Team:Valencia UPV/Notebook/Content

Valencia UPV iGEM 2015

Daily notebook

Here we present you all the procedures we did to develop our project. On this page you can find the notebook contents. If preferred, you can go directly to the protocols, the experiments on Nicotiana or the protoplasts experiments by pressing in the buttons above or below (after protocols). We hope you enjoy reading our incredible journey!


June

27 May 2015

Starts our work in the lab!

Marta, a lab mate gives us a construction, the red toggle swich (E:PIF6:PhyB:VP16:Etr8:luc), we just have to add the renilla; α2 (GB160) to test it.

Make the ligation (step 2 in the protocol):

hrefhttp://stackoverflow.com/questions/2537285/adjust-table-column-width-to-content-size
E:PIF6:PhyB:VP16:Etr8:luc+ren; Ω1
1µL E:PIF6:PhyB:VP16:Etr8:luc; α1
1µL renilla; apha2
1µL Ω1
6,8µL H2O

28 May 2015

Electroporation (step 3 in the protocol) of E. coli to insert our first construction.

Make a petri dish culture with a LB-Agar plate with streptomycin.


29 May 2015

There is no white colonies, we electroporate again and make petri dish culture.


30 May 2015

There was just one white colony, make the ligation again.

E:PIF6:PhyB:VP16:Etr8:luc+ren; Ω1
0.5µL E:PIF6:PhyB:VP16:Etr8:luc; α1
1µL renilla; apha2
1µL Ω1
7.2µL H2O

1 June 2015

Electroporation of the new ligation.


2 June 2015

There are white colonies. Make 2 liquid cultures of them (Step 4 in the protocol). Add 3.5ml of LB and 3.5µL of spectomycin.

Make liquid culture of just some glycerinates:

  • α1
  • α2
  • Ω1
  • Ω2
  • pUPD2
  • E:PIF6:NLS; pUPD2 (GB0288)
  • E:PIF6:NLS; α1 (GB892)
  • E:PIF6:NLS; Ω2 (GB893)
  • E:PIF6:NLS:luc:PhyB; α1 (GB896)
  • Luc:PhyB; Ω1 (GB890)
  • PhyB:VP16; pUPD2 (GB289)
  • PhyB:VP16; α2 (GB88E)
  • Etr8:CMVmini; pUPD2 (GB1097)
  • OpLexA:mini35S; pUPD2 (GB733)
  • OpLexA:mini35S:luc:Tnos; α2
  • LexABD; pUPD2 (GB0732)
  • LacI for N-Tfusion; pUPD2 (GB858)
  • Linker:LacIBD; pUPD2 (GB704)
  • OpLacI:mini35S:luc:Tnos; α2 (GB152)
  • OpLacI:mini35S; pPUD2 (GB534)

3 June 2015

Al cultures have grown except for Ω2. Make minipreps (Step 5 in the protocol).

Digestion of the minipreps (Step 6 of the protocol).

α1--
α2--
Ω1--
pUPD2--
E:PIF6:NLS; pUPD2 (GB0288)EcoRI3000, 1000
E:PIF6:NLS; α1 (GB892) EcoRI6300, 2500
E:PIF6:NLS; Ω2 (GB893)EcoRV1800, 6600, 900
E:PIF6:NLS:luc:PhyB; α1 (GB896)EcoRI3600, 6300, 5600
Luc:PhyB; Ω1 (GB890)BamHI2300, 6300, 4200
PhyB:VP16; pUPD2 (GB289)EcoRI3000, 2000, 500
PhyB:VP16; α2 (GB88E)HindIII2100, 6300, 1800
Etr8:CMVmini; pUPD2 (GB1097)EcoRI3000, 480
OpLexA:mini35S; pUPD2 (GB733)EcoRI3000, 460
OpLexA:mini35S:luc:Tnos; α2 (GB151)HindIII2500
LexABD; pUPD2 (GB0732)EcoRI3000, 300
LacI for N-Tfusion; pUPD2 (GB858)EcoRI3000, 1000
Linker:LacIBD; pUPD2 (GB704)EcoRI3000, 1000
OpLacI:mini35S:luc:Tnos; α2 (GB152)HindIII2500, 2600
OpLacI:mini35S; pPUD2 (GB534)EcoRI3000, 560
E:PIF6:PhyB:VP16:Etr8:luc+ren; Ω1BamHI3700, 6100, 6600, 4200
E:PIF6:PhyB:VP16:Etr8:luc+ren; Ω1EcoRV11000, 400, 2500, 3000, 4000

Make the gel:

pUPD2AlfAlpha1288289534
okokokokok?
704732733858892896
okokokokokok
1097Alpha288E151152Omega1
okokokokokok
890893Red toggle (C1) (EcoRV)Red toggle (C1) (BamHI)Red toggle (C2) (EcoRV)Red toggle (C2) (BamHI)
okokoknonono

4 June 2015

Ask for the NDronpa sequence. This will be part of our blue toggle.

