A) Generation of expression plasmids
The 50 proteins used as baits were transferred into pDest-DB and pDest-AD vectors in simple single-step reaction by Gateway recombination. Completed recombination reactions are then transformed into Escherichia coli, grown for 18 h, and plasmids are isolated.
Protocol 1A: Gateway LR recombinational cloning
For each bait combine in 96-well PCR plate:
1)
- 1 µl of entry clone (10 ng/µl)
- 1 µl of destination vector ( 100 ng/µl)
- 1 µl clonase buffer 5x
- 1.6 µl TE 1x
- 0,4 µl of LR clonase enzyme mix (Invitrogen) (keep this mix on ice)
2) Homogenize by gently pipetting up and down.
3) Incubate at 25 °C for 18 h
4) Add 2 µl of Proteinase K (2µg/µl) solution
5) Incubate 15 min at 37°C
Protocol 2A: Bacterial transformation
1) Thaw competent DH5a-T1R (Invitrogen) cells on ice
2) Add 100 µl of competent cells into 96-well PCR plate containing 5 µl Gateway LR reaction mix
3) Incubate on ice water for 20 min
4) Heat shock cells in a water bath at 42 °C for 15 sec
5) Incubate on ice water for 5 min
6) Transfer the cells into a 96 deep well plate filled with 900 µl SOC
7) Incubate at 37 °C for 1 h
8) Plate out 300µl of reaction onto LB plates containing 100mg/l Ampicillin
9) Incubate over night at 37°C
10) Pick a single colony from the plate and inoculate a 96 deep well plate filled with 1 ml of LB containing 100mg/l Ampicillin
11) Incubate at 37 °C for 18 h
12) Remove 5 µl for subsequent analysis by PCR
13) Remove 100 µl of the overnight culture, mix with 100 µl of 40% (w/v) autoclaved glycerol and store at 80 °C into a 96-well Low profile microplates
14) Use the remainder of the overnight culture for plasmid isolation
Protocol 3A: Bacterial culture PCR
Dilute 5 µl of bacterial culture into 95 µl of sterile water and mix by pipetting up and down. Keep bacterial cultures at 4 °C until PCR results are determined.
For each reaction, prepare in a 96-well PCR plate on ice:
- 1.1 µl of HiFi Platinum Taq polymerase buffer 10x (Invitrogen)
- 0.4 µl of 50 mM MgSO4 (final concentration 1.8 mM)
- 0.11 µl of 40 mM dNTPs (final concentration 400 nM)
- 0.022 µl of 200 µM AD or DB forward primer (final concentration180 nM)
- 0.022 µl of 200 µM Term reverse primer (final concentration 180 nM)
- 0.066 µl of HiFi Platinum Taq polymerase (Invitrogen)
- 8.28 µl of filter-sterilized water
- Add 1 µl of the diluted bacterial culture as DNA template
Place the PCR plate on a thermocycler and run the following program:
Step 1: Denaturation at 94 °C for 4 min.
Step 2: Denaturation at 94 °C for 30 s.
Step 3: Annealing at 58 °C for 30 s.
Step 4: Elongation at 68 °C for x min (depending on the length of the longer ORF)
Repeat Steps 2-3-4, 34 times.
Step 5: Final elongation at 68 °C for 10 min.
Step 6: Hold at 10 °C.
Primer sequences:
AD: 5’-CGCGTTTGGAATCACTACAGGG-3’
DB: 5’-GGCTTCAGTGGAGACTGATATGCCTC-3’
Term: 5’-GGAGACTTGACCAAACCTCTGGCG-3’
Once PCR reactions are completed, analyze 5 µl of PCR product on a 1% agarose. Successful LR reactions will give rise at ORFs sizes to which ~280 bp of vector sequences are added due to the AD, DB, and Term primer positions (the PCR amplicon from a destination vector containing the Gateway cassette has an expected size _1.9 kb). The remaining of the cultures are used for plasmid isolation.
B) Yeast transformation and identification of autoactivating DB-X hybrid proteins
Isolated destination clones were individually transferred into competent Y8930 (MATα) and Y8800 (MATa) strains by Lithium-Acetate based transformation. Identification of DB-ORF autoactivators is achieved in haploid yeast strain onto a Sc–Leu–His + 3-amino-1,2,4-triazole (3-AT) media.
Protocol 1B: Yeast transformation
1) Streak Y8800 and Y8930 on separate YEPD plates and incubate at 30 °C for 48–72h to obtain isolated colonies.
2) For each strain, inoculate 20 ml of YEPD with 10 isolated colonies. Incubate at 30 °C on a shaker for 14–18 h.
3) Measure and record the OD600, which should be between 4.0 and 6.0. Dilute cells into 100 ml of YEPD media to obtain a final OD600 ~ 0.1.
4) Incubate at 30 °C on a shaker until OD600 reaches 0.6–0.8 (4–6 h).
5) Boil carrier DNA (salmon sperm DNA) for 5 min then place on ice until needed.
6) Harvest cells by centrifugation at 800 x g for 5 min. Discard the supernatant and resuspend cells gently in 10 ml of sterile water.
7) Centrifuge as described in step 6 and discard the supernatant.
8) Resuspend cells in 10 ml of TE/LiAc solution, centrifuge, and discard the supernatant.
9) Resuspend cells in 2 ml of TE/LiAc solution, then add 10 ml of TE/ LiAc/PEG solution supplemented with 200 µl of boiled carrier DNA. Mix the solution by inversion.
10) Dispense 120 µl of this mix into each well of a round-bottom 96-well microtiter plate
11) Add 10 µl of plasmid DNA to the competent yeast and mix by pipetting up and down several times.
12) Incubate at 30 °C for 30 min
13) Subject to heat shock in a 42 °C water bath for 15 min.
14) Centrifuge 5 min at 800 x g. Carefully remove the supernatant
15) Add 100 µl of sterile water and resuspend cell pellets by pipetting up and down.
16) Centrifuge 5 min at 800 x g, then carefully remove 90 µl of water
17) Resuspend cell pellets by vortexing.
18) Spot 5 µl of cell suspension onto an appropriate selective plate (Sc-Trp for AD-Y, Sc-Leu for DB-X agar media).
19) Incubate at 30 °C for 72 h.
20) Pick transformed yeast colonies into a 96-well low profile microplates containing 100 µl of selective media (Sc-Trp for AD-Y, Sc-Leu for DB-X).
21) Incubate on a shaker at 30 °C for 72 h.
22) Remove 5 µl for subsequent lysis and analysis by PCR
23) Prepare archival stocks by combining 100 µl of the yeast culture with 100 µl of 40% (w/v) autoclaved glycerol into a 96-well Low profile microplate. Store at -80 °C.
Protocol 2B: Yeast cell lysis and lysate PCR
1) Prepare lysis buffer by dissolving 2.5 mg/ml zymolase 20T (21,100 U/g, Seikagaku Corp.) in 0.1M sodiumphosphate buffer (pH7.4). Keep on ice.
2) Aliquot 15 µl of lysis buffer into the wells of a 96-well PCR plate. Keep on ice.
3) Add 5 µl of yeast cells in the lysis buffer in the PCR plate
4) Place the PCR plate on a thermocycler and run the following program:
- Step 1: 37 °C for 15 min
- Step 2: 95 °C for 5 min
- Step 3: Hold at 10 °C
5) Add 100 ml of filter-sterilized water to each well.
6) Store at -20 °C
Protocol 3B: Yeast lysate PCR
For each reaction, prepare in a 96-well PCR plate on ice:
- 1.1 µl of HiFi Platinum Taq polymerase buffer 10x (Invitrogen)
- 0.4 µl of 50 mM MgSO4 (final concentration 1.8 mM)
- 0.11 µl of 40 mM dNTPs (final concentration 400 nM)
- 0.022 µl of 200 µM AD or DB forward primer (final concentration180 nM)
- 0.022 µl of 200 µM Term reverse primer (final concentration 180 nM)
- 0.066 µl of HiFi Platinum Taq polymerase (Invitrogen)
- 8.28 µl of filter-sterilized water
- Add 3 µl of the yeast cell lysate as DNA template
Place the PCR plate on a thermocycler and run the same program of Protocol 3A. Once PCR reactions are completed, analyze 5 µl of PCR product on a 1% agarose.
Protocol 4B: Autoactivator removal
1) Thaw glycerol stocks of the 50 individual DB-bait yeast strains and the glycerol stock of the yeast strains transformed with the AD encoding plasmid containing no insert (empty pDESTAD).
2) Using the colony picker Qpix2 XT replica-plate the 96-well Low profile microplate with the individual glycerol stocks of each DB-X yeast strain and the yeast strains transformed with the empty-AD into a new plates filled with 100 µl of fresh Sc-Leu and Sc-Trp media respectively.
3) Incubate at 30 °C for 72 h on a shaker
4) Replica-plate from the Sc-Leu-media and Sc-Trp media plates onto mating plates filled with YEPD media
5) Incubate at 30 °C for 24 h.
6) Replica-plate from mating plates onto Sc-Leu-Trp media plates to select for diploid cells and onto Sc-Leu-Trp-His + 1mM 3AT media.
7) Incubate at 30°C for 72 h. Score growth phenotypes. Non-autoactivating yeast cells are not able to activate the GAL1::HIS3 reporter gene hence should not grow on the Sc-Leu-Trp-His + 1mM 3AT media.
Yeast strains showing a growth phenotype on the Sc-Leu-Trp-His + 1mM 3AT media are considered autoactivators. They are physically removed from the collection of DB-X baits and screened against the 12k_space DB-collection using their AD-construction.
C) Primary Screening
Protocol 1C: Construction of DB baits pool
This protocol describes the construction of one pool of 50 different DB-hybrid constructs transformed into Y8930 yeast strain.
1) Thaw glycerol stocks of the 50 individual DB-bait yeast strains
2) Individually inoculate 50-ml polypropylene conical tubes that contain 5 ml of fresh Sc-Leu selective media, with 5 µl of the thawed glycerol stock.
3) Incubate at 30 °C for 72 h.
4) Transfer the contents of the culture tubes into a sterile trough.
5) Mix thoroughly to ensure equal representation of all DB-bait yeast strains in the pool.
6) On a liquid handling platform or with a multichannel pipette plated into the wells of ten 384-well Low profile microplates
Protocol 2C: 12k_space AD-collection culture
1) Thaw glycerol stocks of the 12k_space AD-collection corresponding to 127 96-well plates.
2) Using the colony picker Qpix2 XT replica-plate into 32 384-well plates filled with 50 µl of fresh Sc-Trp media
3) Incubate at 30 °C for 72 h
Protocol 3C: Mating
1) Using the colony picker Qpix2 XT replica-plate the 32 384-well plates corresponding to the 12k_space AD-collection 72 h cultures into the 384-well mating plates filled with YEPD media
2) Using the colony picker Qpix2 XT replica-plate the DB-baits pool into the 384-well mating plates precedently inoculated with the AD-collection. Use one DB-baits pool plate to inoculate five mating plates.
3) Incubate at 30 °C for 24 h
Protocol 4C: Screening
1) Using the colony picker Qpix2 XT replica-plate onto 384-well screening plates filled with 50 µl of fresh Sc-Leu-Trp-His + 1 mM 3AT media. Only diploid yeast with interacting couples can growth in this media.
2) Incubate at 30 °C for 5 days
3) Using the microplate-reader Tecan Infinite M200 PRO measure the OD600 of the 384-well screening plates in order to identify primary positives
Protocol 5C: AD-interacting proteins re-array and archival stock
1) Thaw glycerol stocks of the 12k_space AD-collection plates corresponding to the identified primary positives AD-interacting proteins
2) Using the colony picker Qpix2 XT re-array the primary positives AD-interacting proteins into 96-well plates filled with 100 µl of fresh Sc-Trp media.
3) Incubate at 30 °C for 72 h
4) Prepare an archival glycerol stock by adding 100 µl of 40% (w/v) autoclaved glycerol
D) Deconvolution and targeted matricial assay
Protocol 1D: DB-baits and AD-interacting proteins cultures
1) Thaw glycerol stocks of the 50 individual DB-bait yeast strains and of the identified primary positives AD-interacting proteins
2) Individually inoculate 50 distinct 50-ml polypropylene conical tubes that contain 10 ml of fresh Sc-Leu selective media, with 5 µl of the 50 individual DB-baits
3) Using the colony picker Qpix2 XT replica-plate the glycerol stocks of the identified primary positives AD-interacting proteins into ten 96-well plates filled with 100 µl of fresh Sc-Trp selective media
4) Incubate at 30 °C for 72 h.
5) On a liquid handling platform or with a multichannel pipette plate the 50 individual DB-bait cultures into the wells of 50 distinct 96-well Low profile microplates
Protocol 2D: Mating
1) Using the colony picker Qpix2 XT replica-plate the identified primary positives AD-interacting proteins cultures into the 96-well mating plates filled with 100 µl of YEPD media. Use one AD-interacting proteins plate to inoculate five mating plates.
2) Using the colony picker Qpix2 XT replica-plate the individual DB-baits cultures into the 96-well mating plates precedently inoculated with the AD-interacting proteins.
3) Incubate at 30 °C for 24 h
Protocol 3D: Verification and interacting proteins identification
1) Using the colony picker Qpix2 XT replica-plate onto two phenotyping 96-well plates:
- Verification-plates filled with 100 µl of fresh Sc-Leu-Trp-His + 1 mM 3AT media. Only diploid yeast with interacting couples can growth in this media.
- de novo autoactivators-plates filled with 100 µl of fresh Sc-Leu-His + 1 mM 3AT + CHX (1 mg/l) media. Only truncated fragments that can act like transcription factors can growth in this media
2) Incubate at 30 °C for 5 days
3) Using the microplate-reader Tecan Infinite M200 PRO measure the OD600 of the 96-well plates in order to identify positives pairs and de novo autoactivators
4) Using the colony picker Qpix2 XT re-array the identified positives pairs into 96-well plates filled with 100 µl of fresh Sc-Leu-Trp-His + 1 mM 3AT
5) Incubate at 30 °C for 72 h
6) Lyse cells according to Protocol 2B
7) Amplify the inserts of the DB-X and AD-Y inserts of positive cultures by yeast colony PCR according to Protocol 3B for subsequent ORF identification by end-read sequencing.
8) Prepare an archival glycerol stock by adding 100 µl of 40% (w/v) autoclaved glycerol