Monday, 27^th July 2015

OVA2 (pYGG1): PCR colony and gel electrophoresis

Previously (see 2015-07-23), we transformed E. coli with our golden gate products and plated the transformed E. coli onto 2 plates.
We took 8 positive colonies from the plates and resuspend them separately into 20 µl of Luria Bertoni media.

PCR colony mix (20 µl):

1. 10 µl PCR mix (DreamTaq)
2. 1 µl URA F1
3. 1 µl URA R5 2.0
4. 1 µl colony resuspended in Luria Bertoni media
5. 7 µl water

PCR colony program:

1. Step 1 : 95°C - 5 minutes
2. Step 2 : 95°C – 30 seconds
3. Step 3 : 50°C – 30 seconds
4. Step 4 : 72°C – 2 minutes (Repeat step 2-4, 35 times)
5. Step 5 : 72°C – 10 minutes
6. Step 6 : 4°C – Pause

PCR colony products gel electrophoresis (1% agarose)

PCR from New insert1 m1 Cter (PCR001): amplification of AGA2P with primers containing DEC205 overhang

Q5 PCR mix (50µl)

1. 2,5 µl FWD AGA2P
2. 2,5 µl RV AGA2P
3. 1 µl New insert1 m1 Cter
4. 19 µl water
5. 25 µl PCR mix (Q5)

Q5 PCR Program

1. step 1 : 95°C – 30 seconds
2. step 2 : 95°C – 10 seconds
3. step 3 : 60°C
4. step 4 : 72°C – 1 min (repeat steps 2-4 for 31 cycles)
5. step 5 : 12°C - Hold

PCR from New insert1 m2 Cter (PCR002): amplification of DEC205 with primers containing AGA2P overhang

Q5 PCR mix (50µl)

1. 2,5 µl FWD DEC205
2. 2,5 µl RV DEC205
3. 1 µl New insert1 m2 Cter
4. 19 µl water
5. 25 µl PCR mix (Q5)

Q5 PCR Program

1. step 1 : 95°C – 30 seconds
2. step 2 : 95°C – 10 seconds
3. step 3 : 60°C
4. step 4 : 72°C – 1 min (repeat steps 2-4 for 31 cycles)
5. step 5 : 12°C - Hold

PCR001 and PCR002 gel electrophoresis (1% agarose):

PCR clean up and nanodrop of AGA2P and DEC205 products :

1. AGA2P = 72 .3 ng/µl (260/280 = 1.79 ; 260/230 = 0.76)
2. DEC205 = 104.2 ng/µl (260/280 = 1.79 ; 260/230 = 0.42)

Golden gates of :

1. - (pYGG1) + AGA2P + DEC205
2. - (pYGG1) + AGA2P + OVA1 (Gblock)
3. - (pYGG1) + ADH1 (miniprep « ADH1 ») + Malpha GMCSF (B10, see 22/06/15)

Golden gate program:

E. coli transformation with golden gate 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

Tuesday, 28^th July 2015

We sent to sequencing Mat alpha IFN gamma ADH1 (pYGG1) with new primers since previous sequencing results were unconclusive.
Furthermore, in order to check the presence of our desired fragments, we also performed a multiple PCR on A4, C6 and D7 samples.

A4, C6 and D7 samples sequencing :

1. Seq.ID 26EB16 - A41 (URA F1/SR1 lig IFNg)
2. Seq.ID 26EB17 - A41.2 (replicate of A41)
3. Seq.ID 26EB18 - A42 (S2 lig IFNg/SR2 lig IFNg)
4. Seq.ID 26EB19 - A42.2 (replicate of A42)


5. Seq.ID 26EB20 - C61 (URA F1/SR1 lig IFNg)
6. Seq.ID 26EB21 - C61.2
7. Seq.ID 26EB22 - C62 (S2 lig IFNg/SR2 lig IFNg)
8. Seq.ID 26EB23 - C62.2


9. Seq.ID 26EB24 - D71 (URA F1/SR1 lig IFNg)
10. Seq.ID 26EB25 - D71.2
11. Seq.ID 26EB26 - D72 (S2 lig IFNg/SR2 lig IFNg)
12. Seq.ID 26EB27 - D72.2

Multiple PCR using URA F1/SR1 lig IFNg and S2 lig IFNg/SR2 lig IFNg primers :

PCR mix (20 µl):

