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: