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

PCR Program
Step1 95°C - 5 min
Step 2 95°C – 30 s
Step 3 50°C – 30s
Step 4 72°C – 2 min (Repeat step 2-4 35 times)
Step 5 72°C – 10 min
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

  
  Mix (50µl)
  2,5 µl FWD AGA2P
  2,5 µl RV AGA2P
  1 µl New insert1 m1 Cter
  19 µl water
  25 µl PCR mix (Q5)

  
  PCR program
  Step 1 95°C
  Step 2 95°C
  Step 3 60°C
  Step 4 72°C (Repeat step 2-4 31 times)
  Step 5 72°C
  Step 6 12°C

PCR from New insert1 m2 Cter (PCR002)

• amplification of DEC205 with primers containing AGA2P overhang
  Mix (50µl)
  2,5 µl FWD DEC205
  2,5 µl RV DEC205
  1 µl New insert1 m2 Cter
  19 µl water
  25 µl PCR mix (Q5)
  PCR program
  Step 1 95°C
  Step 2 95°C
  Step 3 60°C
  Step 4 72°C (Repeat step 2-4 31 times)
  Step 5 72°C
  Step 6 12°C

  PCR001 and PCR002 gel electrophoresis :

  PCR clean up and nanodrop of AGA2P and DEC205 products :

  • AGA2P = 72 .3 ng/µl (260/280 = 1.79 ; 260/230 = 0.76)
  • 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)

  Program :

  E. coli transformation with golden gate products
  

  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. Shock the mix at 42°C for 45 seconds, transfer the mix on ice for 10 min
  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. Plate 50 µl and 150 µl to plates containing LB agar media with ampificilin
  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 :
  Seq.ID 26EB16 - A41 (URA F1/SR1 lig IFNg)
  Seq.ID 26EB17 - A41.2 (replicate of A41)
  Seq.ID 26EB18 - A42 (S2 lig IFNg/SR2 lig IFNg)
  Seq.ID 26EB19 - A42.2 (replicate of A42)
  Seq.ID 26EB20 - C61 (URA F1/SR1 lig IFNg)
  Seq.ID 26EB21 - C61.2
  Seq.ID 26EB22 - C62 (S2 lig IFNg/SR2 lig IFNg)
  Seq.ID 26EB23 - C62.2
  Seq.ID 26EB24 - D71 (URA F1/SR1 lig IFNg)
  Seq.ID 26EB25 - D71.2
  Seq.ID 26EB26 - D72 (S2 lig IFNg/SR2 lig IFNg)
  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 µl of DNA
  1 µl forward primer
  1 µl reverse primer
  7 µl water
  10 µl PCR mix (Dreamtaq)

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

  E. coli transformation with golden gate products
  

  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. Shock the mix at 42°C for 45 seconds, transfer the mix on ice for 10 min
  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. Plate 50 µl and 150 µl to plates containing LB agar media with ampificilin
  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:

  
  Sample SD2
  Seq.ID 26EB28 (URA F1/URA F1 Rev Seq V2)
  Seq.ID 26EB29 (URA R5 2.0 Fw seq V2/URA R5 2.0)
  Seq.ID 26EB30 (URA F1/ URA R1)
  Seq.ID 26EB31 (URA F2/URA R5 2.0)
  Seq.ID 26EB32 (URA F1/URA F1 Rev Seq V2)
  Seq.ID 26EB33 (URA R5 2.0 Fw seq V2/URA R5 2.0)
  Seq.ID 26EB34 (URA F1/ URA R1)
  Seq.ID 26EB35 (URA F2/URA R5 2.0)

  
  Sample SD1
  Seq.ID 26EB36 (URA F1/URA F1 Rev Seq V2)
  Seq.ID 26EB37 (URA R5 2.0 Fw seq V2/URA R5 2.0)
  Seq.ID 26EB38 (URA F1/ URA R1)
  Seq.ID 26EB39 (URA F2/URA R5 2.0)
  Seq.ID 26EB40 (URA F1/URA F1 Rev Seq V2)
  Seq.ID 26EB41 (URA R5 2.0 Fw seq V2/URA R5 2.0)
  Seq.ID 26EB42 (URA F1/ URA R1)
  Seq.ID 26EB43 (URA F2/URA R5 2.0)

  Multiple PCR
  PCR mix (20 µl)
  1 µl of DNA
  1 µl forward primer
  1 µl reverse primer
  7 µl water
  10 µl PCR mix (Dreamtaq)

  Golden gates of :
  

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

  E. coli transformation with golden gate products
  

  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. Shock the mix at 42°C for 45 seconds, transfer the mix on ice for 10 min
  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. Plate 50 µl and 150 µl to plates containing LB agar media with ampificilin
  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
  1 µl of resuspended colony in 20 µl LB
  1 µl URA F1
  1 µl URA R5 2.0
  7 µl water
  10 µl PCR mix (Dreamtaq)
  
  PCR Program
  Step1 95°C - 5 min
  Step 2 95°C – 30 s
  Step 3 50°C – 30 s
  Step 4 72°C – 2 min (repeat step 2-4 30 times)
  Step 5 72°C – 10 min
  Step 6 4°C – Pause

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

  Yeast transformation with:

  • AGA2P OVA1 DEC205 (SD1)
  • AGA1
  • AGA2P OVA1 DEC205 (SD2)

    Protocol:
    

    1. From the W303 yeast culture at DO=1, harvest in sterile tube at 5000 rpm for 5 min
    2. Pour off the medium, resuspend the cells in 25 ml of sterile water and centrifuge again
    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. Pellet the cells at top speed for 15 sec and remove the LiAc with a micropipette
    5. Resuspend the cells with 0.1 LiAc to a final volume of 500 µl
    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. 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)
    • Vortex each tube vigorously until the cell pellet has been completely mixed. Usually takes about 1 min
    • Incubate at 30°C for 30 min
    • Heat shock in a water bath at 42°C for 15 minute
    • Ice for 2 minutes
    • Centrifuge at 5000 rpm for 15 sec and remove the transformation mix with a micropipette
    • Pipette 1 ml of sterile water into each tube and resuspend the pellet by pipetting it up and down gently
    • Plate 50 µl and 150 µl of the transformation mix onto plates with corresponding media
    • 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
    

    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. Shock the mix at 42°C for 45 seconds, transfer the mix on ice for 10 min
    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. Plate 50 µl and 150 µl to plates containing LB agar media with ampificilin
    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 coµld 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:

    • T4: ADH1 Ma IFNg (pYGG1)li

    Protocol:
    

    1. From the W303 yeast culture at DO=1, harvest in sterile tube at 5000 rpm for 5 min
    2. Pour off the medium, resuspend the cells in 25 ml of sterile water and centrifuge again
    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. Pellet the cells at top speed for 15 sec and remove the LiAc with a micropipette
    5. Resuspend the cells with 0.1 LiAc to a final volume of 500 µl
    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. 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)
    • Vortex each tube vigorously until the cell pellet has been completely mixed. Usually takes about 1 min
    • Incubate at 30°C for 30 min
    • Heat shock in a water bath at 42°C for 15 minute
    • Ice for 2 minutes
    • Centrifuge at 5000 rpm for 15 sec and remove the transformation mix with a micropipette
    • Pipette 1 ml of sterile water into each tube and resuspend the pellet by pipetting it up and down gently
    • Plate 50 µl and 150 µl of the transformation mix onto plates with corresponding media:
      
      • pYGG1 => URA-
      • pYGG2 => TRP-
      • pYGG1 + pYGG2 => TRP- URA-
    • Incubate at 30°C for 3 days

    Friday, 31^st July 2015

    Since on the 07/30, we coµld 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 :
    AGA1P.1 = 44.2 ng/µl

    Yeast transformations:

    • T1: OVA2 (pYGG2)
    • T2: AGA2P OVA1 (pYGG1) + AGA1P (pYGG2)
    • T3: AGA2P OVA1 DEC205 (pYGG1) + AGA1P (pYGG2)
    • T5: AGA2P/GFP (pYGG1) + AGA1P (pYGG2)
    • T6: AGA2P GFP (pYGG1)
    • T7: AGA2P OVA DEC205 (pYGG1)
    • T8: AGA2P OVA1 (pYGG1)

    Protocol:
    

    1. From the W303 yeast culture at DO=1, harvest in sterile tube at 5000 rpm for 5 min
    2. Pour off the medium, resuspend the cells in 25 ml of sterile water and centrifuge again
    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. Pellet the cells at top speed for 15 sec and remove the LiAc with a micropipette
    5. Resuspend the cells with 0.1 LiAc to a final volume of 500 µl
    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. 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)
    • Vortex each tube vigorously until the cell pellet has been completely mixed. Usually takes about 1 min
    • Incubate at 30°C for 30 min
    • Heat shock in a water bath at 42°C for 15 minute
    • Ice for 2 minutes
    • Centrifuge at 5000 rpm for 15 sec and remove the transformation mix with a micropipette
    • Pipette 1 ml of sterile water into each tube and resuspend the pellet by pipetting it up and down gently
    • Plate 50 µl and 150 µl of the transformation mix onto plates with corresponding media:
      
      • pYGG1 => URA-
      • pYGG2 => TRP-
      • pYGG1 + pYGG2 => TRP- URA-
    • Incubate at 30°C for 3 days

    Monday, 3^rd August 2015

    Colony PCR of yeast transformants with following primers :
    
    AGA2P/DEC205
    P1-4 => URA F1/ URA R1
    P5-8 => URA R2/ URA R5 2.0

    SD1
    R1-4 => URA F1/URA R5 2.0
    R5-6 => URA F1/URA R1
    R7-8 => URA F2/URA R5 2.0

    SD2
    T1-4 => URA F1/ URA R5 2.0
    T5-6 => URA F1/URA R1
    T7-8 => URA F2/URA R5 2.0

    Library
    "130" P2 (AGA2P/DEC205)
    "131" P4 (AGA2P/DEC205)
    "132" R4 (SD1)
    "133" R5 (SD1)
    "134" R6 (SD1)
    "135" T1 (SD2)
    "136" T3 (SD2)
    "137" T4 (SD2)
    "138" T5 (SD2)

    Monday, 3^rd August 2015

    Colony PCR of yeast transformants with following primers :
    GMCSF
    Q1-8 => URA F1/URA R5 2.0

    IFNgamma
    S1-4 => URA F1/SR1 lig IFN gamma
    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
    Colony PCR mix :
    10 µl dreamtaq master mix
    7 µl water
    1 µl reverse primer
    1 µl forward primer

    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 with following primers :
    Colony PCR mix (20 µl):
    3.5 µl water
    0.5 µl primer forward
    0.5 µl primer reverse
    0.5 µl of e.coli resuspended in 10 µl water

    PCR program :
    step 1 : 95°C
    step 2 : 95°C
    step 3 : 72°C
    step 4 : 60°C (URA F1/ URA R5 2.0) or 53°C (others)
    step 5 : 72°C

    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.

    Colony PCR of T4 (see 31/07)
    Colony PCR mix (20 µl):
    10 µl PCR mix (Dreamtaq)
    qsq µl water
    2.5 µl primer forward
    2.5µl primer reverse
    yeast resuspended in 10 µl water (separated in two parts table )

    PCR program :
    step 1 : 95°C – 5 min
    step 2 : 95°C – 30 seconds
    step 3 : 60°C – 30 seconds
    step 4 : 72°C – 4 min (repeat step 2-4 45 times)
    step 5 : 4°C – Pause

    Golden gate of ADH1 mat alpha GMCSF (pYGG1) construction :
    Mix (20 µl) :
    0.849 µl ADH1
    3.243 µl Mat alpha GMCSF
    3.1 µl PYGG1
    2 µl T4 ligase buffer
    0.5 µl T4 ligase
    0.5 µl BSA I

    Program :

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

    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. Shock the mix at 42°C for 45 seconds, transfer the mix on ice for 10 min
    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. Plate 50 µl and 150 µl to plates containing LB agar media with ampificilin
    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)
    Colony PCR mix (20 µl):
    10 µl PCR mix (Dreamtaq)
    qsq µl water
    2.5 µl primer forward
    2.5µl primer reverse
    yeast resuspended in 10 µl water (separated in two parts table )

    PCR program :
    step 1 : 95°C – 5 min
    step 2 : 95°C – 30 seconds
    step 3 : 60°C – 30 seconds
    step 4 : 72°C – 4 min (repeat step 2-4 45 times)
    step 5 : 4°C – Pause

    Thursday, 6^th August 2015

    Yeast transformation without OVA1 Protocol:
    

    1. From the W303 yeast culture at DO=1, harvest in sterile tube at 5000 rpm for 5 min
    2. Pour off the medium, resuspend the cells in 25 ml of sterile water and centrifuge again
    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. Pellet the cells at top speed for 15 sec and remove the LiAc with a micropipette
    5. Resuspend the cells with 0.1 LiAc to a final volume of 500 µl
    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. 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)
    • Vortex each tube vigorously until the cell pellet has been completely mixed. Usually takes about 1 min
    • Incubate at 30°C for 30 min
    • Heat shock in a water bath at 42°C for 15 minute
    • Ice for 2 minutes
    • Centrifuge at 5000 rpm for 15 sec and remove the transformation mix with a micropipette
    • Pipette 1 ml of sterile water into each tube and resuspend the pellet by pipetting it up and down gently
    • Plate 50 µl and 150 µl of the transformation mix onto plates with corresponding media:
      
      • pYGG1 => URA-
      • pYGG2 => TRP-
      • pYGG1 + pYGG2 => TRP- URA-
    • Incubate at 30°C for 3 days

    Friday, 7^th August 2015

    PCR colony yeast and E. coli

    Saturday, 8^th August 2015

    GMCSF E.coli PCR colony

    IFNg E.coli PCR colony

    Monday, 10^th August 2015

    Golden gate ADH1 matalpha GMCSF (pYGG1)
    Mix (20µl):
    Mat alpha GMCSF (gblock)
    pYGG1
    ADH1
    
    2 µl DNA T4 ligase buffer
    0.5 µl DNA ligase
    0.5 µl BSA I
    qsp water

    Program :

    Tuesday, 11^th August 2015

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

    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. Shock the mix at 42°C for 45 seconds, transfer the mix on ice for 10 min
    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. Plate 50 µl and 150 µl to plates containing LB agar media with ampificilin
    5. Put plates in growth incubators at 37°C for 24 hours

    Golden gate ADH1 mat alpha IFNgamma (pYGG1)
    Mix :
    0.033 (x 3) µl Sample « B8 » (M :atalpha IFNgamma) (114 ng/µl)
    0.058 (x 3) µl Sample « A6 » (IFNgamma) (114 ng/µl)
    0.274 µl Sample « ADH1 » (75 ng/µl)
    0.929 µl pYGG1
    2 µl DNA T4 ligase buffer
    0.5 µl DNA ligase
    0.5 µl BSA I

    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. Discard media after centrifugation at 3000 rpm for 4 minutes
    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. 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)
    

    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. Shock the mix at 42°C for 45 seconds, transfer the mix on ice for 10 min
    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. Plate 50 µl and 150 µl to plates containing LB agar media with ampificilin
    5. Put plates in growth incubators at 37°C for 24 hours

    Colony PCR of pYGG1 ADH1 matalpha GMCSF with following set of primers :
    - URA F1 / URA R5 2.0
    - URA F1 / SR1 lig IFNgamma
    - S2 lig IFNgamma / SR2 lig GMCSF

    Thursday, 13^th August 2015

    Colony PCR of ADH1 Mat alpha IFN gamma (pYGG1) with folllowing set of primers :
    - URA F1 / URA R5 2.0
    - URA F1 / SR1 lig IFNgamma
    - S2 lig IFNgamma / SR2 lig IFNgamma

    Mix (10 µl) :
    5 µl dreamtaq master mix
    0.5 µl colony resuspended in 19 µl Luria Bertoni
    0.5 µl reverse primer
    0.5 µl forward primer
    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 Program
    Step1 95°C - 5 min
    Step 2 95°C – 30 s
    Step 3 57/60/63 °C – 30s
    Step 4 72°C – 2 min (repeat step 2-4, 41 cycles)
    Step 5 72°C – 10 min
    Step 6 4°C – Pause

    T1/2/3 Colony PCR gel electrophoresis (1% agarose)
    Expected sizes :
    T1 => OVA2 (290 bp)
    T2 => AGA1P (2225 bp) & AGA2P OVA1 (461 bp)
    T3 => AGA1P (2285 bp) & AGA2P OVA1 DEC205 (1381 bp)

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

    • IFN 1 = 343.5 ng/µl
    • IFN 4 = 290 ng/µl
    • IFN 6 = 265 ng/µl
    • 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 Program
    Step1 95°C - 5 min
    Step 2 95°C – 30 s
    Step 3 57/60/63 °C – 30s
    Step 4 72°C – 2 min (repeat step 2-4, 41 cycles)
    Step 5 72°C – 10 min
    Step 6 4°C – Pause

    T1/2/3 Colony PCR gel electrophoresis (1% agarose) primers used :
    T1/T2/WT : URA F1/R5 2.0
    T3.1 : URA R5 2.0/F1
    T3.2 : URA F1/R1
    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 primers:
    

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

    
    Mat alpha IFN gamma PCR
    PCR1 primers :

    • P007 FW Mat alpha GMCSF/ RV Mat IFN gamma

    Mix (50 µl) :
    25 µl Q5 master mix
    2.5 µl reverse primer
    2.5 µl forward primer
    1 µl DNA
    19 µl water

    Q5 PCR Program
    step 1 : 98°C – 30 seconds
    step 2 : 98°C – 10 seconds
    step 3 : 57, 60 or 63°C
    step 4 : 72°C – 1 min (repeat steps 2-4 for 40 cycles)
    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 constructions :