1. 1 µl of DNA
2. 1 µl forward primer
3. 1 µl reverse primer
4. 7 µl water
5. 10 µl PCR mix (Dreamtaq)

Multiple PCR gel electrophoresis (1% agarose)

Golden gate of ADH1 (miniprep « ADH1 ») Mat alpha GMCSF (B10, see 22/06/15) (pYGG1)

E. coli transformation with golden gate 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

Tuesday, 28^th July 2015

We sent to sequencing AGA2P OVA1 DEC205 (pYGG2) with new primers since previous sequencing results were unconclusive.
Furthermore, in order to check the presence of our desired fragments, we also performed a multiple PCR on SD1 and SD2 samples.

SD1 and SD2 samples sequencing:

Sequencing ID and primers used for sample "SD2":

1. Seq.ID 26EB28 (URA F1/URA F1 Rev Seq V2)
2. Seq.ID 26EB29 (URA R5 2.0 Fw seq V2/URA R5 2.0)
3. Seq.ID 26EB30 (URA F1/ URA R1)
4. Seq.ID 26EB31 (URA F2/URA R5 2.0)


5. Seq.ID 26EB32 (URA F1/URA F1 Rev Seq V2)
6. Seq.ID 26EB33 (URA R5 2.0 Fw seq V2/URA R5 2.0)
7. Seq.ID 26EB34 (URA F1/ URA R1)
8. Seq.ID 26EB35 (URA F2/URA R5 2.0)

Sequencing ID and primers used for sample "SD1":

1. Seq.ID 26EB36 (URA F1/URA F1 Rev Seq V2)
2. Seq.ID 26EB37 (URA R5 2.0 Fw seq V2/URA R5 2.0)
3. Seq.ID 26EB38 (URA F1/ URA R1)
4. Seq.ID 26EB39 (URA F2/URA R5 2.0)


5. Seq.ID 26EB40 (URA F1/URA F1 Rev Seq V2)
6. Seq.ID 26EB41 (URA R5 2.0 Fw seq V2/URA R5 2.0)
7. Seq.ID 26EB42 (URA F1/ URA R1)
8. Seq.ID 26EB43 (URA F2/URA R5 2.0)

Multiple PCR

PCR mix (20 µl):

1. 1 µl of DNA
2. 1 µl forward primer
3. 1 µl reverse primer
4. 7 µl water
5. 10 µl PCR mix (Dreamtaq)

Multiple PCR gel electrophoresis (1% agarose)

Golden gates of :

1. - Golden 1 : pYGG1 + AGA2P + DEC205
2. - Golden 2 : PYGG1 + AGA2P + OVA1 (Gblock) (dec 205 v2)

E. coli transformation with golden gate 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

Wednesday, 29^th July 2015

Results of E.coli transformed with AGA2P OVA1 (Gblock "dEC205 - v2") (pYGG1) and AGA2P DEC205 (pYGG1)

We got a few colonies for AGA2P OVA1 but none for AGA2P DEC2065 so we performed a PCR colony for AFGA2P OVA1 and a second transformation for AGA2P DEC205 with remaining golden gate (see 28/07).

AGA2P OVA1: PCR colony

PCR colony mix (20 µl):

1. 10 µl PCR mix (DreamTaq)
2. 1 µl URA F1
3. 1 µl URA R5 2.0
4. 1 µl colony resuspended in Luria Bertoni media
5. 7 µl water

PCR colony program :

1. Step 1 : 95°C - 5 minutes
2. Step 2 : 95°C – 30 seconds
3. Step 3 : 50°C – 30 seconds
4. Step 4 : 72°C – 2 minutes (Repeat step 2-4, 30 times)
5. Step 5 : 72°C – 10 minutes
6. Step 6 : 4°C – Pause

AGA2P OVA1: PCR colony gel electrophoresis (1% agarose)

Yeast transformations with following golden gate constructions :

1. - AGA2P OVA1 DEC205 (SD1)
2. - AGA1
3. - AGA2P OVA1 DEC205 (SD2)

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

Wednesday, 29^th July 2015

Results of E.coli transformed with ADH1 (miniprep « ADH1 ») + Malpha GMCSF (B10, see 22/06/15) (pYGG1)

We got no colonies, so we performed a second E.coli transformation with remaining golden gate (see 28/07).

