cells. Overnight cultures of P. pastoris GS115
and PP-Orco in 5 mL of YPD were centrifuged at 2,000 rcf for 15 minutes
at room temperature and resuspended in Hank’s buffer with 1 mM
Ca2+ to achieve a final optical density of 0.4. We
then added fluo-4-AM to the cell suspensions to achieve a final
concentration of 2.5 μM and incubated the solutions for 45 minutes at
37oC and in a darkened chamber. Next, the cell
suspensions were centrifuged at 2,000 rcf for 15 minutes at room
temperature and the resultant pellet was resuspended in fresh PBS. We
repeated the centrifugation and resuspension in PBS two more times. 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.
2.6. Assay for assessment of thePichiabiosensor
The PP-Orco cultures were initially propagated in 30 mL of BMGY medium
for 24 hours. When the optical density of the cultures reached 1, they
were centrifuged for 5 minutes at 2,000 rcf and room temperature. The
cell pellets were resuspended in 30 mL BMMY media containing 0.1%
methanol and propagated for 48 hours. The cultures were later
centrifuged for 5 minutes at 5,000 rcf and room temperature and the
resultant pellets were resuspended in a 1:1 volumetric mixture of BMMY
media and PBS to a final optical density of 0.4. Fluo-4-AM was added to
the solutions to a concentration of 2.5 μM and the cells were incubated
with the dye for 45 minutes at 37oC in a darkened
chamber. The cell suspensions were later centrifuged at 2,000 rcf for 15
minutes at room temperature and the resultant pellet was resuspended in
fresh PBS. We repeated the centrifugation and resuspension in PBS two
more times. After the final PBS wash, the cells were resuspended in
Hank’s buffer to achieve an optical density of 0.4, and 200 μL of this
suspension was pipetted in each well of a 96-well plate. Hank’s buffer
is a calcium-containing medium. We evaluated the biosensor in Hank’s
buffer containing 1 mM and 5 mM of Ca2+.
After allowing the basal fluorescence in each well to equilibrate, which
takes approximately 6 minutes, we injected the odorants into each well.
We tested VUAA1 (0.125, 0.25, 0.50, 1, 1.50 and 2 mM), citronella oil
(1:15, 1:7, 1:3 and 1:1 volumetric dilution in 1 μL solution with DMSO)
and oct-1-en-3-ol (1:7, 1:3 and 1:1 volumetric dilution in 1 μL solution
with DMSO) individually. The fluorescence emitted by each well was
recorded over 6 minutes using excitation and emission wavelengths of 485
and 520 nm, respectively. We also assessed the behavior of the sensor
when it is sequentially exposed to citronella oil followed by VUAA1. We
added VUAA1 to the well 2 minutes after addition of citronella oil.
Normalization of the fluorescence recordings was performed as follows: