Team:Valencia UPV/Notebook

Valencia UPV iGEM 2015

The project
Notebook

CONSTRUCTIONS


5 June 2015

We had 2 cultures from the last day, corresponding to other 2 colonies of ligation.

Agrobacterium culture of promoter less: Luciferase + Renilla

Minipreps

Digestion with BamHI and EcoRV

Agarose gel 1%

How to ask and make primers?

  • Select the sequence to amplify and save in FASTA format.
  • gbCloning, go to Tools-Domesticator-1º Category
  • Add FASTA and select parts.
  • On the protocol we have the primers
  • The oligos they give us:
    • 4 first nucleotides: so the enzyme can recognize without problems
    • 6 following bingind sites.
    • 1 extra nucleotide.
    • 4 overhangs.

Meeting with Daniel Ramón (Biopolis).

Ligation with part 2 and 24 of task sheet.

PIF6 + PhyB; ?1Etr8 CMV_Bxb1_T35S
1µL 892 (PIF α1)1µL 1097 (Etr8 CMV) Pupd2
1µL 88E (Phy α2)1µL Bxb1 (PuPD)
1µL ?1 1µL Tnos PuPD
1.2µL Buffer ligase1µL α1
1µL Bsmb15.8µL H2O
6.8µL H2O

If we make a digestion of 160 (35S:Renilla:tNOS-35S:P19:tNOS) with EcoRV, we obtain: 2475, 381, 4601 pb.

If we make a digestion of 896 (Luc:PIF6:PhyB)with EcoRV, we obtain: 11608, 3942 pb.


June 2015

Transform to E.coli from PIF+Phy and Bxb1

  • 1.5µL of ligation
    • Cuvette on ice
    • Competent cells + 1.5µL of ligation
    • Pulse (electroporator) at 1500V
    • Add 300µL shock medium and put Eppendorf 1h at 37ºC

Culture on petri dishes the ligations.

Digest of 160, 289 and the two ligations, PIF+phy and Etr8+BxbI.

Agarose gel.


7 June 2015

We’ve got white colonies! (from PIF+Phy and Bxb1)

Pick two colonies from each construction.

4 tubes

  • 3.5µL LB each tube

2) 2 tubes + 3.5µL Kanamycin (K)


8 June 2015

Minipreps of the 4 liquid cultures and digestion to see the band patterns.

Digestion:

Etr8(CMV):Bxb1:Tnos; ?1EcoRI6345, 238
EPIF6 + PhyB-PV16; ?1BamHI6686, 1439, 2685, 2237

Agarose gel was made:

Bxb1 (C1)Bxb1 (C2)EPIF6 + PhyB-PV16 (C1)EPIF6 + PhyB-PV16 (C2)
okok

Repeat digestion (errors).

We don’t have sure the toggle, so we decide to repeat the digestion with other enzyme tomorrow, noticing that the colony 2 has better bands pattern.

Optimized ligation of PIF-Phy-Lac-Renilla-P19

  • 1 µL vector
  • 0.8 µL dilution ½ 160
  • 1.7 µL big part
  • 1.2 µL BSA
  • 1.2 µL buffer
  • 1 µL BsbmI
  • 1 µL ligase
  • 4.15 µL H2O
  • Ratio 1:2 vector insert

As Bxb1 was good at the digestion we put 1 µL of LB and 1 µL of Kanamicyne on the tube where it had grown and store at 37ºC to glycerinate later.

We design primers to binding domain (BD) and PIF.

  • Problem: domesticator is introduced in an old pUPD. The new one has different bases.
  • Change manually the pUPD bases in the program (Benchling).

9 June 2015

Digestion of the ligation of yesterday containing: EPIF6-PhyB-VP16 (C1 y C2)

EPIF6-PhyB-VP16PvuII (green buffer)3663, 9472pb

Agarose gel 1%:

EPIF6-PhyB-VP16 (C1)EPIF6-PhyB-VP16 (C2)EPIF6-PhyB-VP16 (C3)
nonono

We see three bands: 7000, 4000, 1900pb

Transform optimized ligation (yesterday 8/6)

  • 3.5 µL ligation product (EPIF6-PhyB-VP16 + Luciferase + Renille + P19)
  • 40 µL electrocompetent cells.
  • Pulse of 1500V
  • Store 1h at 37ºC
  • Plate culture at agar with Spectinomycin: 50 µL of the transformation.