E. coli transformation with golden gate 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

AGA1P sequencing

Friday, 31^st July 2015

Since on the 07/30, we could not perform all yeast transformations (transformation 5 or T5), due to lack of samples for AGA1P, we transformed all the constructions into yeast again.
The yeasts that were transformed the day before are already growing except for the plate CoT2 that did not contain enough substrate for the yeast to feed off.

Yeast transformation:

1. T4 : ADH1 Ma IFNg (pYGG1)

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:


8. - 240 µl PEG 50%
9. - 36 µl 1 M LiAc
10. - 25 µl Salmon sperm DNA (2 mg/ml)
11. - 50 µl water and plasmid (10 ug)
12. 8) Vortex each tube vigorously until the cell pellet has been completely mixed. Usually takes about 1 min
13. 9) Incubate at 30°C for 30 min
14. 10) Heat shock in a water bath at 42°C for 15 minute
15. 11) Ice for 2 minutes
16. 12) Centrifuge at 5000 rpm for 15 sec and remove the transformation mix with a micropipette
17. 13) Pipette 1 ml of sterile water into each tube and resuspend the pellet by pipetting it up and down gently
18. 14) Plate 50 µl and 150 µl of the transformation mix onto plates with corresponding media:
• pYGG1 => URA-
• pYGG2 => TRP-
• pYGG1 + pYGG2 => TRP- URA-
19. 15) Incubate at 30°C for 3 days

Friday, 31^st July 2015

Since on the 07/30, we could not perform all yeast transformations (transformation 5 or T5), due to lack of samples for AGA1P, we transformed all the constructions into yeast again.
The yeasts that were transformed the day before are already growing except for the plate CoT2 that did not contain enough substrate for the yeast to feed off.

AGA1P minipreped using NucleoSpin Plasmid (LOT 1306/003 Macherey-Nagel) and nanodroped :

1. AGA1P.1 = 44.2 ng/µl

Yeast transformations:

1. T1: OVA2 (pYGG2)
2. T2: AGA2P OVA1 (pYGG1) + AGA1P (pYGG2)
3. T3: AGA2P OVA1 DEC205 (pYGG1) + AGA1P (pYGG2)
4. T5: AGA2P/GFP (pYGG1) + AGA1P (pYGG2)
5. T6: AGA2P GFP (pYGG1)
6. T7: AGA2P OVA DEC205 (pYGG1)
7. T8: AGA2P OVA1 (pYGG1)

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:
• pYGG1 => URA-
• pYGG2 => TRP-
• pYGG1 + pYGG2 => TRP- URA-
15. 15) Incubate at 30°C for 3 days

Monday, 3^rd August 2015

Colony PCR of yeast transformants with following primers :

AGA2P/DEC205

1. P1-4 => URA F1/ URA R1
2. P5-8 => URA R2/ URA R5 2.0

SD1

1. R1-4 => URA F1/URA R5 2.0
2. R5-6 => URA F1/URA R1
3. R7-8 => URA F2/URA R5 2.0

SD2

1. T1-4 => URA F1/ URA R5 2.0
2. T5-6 => URA F1/URA R1
3. T7-8 => URA F2/URA R5 2.0<:li>

Library

1. - "130" P2 (AGA2P/DEC205)
2. - "131" P4 (AGA2P/DEC205)
3. - "132" R4 (SD1)
4. - "133" R5 (SD1)
5. - "134" R6 (SD1)
6. - "135" T1 (SD2)
7. - "136" T3 (SD2)
8. - "137" T4 (SD2)
9. - "138" T5 (SD2)

PCR Colony mix:

1. 1 µl of resuspended colony in 20 µl LB
2. 1 µl URA F1
3. 1 µl URA R5 2.0
4. 7 µl water
5. 10 µl PCR mix (Dreamtaq)

PCR colony Program:

1. Step 1 : 95°C - 5 min
2. Step 2 : 95°C – 30 s
3. Step 3 : 50°C – 30s
4. Step 4 : 72°C – 2 min (repeat step 2-4, 45 times)
5. Step 5 : 72°C – 10 min
6. Step 6 : 4°C – Pause

Monday, 3^rd August 2015

Colony PCR of yeast transformants with following primers :

GMCSF

1. Q1-8 => URA F1/URA R5 2.0

IFNgamma

1. S1-4 => URA F1/SR1 lig IFN gamma
2. S5-8 => S2 lig IFN gamma/SR2 lig IFN gamma

Tuesday, 4^th August 2015

Colony PCR of transformed E. coli with ADH1 Mata GMCSF and ADH1 Mata IFNg

PCR colony mix (20 µl):

1. 10 µl PCR mix (DreamTaq)
2. 1 µl URA F1
3. 1 µl URA R5 2.0
4. 1 µl colony resuspended in Luria Bertoni media
5. 7 µl water

PCR colony program:

1. Step 1 : 95°C - 5 minutes
2. Step 2 : 95°C – 30 seconds
3. Step 3 : 50°C – 30 seconds
4. Step 4 : 72°C – 2 minutes (Repeat step 2-4, 35 times)
5. Step 5 : 72°C – 10 minutes
6. Step 6 : 4°C – Pause

Colony PCR gel electrophoresis (1% agarose)

Colony PCR yeast gel electrophoresis (1% agarose)

Wednesday, 5^th August 2015

Gel electrophoresis of ADH1 Mata GMCSF and ADH1 Mata IFNg

Colony PCR2 of ADH1 Mata GMCSF and ADH1 Mata IFNg :

PCR colony mix (10 µl):

1. 5 µl PCR mix (DreamTaq)
2. 0.5 µl URA F1
3. 0.5 µl URA R5 2.0
4. 0.5 µl colony resuspended in Luria Bertoni media
5. 3.5 µl water

PCR colony program:

1. Step 1 : 95°C - 5 minutes
2. Step 2 : 95°C – 30 seconds
3. Step 3 : 72°C – 30 seconds
4. Step 4 : 60°C/53°C– 2 minutes (Repeat step 2-4, 35 times)
5. Step 5 : 72°C – 10 minutes
6. Step 6 : 16°C – Pause

Miniculture of ADH1 Mata GMCSF and ADH1 Mata IFNg

The remaining resusepended E.coli was put into 4 mL of Luria Bertoni media containing with 4 µl ampicilin.

PCR colony of T4 (see 31/07)

PCR Colony mix (20 µl):

1. 1 µl of resuspended colony in 20 µl Luria Bertoni media
2. 1 µl URA F1
3. 1 µl URA R5 2.0
4. 7 µl water
5. 10 µl PCR mix (Dreamtaq)

PCR colony program:

1. Step 1 : 95°C - 5 minutes
2. Step 2 : 95°C – 30 seconds
3. Step 3 : 60°C – 30 seconds
4. Step 4 : 72°C – 2 minutes (repeat step 2-4, 45 times)
5. Step 5 : 72°C – 10 minutes
6. Step 6 : 4°C – Pause

Golden gate of ADH1 mat alpha GMCSF (pYGG1) construction :

Golden gate mix (20 µl):

1. 0.849 µl ADH1
2. 3.243 µl Mat alpha GMCSF
3. 3.1 µl PYGG1
4. 2 µl T4 ligase buffer
5. 0.5 µl T4 ligase
6. 0.5 µl BSA I

Golden gate program :

E.coli transformation with golden gate ADH1 mat alpha GMCSF (pYGG1)

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

Wednesday, 5^th August 2015

Colony PCR of T1/2/3/5/6/7/8 (see 31/07)

PCR Colony mix (20 µl):

1. 1 µl of resuspended yeast colony in 10 µl water
2. 1 µl URA F1
3. 1 µl URA R5 2.0
4. 7 µl water
5. 10 µl PCR mix (Dreamtaq)

PCR colony program :

1. Step 1 : 95°C - 5 minutes
2. Step 2 : 95°C – 30 seconds
3. Step 3 : 60°C – 30 seconds
4. Step 4 : 72°C – 4 minutes (repeat step 2-4, 45 times)
5. Step 5 : 4°C – Pause

Thursday, 6^th August 2015

Yeast transformation without OVA1

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:
• pYGG1 => URA-
• pYGG2 => TRP-
• pYGG1 + pYGG2 => TRP- URA-
15. 15) Incubate at 30°C for 3 days

Friday, 7^th August 2015

PCR colony yeast and E.coli

PCR colony gel electrophoresis (1% agarose) :

Saturday, 8^th August 2015

E.coli PCR colony of GMCSF and IFNg

PCR colony gel electrophoresis (1% agarose) :

Monday, 10^th August 2015

Golden gate ADH1 matalpha GMCSF (pYGG1)

Golden gate mix (20 µl):

1. 0.849 µl ADH1
2. 3.243 µl Mat alpha GMCSF
3. 3.1 µl PYGG1
4. 2 µl T4 ligase buffer
5. 0.5 µl T4 ligase
6. 0.5 µl BSA I

Golden gate program:

Tuesday, 11^th August 2015

E.coli transformation of ADH1 Mat alpha GMCSF (pYGG1) (again)

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

Golden gate of ADH1 mat alpha IFNgamma (pYGG1)

Golden gate mix (20 µl):

1. 0.033 (x 3) µl Sample « B8 » (M :atalpha IFNgamma) (114 ng/µl)
2. 0.058 (x 3) µl Sample « A6 » (IFNgamma) (114 ng/µl)
3. 0.274 µl Sample « ADH1 » (75 ng/µl)
4. 0.929 µl pYGG1
5. 2 µl T4 ligase buffer
6. 0.5 µl T4 ligase
7. 0.5 µl BSA I

Golden gate program :

Tuesday, 11^th August 2015

Culture of T1, T2 T3 and WT yeast from plates in glucose (without induction)

Wednesday, 12^th August 2015

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

Wednesday, 12^th August 2015

E.coli transformation of ADH1 Mat alpha IFNgamma (pYGG1)

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

Colony PCR of pYGG1 ADH1 matalpha GMCSF with 3 sets of primers :

1. - URA F1 / URA R5 2.0
2. - URA F1 / SR1 lig IFNgamma
3. - S2 lig IFNgamma / SR2 lig GMCSF

Thursday, 13^th August 2015

Colony PCR of ADH1 Mat alpha IFN gamma (pYGG1) with 3 sets of primers :

1. - URA F1 / URA R5 2.0
2. - URA F1 / SR1 lig IFNgamma
3. - S2 lig IFNgamma / SR2 lig IFNgamma

PCR colony mix (10 µl):

1. 5 µl dreamtaq master mix
2. 0.5 µl colony resuspended in 20 µl Luria Bertoni
3. 0.5 µl reverse primer
4. 0.5 µl forward primer
5. 3.5 µl water

Miniprep of ADH1 Mat alpha GMCSF (pYGG1) using NucleoSpin Plasmid (LOT 1306/003 Macherey-Nagel) :

• AGA2P = 72 .3 ng/µl (260/280 = 1.79 ; 260/230 = 0.76)
• GMCSF (1) = 304.2 ng/µl (280/260 = 1.82 ; 260/230 = 1.71)

Monday, 17^th August 2015

Yeast Colony PCR on T1, T2 and T3 (induced/non induced)

1. Resuspend colony in 10 µl water
2. Microwave 8 minutes 5 µl of resuspended colony (900W)
3. Add 10 µl of mix dreamtaq
4. Add 2.5 µl primer (URA R5 2.0/ URA F1)

PCR yeast colony program:

1. Step 1 : 95°C - 5 minutes
2. Step 2 : 95°C – 30 seconds
3. Step 3 : 57/60/63°C – 30 seconds
4. Step 4 : 72°C – 2 minutes (repeat step 2-4, 41 cycles)
5. Step 4 : 72°C – 10 minutes
6. Step 5 : 4°C – Pause

T1/2/3 Colony PCR gel electrophoresis (1% agarose) :

Expected sizes :

1. T1 => OVA2 (290 bp)
2. T2 => AGA1P (2225 bp) & AGA2P OVA1 (461 bp)
3. T3 => AGA1P (2285 bp) & AGA2P OVA1 DEC205 (1381 bp)

Miniprep of ADH1 Mat alpha IFN gamma using NucleoSpin Plasmid (LOT 1306/003 Macherey-Nagel) :

1. IFN 1 = 343.5 ng/µl
2. IFN 4 = 290 ng/µl
3. IFN 6 = 265 ng/µl
4. IFN 7 = 377 ng/µl

Tuesday, 18^th August 2015

We decided to remake the yeast colony PCR from 17/08 using same protocol to check our results a second time.
We tested plates of the same construction, T1, T2 and T3, made by different experimentators. For T1, we tested 3 different plates, each at 2 different temperatures : 53 and 60°C.
We also tested T3 with 3 different sets of primers (see gel picture below). Yeast Colony PCR on T1, T2 and T3 (induced/non induced)