This piece is known by reading the paper ‘Reversible photoswichable Dronpa-1 monitors nucleocytoplasmic transport of an RNA-binding protein in transgenic plants?(Doi: 10.111/j.1600-0854.2011.01180.lambda.).

The sequence of NDronpa is plant optimised and avoid cryptic sequences. We have domesticated this sequence with a linker in N-terminal to allow us to join it to a binding domain and also we had a NLS in the C-terminal to transport itself to the nucleus. It is domesticated as B5 part for Golden Braid assembling.

After obtaining the sequence we compare the protein in Uniprot and we can observed that our sequence add a V in the position 2. We compare this results with other papers and none of them has this addition. When we compare this sequence with the paper ?Optical control protein activity by fluorescent protein domains?(Doi: 10.1126/science.1226854) we observed that our position 146 is the position 145 and as what we want is the interaction caused by the N145-K145, we eliminate the V. We also eliminate a pair of amino acids at the end of the sequence following the same criteria.

Once obtained both variants of Dronpa, we decided to add the binding domain to KDronpa and the activation to NDronpa as this last one tetramerizes and all operator sequence are repeated in our promoters.


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-1?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).

Ligations:

PIF6 + PhyB; Ω1Etr8(CMV)+BxbI:T35S; α1
1µL (GB892) PIF; α11µL (GB1097) Etr8(CMV); pUPD2
1µL (GB88E) PhyB; α21µL BxbI; pUPD2
1µL Ω1 1µL Tnos pUPD2
6.8µL H2O1µL α1
5.8µL H2O

Digestions:

(GB160) 35S:Renilla:tNOS-35S:P19:tNOSEcoRV2475, 381, 4601
(GB896) Luc:PIF6:PhyBEcoRV11608, 3942

6 June 2015

Transform to E. coli from PIF+Phy and BxbI and make petri dish cultures.

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

Agarose gel.

GB160289PIF+PhyBBxbI
okno??

7 June 2015

We’ve got white colonies from PIF+Phy and BxbI!

Pick two colonies from each construction.


8 June 2015

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

Digestion:

Etr8(CMV):Bxb1:Tnos; α1EcoRI6345, 238
EPIF6 + PhyB-PV16; Ω1BamHI6686, 1439, 2685, 2237

Agarose gel was made:

BxbI (C1)BxbI (C2)E:PIF6+PhyB-VP16 (C1)E:PIF6+PhyB-PV16 (C2)
okoknono

Repeat digestion because we are not sure of the last digestions.

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:

PIF-PhyB-Luc-Renilla-P19
1 µL vector
0.8 µL dilution ?GB160
1.7 µL PIF:PhyB
4.15 µL H2O
Ratio 1:2 vector insert

As BxbI 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 pUPD2. The new one has different bases.
  • Change manually the pUPD2 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)
nono

We see three bands: 7000, 4000, 1900pb

Transform optimized ligation PIF-Phy-Luc-Renilla-P19 and make petri dish cultures.


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.

  • 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:VP16PvuII (buffer green 10x)3663, 9472
PIF+Phy:VP16BamHI1939, 2685, 2337, 6674
  • Agarose gel 1%:
PIF + Phy (PvuII) C3PIF + Phy (PvuII) C4PIF + Phy (PvuII) C5PIF + Phy (PvuII) C6
nooknoNo
PIF + Phy (BamHI) C3PIF + Phy (BamHI) C4PIF + Phy (BamHI) C5PIF + Phy (BamHI) C6
nooknoNo
  • Transformation into AgrobacteriumEPIF6-PhyB-VP16 + luciferase (GB896) and make petri dish culture. We are not going to have the positive control (renilla+P19) and we won’t be able to quantify and make ratios.

11 June 2015

  • Minipreps of the culture:
  • Digestion:
E:PIF6:PhyB:VP16:luc:renBamHI4209, 3756, 6100, 6674
EcoRV3942, 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)
nononono

Transformation into Agrobacteriumof Renilla (GB160) because we could not join this construction with PIF:PhyB and so we will do a cotransfection of both plasmids.Make petri dish culture.


12 June 2015

The petri dish with PIF:PhyB: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).
BxbI; α1+PhyB; α2
1µl BxbI
1 µl PhyB
1 µl Ω2
4.6 µl H2O
  • Transform renilla (160) with pSub plasmid into agrobacterium and make petri dish culture.

15 June 2015

  • Repeat the ligation BxbI+35S:E-PIF6:tnos because PIF was Ω2
BxbI + 35S:E-PIF6:tnos; Ω1
1µl BxbI
1 µl PhyB
1 µl Ω1
4.6µl H2O
  • KDronpa has arrived:
    • Centrifuge it 2-5sec at maximum velocity.
    • Add 50 µl to have a concentration of 20ng/µl
    • Mix it with the vortex and spin.
  • Ligation:
KDronpa; pUPD2
1 µl KDronpa
1 µl pUPD2
5.6 µl H2O
  • It was not possible to pick colonies of the Agrobacterium transformed with renilla 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; Ω1, into E. coli.Make petri dish culture.

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 following the temperature gradient.
    • 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:
Template1+25+67+8PIF7+8VP169+1011+12
Band pattern1017284391478464290
Gel resultokokokokNo DNAok
  • Transformation in E. coli of the correct ligations and make petri dishes cultures:
    • 1+2, 5+6, 7+8PIF, 7+8VP16, 11+12

18 June 2015

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

KDronpa EcoRI 2800

  • Gel:
Kdronpa C1Kdronpa C2Kdronpa C3Kdronpa C4
nonookno
Etr8:BxbI:phyB C1Etr8:BxbI:phyB C2Etr8:BxbI:phyB C3
Nonono

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

  • Take glicerynates out:
    • Gal4; pUPD2 (GB731)
    • Ω2
    • NoATGPromoter (GB00552)
    • Renilla (GB160)(GB159)(GB109)
  • 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
31.5µl H2O
  • Ligations:
Etr8:BxbI:T35S; α1Template PCR; pUPD2
1 µlEtr80.5µl template
1 µl BxbI1µl pUPD2
1 µl T35S6.1µl H2O
1 µl α1
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.
    • Gal4; pUPD2 (GB731)
    • Ω2
    • NoATGPromoter (GB00552)
    • Renilla (GB160)(GB159)(GB109)
  • Do the glycerinates digestions:
Minipreps:EnzimeBand pattern
(GB159) pDGB1_Ω2 renillaEcoRV2909, 2475,882, 812, 381
Entry vector, Ω2EcoRV6652, 621
(GB552) pP35s NoATG; pUPD2EcoRI2997, 1090
(GB160) renilla pDGB1, α2 EcoRV4601, 2475, 381
(GB731) Gal4BD (CDS); pUPD2EcoRI2997, 2493
(GB109)355:renilla:Tnos; α1EcoRI2580, 2493
  • We make an agarose gel with the digestions made before and the PCR of KDronpa.
159160Ω25527311099+109+1211+12
okokokokokoknookok
  • Transformation into E. coli of ligations:

1+2, 5+6, 7+8PIF, 7+8VP16 all in pUPD2

  • We made an stack of Cloranfenicol petri dishes
    • 250ml LB agar
    • X-Gal (1:500): 500 µl
    • IPTG (1:1000): 250 µl
    • Cloranfenicol (1:2000): 125 µl

20 june 2015

We have white colonies of renilla! Also of Etr8+BxbI; α1

We have also pUPD2 colonies but they are so close to the blue ones that we can’t pick anyone.So we make strakes.

  • We make a liquid culture of Agrobacteriumof Renilla (rif/kan/tetr).

21 June 2015

  • Pick colonies and make liquid culure of (all colonies are in pUPD2):
    • Plates : PIF (17/06/15) (C1 and C2)
    • VP16 (C1 and C3)
    • LacI (C1-C3)
    • Plates (19/06/15): BxbI (C1, C2, C3),
    • VP16 (C4, C5)
    • LacI (C1, C2)
    • PIF (C1-C5)
    • LexA (C1, C2)
  • We take out two glicerynates of GFP and BFP (of the Alfredo’s box)

22 June 2015

  • We made minipreps of the liquid culture of the day before:
    • LacIBD; pUPD2 (C1-C5)
    • LexABD; pUPD2 (C1, C2)
    • Etr8(CMV):Bxb1 (C1-C3)
    • PIF6; pUPD2 (C1-C5)
    • VP16; pUPD2 (C1, C4, C5)
  • Make the digestions of all the minipreps:
LacIBD, pUPD2NotI2046, 1053
LexABD, pUPD2 NotI2046, 321
Etr8(CMV):Bxb1 NotI1532, 1290, 5896
PIF6,pUPD2 NotI2046, 407
VP16, pUPD2 NotI2046, 500
  • Refresh the viral system that a lab mate borrow to us. This is going to be use to agroinfiltrate some plants to make some cool draws to sent to a TV programm so they can watch what are we doing. This cultures consist of three parts divided in three Agrobacteriumcolonies. They are the citoplasm, the fluerescent protein (GFP, DsRed or YFP) and the integrase, in our case PhiC31.
  • We received the reporter BxbI (RepBxbI)!
    • 500ng of sample
    • Centrifuge at 3000rpm for 5 seconds (spin).
    • Add 50 µl H2O
    • Shake it and let at 50ºC for 20min
  • Make a PCR of Gal4 and NDronpa (9-10), the primers of NDronpa are aliquoted.

Make an agarose gel with all the digestions:

LacI C1LacI C2LacI C3LacI C4LacI C5LexA C1LexA C2BxbI C1BxbI C2BxbI C3
Okokokokoknonookokno
PIF C1PIF C2PIF C3PIF C4PIF C5VP16 C1VP16 C4VP16 C5
Nono-okokokokok
Gal4NDronpa 1NDronpa 2

FOTO

  • We make ligations of:
    • Etr8(CMV):BxbI; α1 + PhyB:VP16;α2; Ω1
    • LacIBD;pUPD2 + PIF6BDPless; pUPD2; α1
    • KDronpa;pUPD2 + LacIBD; pUPD2; α1
    • Gal4BD; pUPD2
    • Reporter of BxbI; pUPD2
  • Tomorrow we have to take out pUPD2 of constitutive promoters, terminators and GFP (CDS).

23 June 2015

  • Transform into E.Coli the 5 ligations done yesterday and two more transformations of 5+6(1) and 5+6(2) which are the ligations in pUPD2 of the 18/06.
    • Etr8(CMV):BxbI; α1 + PhyB:VP16;α2; Ω1
    • LacIBD;pUPD2 + PIF6BDPless; pUPD2; α1
    • KDronpa;pUPD2 + LacIBD; pUPD2; α1
    • Gal4BD; pUPD2
    • Reporter of BxbI; pUPD2
    • LexABD (5+6), pUPD2 (1 and 2)
  • We have taken out of the -80ºC fridge the glycerinate of GFP; pUPD2 (GB0059)/ampicilin.
  • The liquid culture of Renilla (ryfampicin/kanamycin/tetracyclin) does not grow after the two days required. So we decide to refresh two new colonies, one of them in a tube with the three antibiotics and another with rifampicina and kanamicine. Asun says that the tetracycline slow down the growth of Agro.
  • The 4 liquid cultures of LexA+IPTG/+gal are all blue: throw them.
  • We ordered again the primer n?0 (NDronpa R1). Changing one codon in 3?and delete another in 5?

24 June 2015

Pick colonies of the plates done yesterday and pass them into a liquid medium:

  • LacIBD+PIF; α1 (C1, C2)
  • Gal4BD; pUPD2 (C1)
  • RepBxb1; pUPD2 (C1-C3)
  • LacIBD+KDonpa; α1 (C1, C2)
  • Etr8(CMV)+BxbI+PhyB+VP16; Ω1 (C1)
  • LexABD1; pUPD2 (C1-C4)
  • LexABD2; pUPD2. No colonies.

The viral systems of Agrobacteriumcultures to make the color mosaics are ready after 2 days at 28ºC. We can make the agroinfiltration.

Protocol to prepare solution to agroinfiltrate in the protocols notebook part.

  • Ligation:
ETR8(CMV):BxbI; α1+PhyB:VP16; α2; Ω1 Gal4BD(pcr) + pUPD2
1.5 µl Etr8:BxbI1 µl Gal4 PCR
1.5 µl 88E (PhyB:VP16)1 µl pUPD2
1 µl Ω15,6 µl H2O
3.6µl H2O

Quantification of DNA:

  • GFP (GB0059); pUPD2: 249 ng/µl
  • Ω2: 238 ng/µl
  • Alfredo’s pUPD2, domesticator: 102 ng/µl
  • iGEM704: 405 ng/µl
  • iGEM735: 403 ng/µl
  • 552 AMP 35S noATG: 45 ng/µl
  • PIF (C5), pUPD2: 119 ng/µl
  • pD6B3, Ω2 (22/06): 158 ng/µl
  • LacIBD (C1); pUPD2 (22/06): 129 ng/µl
  • 109 renillaDC: 49 ng/µl
  • IGEM 534: 13.6 ng/µl
  • VP16 (C1); pUPD2:102 ng/µl
  • IGEM 1097: 409 ng/µl
  • KDronpa (C3); pUPD2 (18/06): 174 ng/µl
  • IGEM 858: 487 ng/µl
  • 731AMP Gal4 (19/06): 81 ng/µl
  • IGEM pUPD2 domesticator: 87 ng/µl
  • PIF+PhyB (C1) (08/06): 108 ng/µl
  • 160 renilla, α2 (19/06): 46 ng/µl
  • 159 renilla, Ω2 (19/06): 149 ng/µl
  • Etr8:BxbI (C1)(22/06): 149 ng/µl
  • IGEM 732: 422 ng/µl

25 June 2015

Minipreps of the liquid culture:

  • We don’t observed growth in LacIBD+PIF and LacIBD+KDronpa.

Digestion of the minipreps and do the gel:

Gal4BD; pUPD2NotI2046, 282
RepBxbI; pUPD2NotI2046, 460
Etr8(CMV):BxbI:PhyB; α1BamHI6674, 2237, 2806, 1174
LexABD; pUPD2NotI2046, 321
9+10; pUPD2NotI464

Gel:

Etr8:BxbILexA C1LexA C2LexA C3LexA C4RepBxbI C1RepBxbI C2RepBxbI C3Gal4 C1PCR 9+10
nononononookokoknook
  • We make a PCR of the Fusion Taq pH (proof-reading) to prove that the primer received number 10. This new one works! Amplify the sequence of NDronpa (R1).
  • Refresh the cultures of Agrobacteriumwith the viral system. Add only ryfampicin and kanamycin.
  • Ligations:
N-dronpa; pUPD2RepBxbI; α1Gal4BD, pUPD2LexABD; pUPD2
1 µl PCR 9+101 µl Rep Bxb11 µl PCR 3+41 µl PCR 5+6
1 µl PCR11+121 µl Promoter without ATG1 µl pUPD21 µl pUPD2
1 µl pUPD21 µl Tnos
1 µl α1
4,6 µl H2O3,6 µl H2O5,6 µl H2O5,6 µl H2O
Etr8:BxbI+PhyB; Ω1
1 µl Etr8:BxbI
1 µl 88E
1µl Ω1
3,6 µl H2O

Transform ligations into E.Coli and make petri dish cultures with cloranfenicol for all of them except the ligation of Etr8:Bxb1+PhyB that goes with streptomycin.


26 June 2015

Do ligations:

RepBxbI+GFP; α2LacIBD+PIF6; α1
1 µl RepBxbI1 µl LacIBD, pUPD2
1 µl promoter without ATG1 µl PIF6, pUPD2
1 µl Tnos1 µl promoter
1µl GFP (0059)1 µl T35
1 µl α21 µl α1
2.6 µl H2O2.6 µl H2O

Digestion:

LacIBD+PIF6; α1EcoRI6345, 1997, 641

Gel:

LacIBD+PIF C1LacIBD+PIF C2
nono

Both of them present the fragment of the vector at 6000 pb but none of them at 2000bp which is the insert one.

Measurement of the ODs of PhyB:PIF6:luc and renilla+P19.

PhyB:PIF6:luc: 0.35 (1:2)0.351.429 µl
Ren+P19: 0.34 (1:2)0.341.412 µl
  • Ligation of:
LacIBD; pUPD2+KDronpa; pUPD2; α1
1 µl 35S
1 µl LacIBD;pUPD2
1 µl KDronpa; pUPD
1 µl T35S
1 µl α1
2.6 µl H2O

1?EXPERIMENT. Red toggle. E:PIF6:PhyB and renilla. For more info, click here.


27 June 2015

Transformation into E. coli of LacIBD+KDronpa; α1 and make petri dish culture.

Make petri dish culture of LexABD and Etr8(CMV):Bxb1:GFP.

We make liquid culture of:

  • RepBxbI:GFP (C1-C4)
  • LacIBD+PIF6 (C1-C5)
  • NDronpa (C1-C4)
  • Gal4BD (C1-C5)
  • LexABD (C1-C3)

28 June 2015

Do the minipreps of the liquid cultures that have grown.