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

    Mix :
    2 µl NEB 2.1 Buffer
    1 µl Hind III
    2 µl DNA
    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 constructions :

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

    Mix :
    2 µl NEB 2.1 Buffer
    1 µl Hind III
    2 µl DNA
    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 :

    
    Mix (50 µl) :
    25 µl Q5 master mix
    2.5 µl reverse primer
    2.5 µl forward primer
    1 µl DNA
    19 µl water

    Q5 PCR Program
    step 1 : 98°C – 30 seconds
    step 2 : 98°C – 10 seconds
    step 3 : 57, 60 or 63°C
    step 4 : 72°C – 1 min (repeat steps 2-4 for 40 cycles)
    step 5 : 16°C - Hold

    Mutagenesis of IFNgamma and Mat alpha IFN gamma: PCR2

    IFN gamma
    PCR2 primers :
    

    • P015 FW IFN gamma/P016 RV IFNgamma

    Mat alpha IFN gamma
    PCR2 primers :
    

    • P015 FW IFN gamma / P016 RV IFN gamma

    Mix (50 µl) :
    25 µl Q5 master mix
    2.5 µl reverse primer
    2.5 µl forward primer
    1 µl DNA
    19 µl water

    Q5 PCR Program
    step 1 : 98°C – 30 seconds
    step 2 : 98°C – 10 seconds
    step 3 : 57, 60 or 63°C
    step 4 : 72°C – 1 min (repeat steps 2-4 for 40 cycles)
    step 5 : 16°C - Hold

    Wednesday, 26^th August 2015

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

    • pYGG1 = 68 ng/µL
    • pYGG2 = 127 ng/µL
    • AGA1P (pYGG2) = 32 ng/µL

    Wednesday, 26^th August 2015

    PCR Clean up of biobrick Mata-IFNg and biobrick IFNg
    

    • BB Mata-IFNg = 50 ng/µL
    • 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
    

    • 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
    

    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. Shock the mix at 42°C for 45 seconds, transfer the mix on ice for 10 min
    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. Plate 50 µl and 150 µl to plates containing LB agar media with ampificilin
    5. Put plates in growth incubators at 37°C for 24 hours

    Thursday, 27^th August 2015

    PCR of biobrick OVA1

    Mix (50 µl) :
    25 µl Q5 master mix
    2.5 µl reverse primer (P012 RV OVA1)
    2.5 µl forward primer (P012 FW OVA1)
    1 µl DNA (SD1)
    19 µl water

    Q5 PCR Program
    step 1 : 98°C – 30 seconds
    step 2 : 98°C – 10 seconds
    step 3 : 57, 60 or 63°C
    step 4 : 72°C – 1 min (repeat steps 2-4 for 40 cycles)
    step 5 : 16°C - Hold

    Gel electrophoresis (2% agarose)

    Digestion of the following biobricks using NEB kit:
    

    • BB IFNg
    • BB Mata-IFNg
    • BB DEC 205
    • BB OVA1
    • BB OVA2
    • pSB1C3

    Digestion mix:
    2µL NEB Buffer 2.1
    0.5µL Eco RI
    0.5 µL PstI
    500 ng DNA
    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: ul>

    BB IFNg D = 13 ng/µL

    BB Mata-IFNg D = 10 ng/µL

    BB DEC 205 D = 15.4 ng/µL

    BB OVA1 D = 9 ng/µL

    BB OVA2 D = 14 ng/µL

    pSB1C3 D = 7.4 ng/µL

    Ligation of digested products into pSB1C3 with T4 Ligase (ratio 3:1):
    2µL T4 DNA Ligase Buffer
    50ng pSB1C3
    3:1 insert
    water qsp 20µL
    1µL T4 DNA Ligase
    
    We let the mix at room temperature for 30 minutes and at 65°C for 10 minutes.

    E.coli transformation with ligated products:
    

    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. Shock the mix at 42°C for 45 seconds, transfer the mix on ice for 10 min
    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. Plate 50 µl and 150 µl to plates containing LB agar media with chloramphenicol
    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 µl of resuspended colony in 20 µl LB
    1 µl URA F1
    1 µl URA R5 2.0
    7 µl water
    10 µl PCR mix (Dreamtaq)

    
    PCR Program
    Step1 95°C - 5 min
    Step 2 95°C – 30 s
    Step 3 50°C – 30 s
    Step 4 72°C – 2 min (repeat step 2-4 30 times)
    Step 5 72°C – 10 min
    Step 6 4°C – Pause

    Gel electrophoresis (1% agarose)

    Miniculture of biosensor 2 and 3

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