10 June 2015

  • Check the primers and order LexA, Gal4, PIF6, LacI, Dronpa.
  • Check linker VP16 (88E) and make a primer for it.
  • Take out glycerinate of Ω2.

Alfredo’s part is not working.

  • Pick colonies of transformation and make liquid culture of E:PIF6:PhyB:VP16:luc:ren (C1-C3).
  • Minipreps of liquid culture (PIF + Phy), colonies C3, C4, C5, C6
  • Digestion:

PIF + Phy:VP16 PvuII (buffer green 10x) 3663, 9472

PIF + Phy:VP16 BamHI 1939, 2685, 2337, 6674

  • Agarose gel 1% (10 samples, 8 + 2 molecular markers)

PIF + Phy (PvuII) C3 PIF + Phy (PvuII) C4 PIF + Phy (PvuII) C5 PIF + Phy (PvuII) C6

no ok no No

PIF + Phy (BamHI) C3 PIF + Phy (BamHI) C4 PIF + Phy (BamHI) C5 PIF + Phy (BamHI) C6

no ok no No

  • Transformation of Agrobacterium (C58) of the 896 construction (EPIF6-PhyB-VP16 + luciferase). We are not going to have the positive control and we won’t be able to quantify (we don’t have Renilla + P19).

11 June 2015

  • Minipreps of the culture:
  • Digestion:

E:PIF6:PhyB:VP16:luc:ren BamHI 4209, 3756, 6100, 6674

EcoRV 3942, 2989, 2475, 381, 10952

Gel:

PIF6:PhyB:VP16:luc:
ren C1 (BamHI)PIF6:PhyB:VP16:luc:
ren C3 (BamHI)PIF6:PhyB:VP16:luc:ren
C1 (EcoRV)PIF6:PhyB:VP16:luc:ren
C3 (EcoRV)
??

Transformation in Agrobacterium of Renilla (160) due to that we could not join this with PIF:phyB and so we will do a cotransfection of both plasmids. Make petri dish culture with kanamicyn and rifampicin.


12 June 2015

The petri dish with PIF:phy:luc was taken out the 37ºC room and put into the fridge to pick colonies tomorrow.


13 June 2015

  • Pick colonies to make liquid culture:
    • Renilla in agrobacterium: just one colony, it was made liquid culture but check carefully the gel.
    • It was noticed that the piece 160, renilla, needs a pSub plasmid to replicate itself so we will transform 160 into a agrobacterium with this plasmid (C58 pSub).
  • Ligation:

BxbI; alfa1+phyB; alfa2

1µl BxbI

1 µl phyB

1 µl ?2

4.6 µl H2O

  • Transform renilla (160) into agrobacterium and make

15 June 2015

  • Repeat the ligation BxbI+35S:E-PIF6:tnos because PIF was ?2

BxbI + 35S:E-PIF6:tnos; ?1

1µl BxbI

1 µl phyB

1 µl ?1

4.6 µl H2O

  • KDronpa has arrived:
    • Centrifuge it 2-5sec at max velocity.
    • Add 50 µl to have a concentration of 20ng/µl
    • Mix it with the vortex and spin.
  • Ligation: en la libreta pone que se liga a pUPD2

KDronpa; pUPD2

1 µl KDronpa

1 µl pUPD2

5.6 µl H2O

  • It was not possible to pick colonies of the Agrobacterium transformed because they did not grow. Maybe the problem is that with tetraciclyn bacterias grow slowly. Wait 1 day more.
  • Transformation of the ligation, BxbI + 35S:E-PIF6:tnos; ?1, into E.coli.
  • Make an agar culture in petri dish and let grow 16h at 37ºC.