1. Resuspend colony in 10 µl water
2. Microwave 8 minutes 5 µl of resuspended colony (900W)
3. Add 10 µl of mix dreamtaq
4. Add 2.5 µl primer (URA R5 2.0/ URA F1)

PCR yeast colony program :

1. Step 1 : 95°C - 5 minutes
2. Step 2 : 95°C – 30 seconds
3. Step 3 : 57/60/63°C – 30 seconds
4. Step 4 : 72°C – 2 minutes (repeat step 2-4, 41 cycles)
5. Step 4 : 72°C – 10 minutes
6. Step 5 : 4°C – Pause

T1/2/3 Colony PCR gel electrophoresis (1% agarose):

Primers used :

1. T1/T2/WT : URA F1/R5 2.0
2. T3.1 : URA R5 2.0/F1
3. T3.2 : URA F1/R1
4. T3.3 : URA F2/ R5.2.0

Wednesday, 19^th August 2015

We started taking care of our biobricks ! We planned to deposite four biobricks : DEC205, OVA2, IFNgamma and Mat alpha IFN gamma. For OVA2 and DEC205, we just amplified the fragments with primers designed to make the fragment fit into the pSB1C3 plasmid. For IFN gamma and Mat alpha IFN gamma, as IFN gamma presented a restriction site, we proceeded to do a site directed mutagenenis to remove it. Mutagenesis of IFNgamma and Mat alpha IFN gamma: We performed two consecutive PCR, the first one (PCR1) to introduce a mutation at the desired site and the second one (PCR2) to amplify the entire fragment (see 21/08). In between, PCR clean-ups were done using Nucleospin Gel and PCR Clean-up (LOT :1504/001).

IFNgamma PCR1

PCR1 primers:

• - P005 FN IFNgamma/RV Mat IFNgamma
• - FW Mat IFNgamma/P006 RV IFNgamma

Q5 PCR mix (50µl):

1. 2,5 µl forward primers
2. 2,5 µl reverse primers
3. 1 µl New insert1 m2 Cter
4. 19 µl water
5. 25 µl PCR mix (Q5)

Q5 PCR Program

1. step 1 : 98°C – 30 seconds
2. step 2 : 98°C – 10 seconds
3. step 3 : 57, 60 or 63°C
4. step 4 : 72°C – 1 min (repeat steps 2-4 for 40 cycles)
5. step 5 : 16°C - Hold

Mat alpha IFN gamma PCR1

Q5 PCR mix (50µl):

1. 2,5 µl P007 FW Mat alpha GMCSF
2. 2,5 µl P007 RV Mat IFN gamma
3. 1 µl New insert1 m2 Cter
4. 19 µl water
5. 25 µl PCR mix (Q5)

Q5 PCR Program

1. step 1 : 98°C – 30 seconds
2. step 2 : 98°C – 10 seconds
3. step 3 : 57, 60 or 63°C
4. step 4 : 72°C – 1 min (repeat steps 2-4 for 40 cycles)
5. step 5 : 16°C - Hold

Thursday, 20^th August 2015

Mutagenesis of IFNgamma and Mat alpha IFN gamma: Gel electrophoresis (2% agarose) for the PCR1 products

Friday, 21^th August 2015

We checked by digestion the following samples :

• - ADH1 Mat alpha IFN gamma (pYGG1) (sample « IFNgamma)
• - pYGG1
• - water

Digestion mix :

1. 2 µl NEB 2.1 Buffer
2. 1 µl Hind III
3. 2 µl DNA
4. 15 µl water

We let the mix, 1 hour at 37°C and 500 rpm.

Digestion gel electrophoresis (agarose 1%) :

Friday, 21^th August 2015

We checked by digestion the following samples :

• - AGA2P OVA1 DEC205 pYGG1 (sample « SD »)
• - AGA2P OVA1 pYGG1 (sample « DEC -»)
• - OVA2 pYGG2 (sample « OVA2 »)
• - pYGG2
• - water

Digestion mix :

1. 2 µl NEB 2.1 Buffer
2. 1 µl Hind III
3. 2 µl DNA
4. 15 µl water

We let the mix, 1 hour at 37°C and 500 rpm.

