Protocols

Western blot protocol

• - Grow cells to mid-log phase (OD600 = 0.5-0.6), spin 5 ml culture, obtaon pellet.
• - Resuspend pellet in 250 ul lysis mix
• - BCA pf protein
• - Add loading dye and boil samples 5 minutes
• - Load gel, run at 50V till through stacking gel, then turn up to 100V
• - Put gel in transfer buffer to equilibrate 5 minutes
• - Transfer at 400mA for 60 minutes
• - Wash blot 5 minutes in TBS to remove MeOH
• - Block membrane 1 hour at room temperature or O.N. 4°C
• - Incubate with primary antibody diluted in blockin buffer for 1 hour shaking at room temperature
• - Wash blot 4 times with TBST 5 minutes
• - Incubate with secondary antibody diluted in blockin buffer for 1 hour shaking at room temperature
• - Wash blot 4 times with TBST 5 minutes
• - Mix Supersignal West Dura solutions 1:1 and put on membrane, incubate at room temperature for 5 minutes
• - Remove excess solution, seal and take pictures

E. coli transformation with golden gates G1, G2, G3, G4 products

Protocol :

1. 1) Add 10 µl plasmids to 50 µl E. coli competent cells on ice and let the mix rest on ice for 15 min
2. 2) Shock the mix at 42°C for 45 seconds, transfer the mix on ice for 10 min
3. 3) Add 800 µl of Luria Bertoni (LB) media to the mix and let it recover at 37°C and 750 rpm on shakers for 1 hour
4. 4) Plate 50 µl and 150 µl to plates containing LB agar media with ampificilin
5. 5) Put plates in growth incubators at 37°C for 24 hours

Bacterial strains

E. coli DH5alpha was used for cloning purpose in all experiments. Two shuttle plasmids for yeast expressing were transformed, pYGG1 containing URA selection and Amp resistance, pYGG1 containing TRP selection marker and Amp resistance. Those plasmids are high copy episomal plasmids, they contain a 2micron origin and GAL1 galactose inducible promoter. Inserts were cloned by Golden Gate assembly.

Yeast culture

Strains used was yeast W303 auxotroph for URA, TRP, HIS, LEU and ADE. A complemented media without amino-acids was used to select and maintain the recombinant yeasts. Yeast was grown to exponential mid-log phase for 12 hours, then resuspended in galactose media for 30h of induction at 25°C. In some experiments, yeast was pre-fixed in 0.5 % paraformaldehyde for 30 minutes followed by washing in PBS before DC loading.

Cell culture

The T cell hybridoma cell line, B3Z, specific for the OVA 257-264 peptide (SIINFEKL) in the context of Kb, was a gift from the Curie Institute (Paris V). B3Z were maintained in a medium (RP-10) consisting of RPMI 1640 supplemented with 10% FCS, Glutamax, HEPES, 50 μM 2-mercaptoethanol, 50 U/ml penicillin, and 50 μg/ml streptomycin. All cells were incubated at 37°C with 5% CO2.

Miniculture
19 µl colony resuspended Luria Bertoni media in 4 mL of Luria Bertoni media complemented with 4 µl of ampicilin (100 mg/µl) and put to incubate at 37°C overnight.

Yeast transformation protocol :