16 June 2015

  • Transformation of the ligation, KDronpa, into E.coli.
  • Pick colonies of BxbI:E-PIF6 and make liquid culture (C1-C3).
  • Primers had arrived, it has been done the resuspension (dilution 1:10)of all of them.
PrimersCode TemplateWorking temperature? ºC
LacI F1LacI (858)69.7
LacI R 2
Gal4 F3We did not take out the glicerynate.63.2
Gal4 4
LexA F5LexA (732)62.7
LexA R6
PIF:VP16 F7PIF6 (288)60.1
PIFVP16 R8
NDronpa F19Kdronpa67.7
NDronpa R110
Dronpa F21158.5
NDronpa R212
  • A PCR with all the primers and the fragments was done, the samples were put in order with the temperature.
    • The templates were in dilution 1:50, exception of KDronpa that was dilution 1:5 and the primers 1:10.
PCR Fusion Taq (50µl)
DNA template (10 µg/µl)
0.5 µl fusion taq
2.5 µl primer F
2.5 µl primer R
2 µl NTPs
31.5 µl H2O
17 June 2015
  • Pick colonies and make liquid culture of:
    • KDronpa (C1-C4)
    • Ligations with the PCR’s products:
    • Templates PCR: 1+2, 5+6, 7+8PIF, 7+8VP16, 9+10, 11+12.
Template PCR; pUPD2
0.5µl template
1µl pUPD2
6.1µl H2O
  • Minipreps of liquid cultures:
    • BxbI:E-PIF6 (C1-C3)
  • Agarose gel with the PCRs:

Template 1+2 5+6 7+8PIF 7+8VP16 9+10 11+12

Band pattern 1017 284 391 478 464 290

Gel result ok ok ok ok No DNA ok

  • Transformation in E.coli of the correct ligations:
    • 1+2, 5+6, 7+8PIF, 7+8VP16, 11+12
    • Put in cloranfenicol petri dishes.

18 June 2015

  • Minipreps of the liquid cultrures:
    • KDronpa (C1-C4)
  • Digestions:

KDronpa EcoRI 2800

  • Gel:

Kdronpa C1 Kdronpa C2 Kdronpa C3 KdronpaC4

no no ok no

Etr8:BxbI:phyB C1 Etr8:BxbI:phyB C2 Etr8:BxbI:phyB C3

No no no

We discovered that the construction with BxbI did not go well because our lab college gives us the wrong piece.

  • Take glicerynates out:
    • Gal4; pUPD (731)
    • ?2
    • PromotersinATG (GB00552)
    • Renilla (160)(159)(109)
  • PCR:

NDronpa

2.5 µl (9+10) primer F

2.5 µl (11+12) primer R

2 µl NTPs

0.2 µl Taq

10 µl Buffer

31.5 µl H2O

  • Ligations:

Etr8:BxbI:T35S; α1 Template PCR; pUPD2

1 µlEtr8 0.5µl template

1 µl BxbI 1µl pUPD2

1 µl T35S 6.1µl H2O

5.8 µl H2O

Templates PCR: 1+2, 5+6, 7+8PIF, 7+8VP16


19 June 2015

  • We do a PCR with the normal Taq polymerase.
    • 1µl of DNA’s template (9+10, 9+12 and 11+12)
    • 2µl of specific buffer
    • 2µl of NTPs
    • 1µl primer forward
    • 1µl primer reverse
    • 0.5 µl of Taq
    • 12.5 µl H2O
    • These quantities multiplied by 3.
  • Minipreps of the yesterday’s glycerinated cultures.
  • To do the digestions we add in each eppendorf.

Minipreps: Enzime Band pattern

159 pDGB1_?2 renilla EcoRV 2909, 2475,882, 812, 381

Entry vector, ?2 EcoRV 6652, 621

552 pP35s NoATG, pUPD EcoRI 2997, 1090

160 renilla pDGB1, a2 EcoRV 4601, 2475, 381

731 pUPD pGal4BD (CDS) EcoRI 2997, 2493

109 GB1_a1 355:renilla:Tnos EcoRI 2580, 2493

  • We make an agarose gel with the digestions made before and the PCR of KDronpa.

159 160 ?2 552 731 109 9+10 9+12 11+12

ok ok ok ok ok ok no ok ok


20 june 2015

We have White colonies of renilla! Also of Etr8 + Bxb1

We have also pUPD colonies but they are so close to the blue ones that we can’t pick anyone.So we make strakes in another plates to have the colonies separated.


21 June 2015


22 June 2015

LacIBD, pUPD NotI 2046, 1053

LexABD, pUPD NotI 2046, 321

Etr8(CMV):Bxb1 NotI 1532, 1290, 5896

PIF6,pUPD NotI 2046, 407

VP16, pUPD NotI 2046, 500