Digestion gel electrophoresis (agarose 1%) :

Monday, 24^th August 2015

PCR of the fragment RFP of pYGG1 for biosensor design using primers 3B FW 2.0 and 3B reverse :

Q5 PCR mix (50µl)

1. 2,5 µl 3B FW 2.0
2. 2,5 µl 3B RV
3. 1 µl New insert1 m2 Cter
4. 19 µl water
5. 25 µl PCR mix (Q5)

Q5 PCR Program

1. step 1 : 95°C – 30 seconds
2. step 2 : 95°C – 10 seconds
3. step 3 : 57, 60 or 63°C
4. step 4 : 72°C – 1 min (repeat steps 2-4 for 40 cycles)
5. step 5 : 12°C - Hold

Mutagenesis of IFNgamma and Mat alpha IFN gamma: PCR2

IFN gamma PCR2 primers :

1. P015 FW IFN gamma/P016 RV IFNgamma

Mat alpha IFN gamma PCR2 primers :

1. P015 FW IFN gamma / P016 RV IFN gamma

Q5 PCR mix (50µl) :

1. 2,5 µl forward primers
2. 2,5 µl reverse primers
3. 1 µl New insert1 m2 Cter
4. 19 µl water
5. 25 µl PCR mix (Q5)

Q5 PCR program :

1. step 1 : 95°C – 30 seconds
2. step 2 : 95°C – 10 seconds
3. step 3 : 57, 60 or 63°C
4. step 4 : 72°C – 1 min (repeat steps 2-4 for 40 cycles)
5. step 5 : 12°C - Hold

Wednesday, 26^th August 2015

Miniprep and nanodrop of pYGG1, pYGG2 and AGA1P-pYGG2 using NucleoSpin Plasmid (LOT 1306/003 Macherey-Nagel):

1. pYGG1 = 68 ng/µL
2. pYGG2 = 127 ng/µL
3. AGA1P (pYGG2) = 32 ng/µL

Wednesday, 26^th August 2015

PCR Clean up of biobrick Mata-IFNg and biobrick IFNg

1. - BB Mata-IFNg = 50 ng/µL
2. - BB IFNg = 35 ng/µL

Gel electrophoresis (1% agarose) of biobricks Mata-IFNg (BB Mata-IFNg) and IFNg (BB IFNg) :

Wednesday, 26^th August 2015

PCR Clean up of Biosensor3

1. - Biosensor 3 = 46 ng/µL

Gel electrophoresis (1% agarose) of biosensor 3

Thursday, 27^th August 2015

E.coli transformation of biosensor 2 and 3

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

Thursday, 27^th August 2015

PCR of biobrick OVA1

Q5 PCR mix (50µl) :

1. 2,5 µl FWD
2. 2,5 µl RV
3. 1 µl New insert1 m2 Cter
4. 19 µl water
5. 25 µl PCR mix (Q5)

Q5 PCR program :

1. step 1 : 95°C – 30 seconds
2. step 2 : 95°C – 10 seconds
3. step 3 : 57, 60 or 63°C
4. step 4 : 72°C – 1 min (repeat steps 2-4 for 40 cycles)
5. step 5 : 12°C - Hold

Gel electrophoresis (2% agarose) :

Digestion of the following biobricks using NEB kit :

1. - BB IFNg
2. - BB Mata-IFNg
3. - BB DEC 205
4. - BB OVA1
5. - BB OVA2
6. - pSB1C3

Digestion mix :

1. 2µL NEB Buffer 2.1
2. 0.5µL Eco RI
3. 0.5 µL PstI
4. 500 ng DNA
5. water qsp 20µL

The mix was left at 1h 37°C under agitation 500rpm and 20 minutes at 80°C.

Friday, 28^th August 2015

PCR clean-up and nanodrop of digested products:

1. - BB IFNg D = 13 ng/µL
2. - BB Mata-IFNg D = 10 ng/µL
3. - BB DEC 205 D = 15.4 ng/µL
4. - BB OVA1 D = 9 ng/µL
5. - BB OVA2 D = 14 ng/µL
6. - pSB1C3 D = 7.4 ng/µL

Ligation of digested products into pSB1C3 with T4 Ligase (ratio 3:1):

1. 2µL T4 DNA Ligase Buffer
2. 1µL T4 DNA Ligase
3. 50ng pSB1C3
4. 3:1 insert
5. water qsp 20µL

We let the mix at room temperature for 30 minutes and at 65°C for 10 minutes.