1. 1) From the W303 yeast culture at DO=1, harvest in sterile tube at 5000 rpm for 5 min
2. 2) Pour off the medium, resuspend the cells in 25 ml of sterile water and centrifuge again
3. 3) Pour off the water, resuspend the cells in 1 ml of 0.1 M LiAc and transfer the suspension to a 1.5 ml microfuge tube
4. 4) Pellet the cells at top speed for 15 sec and remove the LiAc with a micropipette
5. 5) Resuspend the cells with 0.1 LiAc to a final volume of 500 µl
6. 6) Vortex the cell suspension and pipette 50 µl samples into new 1.5 ml tubes. Pellet the cells and remove the LiAc with a micropipette
7. 7) Add the following to the samples in order:
• - 240 µl PEG 50%
• - 36 µl 1 M LiAc
• - 25 µl Salmon sperm DNA (2 mg/ml)
• - 50 µl water and plasmid (10 ug)
8. 8) Vortex each tube vigorously until the cell pellet has been completely mixed. Usually takes about 1 min
9. 9) Incubate at 30°C for 30 min
10. 10) Heat shock in a water bath at 42°C for 15 minute
11. 11) Ice for 2 minutes
12. 12) Centrifuge at 5000 rpm for 15 sec and remove the transformation mix with a micropipette
13. 13) Pipette 1 ml of sterile water into each tube and resuspend the pellet by pipetting it up and down gently
14. 14) Plate 50 µl and 150 µl of the transformation mix onto plates with corresponding media
15. 15) Incubate at 30°C for 3 days

Culture induction of T1/T2/T3 and WT yeast

1. 1) Discard media after centrifugation at 3000 rpm for 4 minutes
2. 2) Resuspend yeast in 10 mL induction media :
   • - galactose 1X without tryptophane for T1
   • - galactose 1X without tryptophane and uracile for T2 and T3
3. 3) Put into incubator and agitation at 25 °C for 48 hours

• 1) 5 spleens from C57BL/6 mice were digested in 5 mL of Collagenase/DNase for 45 minutes à 37°C.
• 2) The reaction was stopped by 0.6 mL of PBS/EDTA.
• 3) The organs digested were filtered 70 μ et centrifuged 10 minutes at 1300 rpm.
• 4) FC receptors were saturated before magnetic separation

The percentage of CD11c+ cells was determined by AUTOMACS. CD11c+ DC were incubated with anti-CD11c-conjugated magnetic cell sorting (MACS) microbeads and purified using magnetic separation columns as indicated by the manufacturer (Miltenyi Biotec). The negative fraction CD11c- was separated and marked for CD11b+ (macrophages).

Surface display yeast immunostaining

We stained yeasts with monoclonal anti-HA antibody conjugated to fluorochrome 640 (Abcam).
We compared yeasts expressing OVA1-DEC205-HA with or without AGA1P co-transformation.
Image settings were identical with 800 ms acquisition time for red fluorescence at 680 nm.

Cross presentation essay protocol :

• 1) Dendritics cells or macrophages were seeded at 1.105 cells per well in U-bottom 96-well plates in complete RPMI medium.
• 2) Recombinant S. cerevesiae or soluble SIINFEKL protein were added at various concentrations.
• 3) After 24h of co-incubation, 1.105 B3Z cells were added for 16 h at 37°C, 5% CO2.
• 4) Plates were washed once with PBS and 􏰁beta-galactosidase activity was assessed by the addition of 120 u􏰃l of lysis buffer (PBS, 9 mM MgCl2, 0.125% NP40 and 0.15 mM chlorophenolred-galactoside (CPRG).
• 5) After the color shift to red, the absorbance at 570 nm was read on a micro-plate reader.

Flow cytometry for yeast surface display and IFN gamma

Acquisition was performed at Genethon, Genpole (Evry) with a flow cytometer.
For surface display staining with HA-tag-680 label, yeasts were grown at 30°C with 300 r.p.m. agitation in SC-auxotrophic complemented media.
When culture reached mid-exponential phase, cells were harvested and fixed with paraformaldehyde with the following protocol : 1 mL sample was cooled to an ice water-bath and centrifuged at 4°, 2000g for 2 min.
Antibodies were added during 30 minutes, then the cells were washed with PBS. Supernatant was removed and pellet was resuspended in 200 uL of 0.5 % paraformaldehyde. The mix was incubated on ice for 30 minutes and subsequently centrifuged at 4°C, 2000g for 2 minutes.

The protocols we used during the competition are regrouped and documented here.