E.coli transformation with ligated 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 chloramphenicol
5. 5) Put plates in growth incubators at 37°C for 24 hours

Friday, 28^th August 2015

Colony PCR of transformed E.coli with biosensor 2 and 3

PCR colony mix :

1. 1 µl of resuspended colony in 20 µl LB
2. 1 µl URA F1
3. 1 µl URA R5 2.0
4. 7 µl water
5. 10 µl PCR mix (Dreamtaq)

PCR colony program :

1. Step 1 : 95°C - 5 min
2. Step 2 : 95°C – 30 seconds
3. Step 3 : 50°C – 30 seconds
4. Step 4 : 72°C – 2 minutes (repeat step 2-4, 45 times)
5. Step 5 : 72°C – 10 minutes
6. Step 6 : 4°C – Pause

Gel electrophoresis (1% agarose)

Miniculture of biosensor 2 and 3

Monday, 31^st August 2015

Colony PCR gel electrophoresis (1% agarose)

Tuesday, 1^st September 2015

Glycerol library of Biosensor 2 and 3 :

7.5 μl 50% glycerol + 7.5 μl miniculture

samples names:

1. "145" : Biosensor 2
2. "146" : Biosensor 3

Miniprep and nanodrop of biosensor 2 and 3 using NucleoSpin Plasmid (LOT 1306/003 Macherey-Nagel) :

1. 2.1 = 380 ng/µL
2. 2.2 = 440 ng/µL
3. 2.3 = 392.8 ng/µL
4. 3.1 = 225 ng/µL
5. 3.2 = 250 ng/µL
6. 3.3 = 85 ng/µL

Tuesday, 1^st September 2015

Gal1 deletion: Q5 PCR of IFNγ and Biosensor 3

Q5 PCR primers :

1. - IFNγ amplification : Gal1- Fwd IFNγ / Gal1- Rv IFNγ
2. - Biosensor 3 amplification : Fwd Gal1- sur ligate RFP / Rv Gal1- sur ligate RFP

Q5 PCR mix (50µl):

1. 2,5 µl FWD
2. 2,5 µl RV
3. 1 µl New insert1 m2 Cter
4. 19 µl water
5. 25 µl PCR mix (Q5)

Q5 PCR program :

1. step 1 : 95°C – 30 seconds
2. step 2 : 95°C – 10 seconds
3. step 3 : 60°C
4. step 4 : 72°C – 1 min (repeat steps 2-4 for 40 cycles)
5. step 5 : 12°C - Hold

Gal1 deletion: Digestion DpnI

Digestion mix :

1. 50μL PCR reaction
2. 5μL NEB 2.1 Buffer
3. 1μL DpnI

We let the mix at 37°C for 1 hour and then at 80°C for 20 minutes

PCR clean-up

Gel electrophoresis (0.7% agarose) :

Wednesday, 2^nd September 2015

Miniprep and nanodrop of biobricks using NucleoSpin Plasmid (LOT 1306/003 Macherey-Nagel):

1. - BB IFNγ-p 1 = 85ng/μL
2. - BB IFNγ-p 2 = 40ng/μL
3. - BB MATα-IFNγ-p 1 = 85ng/μL
4. - BB MATα-IFNγ-p 2 = 100ng/μL
5. - BB DEC 205-p 1 = 105ng/μL
6. - BB DEC 205-p 2 = 90ng/μL
7. - BB OVA1-p 1 = 86ng/μL
8. - BB OVA1-p 2 = 72ng/μL
9. - BB OVA2-p 1 = 50ng/μL
10. - BB OVA2-p 2 = 57ng/μL

The biobricks were sent to sequencing:

Sequencing mix:

1. 2.5μL primer VF2
2. 2.5μL primer VR
3. 5μL DNA (Ci=80-100ng/μL)

Each samples were sent with duplicates, which makes 20 tubes in total.

Thursday, 3^rd September 2015

Gal1 deletion: Phosphorylation

Mix (10µl) :

1. 2μL DNA
2. 1μL T4 DNA Ligase buffer (with 10Mm ATP)
3. 1μL PNK
4. 6μL H20

We let the mix at 37°C for 1 hour.

Gal1 deletion: Ligation

Mix (10µl) :

1. 1μL T4 DNA Ligase buffer
2. 1μL T4 DNA Ligase
3. 8μL H20

We let the mix at room temperature for 1 hour.

Gal1 deletion E.coli transformation with ligated 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 chloramphenicol
5. 5) Put plates in growth incubators at 37°C for 24 hours

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