  • RepBxbI:GFP (C1 and C2)
  • LacIBD+PIF6 (C1-C4)
  • NDronpa (C1-C4)
  • Gal4BD (C1-C5)
  • LexA: didn’t grow

Do the digestions of the minipreps:

LacIBD+PIF; α1EcoRI6345, 1997, 641
RepBxbI:GFP; Ω2HindIII6345, 2683
Gal4BD; pUPD2NotI2681, 644
NDronpa; pUPD2NotI2046, 744

Make the gel.

RepBxbI:GFP C1RepBxbI:GFP C2LacIBD+PIF C1LacIBD+PIF C2LacIBD+PIF C3LacIBD+PIF C4
nononononoNo
Gal4BD C1Gal4BD C2Gal4BD C3Gal4BD C4Gal4BD C5N-Dronpa C1
okokokokokok
N-Dronpa C2N-Dronpa C3N-Dronpa C4
nookok

Take glycerinated:

  • GB0030: p35S
  • GB0036: T35S
  • Make liquid culture of LexABD (C1-C4).
  • We transform again LacIBD:KDronpa and RepBxb1:GFP, adding to the agar plates 100 µl of each transformation.

29 June 2015

Do the minipreps of the 4 colonies of LexABD and both glycerinates, 35S and T35S.

Do the digestion of the minipreps:

LexABD; pPPD2NotI2358, 312
35S; pUPD2NotI2981, 1074
T35S; pPUD2NotI2981, 304

Make the gel:

LexA C1LexA C2LexA C3LexA C4P35ST35S
okokokokOk?Ok?

Make ligations:

LacIBD+KDronpa+promoter+termi; α1Gal4BD+KDonpa+prom+ter; α1LexABD+KDronpa+prom+term; α1
1 µl LacI; pUPD21 µl Gal4; pUPD21 µl Gal4; pUPD2
1 µl KDronpa; pUPD21 µl KDronpa; pUPD21 µl KDronpa; pUPD2
1 µl 35S (GB0030)1 µl 35S (GB0030)1 µl 35S (GB0030)
1 µl T35S (GB0036)1 µl T35S (GB0036)1 µl T35S (GB0036)
2.6 µl H2O2.6 µl H2O2.6 µl H2O
1 µl α11 µl α11 µl α1
NDronpa+VP16; α2Gal4BD+PIF6; α1LacIBD+PIF6; α1
1 µl NDronpa; pUPD21 µl Gal4BD; pUPD21 µl LacIBD; pUPD2
1 µl VP16; pUPD21 µl PIF6; pUPD21 µl PIF6; pUPD2
1 µl 35S (GB0030)1 µl 35S (GB0030)1 µl 35S (GB0030)
1 µl T35S (GB0036)1 µl T35S (GB0036)1 µl T35S (GB0036)
2.6 µl H2O2.6 µl H2O2.6 µl H2O
1 µl α21 µl α11 µl α1
LexABD+PIF6; α1
1 µl LexABD; pUPD2
1 µl PIF6; pUPD2
1 µl 35S (GB0030)
1 µl T35S (GB0036)
2.6 µl H2O
1 µl α2
  • Transform all the ligations into E.Coli. Gal4BD+K-Dronpa and LacIBD+K-Dronpa went wrong and we have to do it again.

Sent N-Dronpa with the primers 9 and 12 to sequence to check if the codon that synthetize for the amino acid K has change to the amino acid N.

Quantification of DNA:

  • RepBxbI:GFP (C1): 163.8 ng/µl
  • NDronpa; pUPD2 (C4):113.1 ng/µl
  • NDronpa (C3): 83.2 ng/µl
  • NDronpa (C1): 116.6 ng/µl
  • Gal4BD (C1): 95.2 ng/µl
  • Gal4BD (C2): 120.7 ng/µl
  • RepBxbI:GFP (C2): 170.6 ng/µl
  • RepBxbI (C1): 80.6 ng/µl

30 June 2015

Transform Gal4+KDronpa and LacI+KDronpa and make petri dish culture.

Miniprep of:

  • RepBxbI+GFP (C1-C3)
RepBxb1+GFP; Ω2HindIII6345, 2683

Gel:

RepBxbI+GFP C1RepBxbI+GFP C2RepBxbI+GFP C3
Nonono

We pick more colonies of RepBxb1+GFP, Ω2 and make liquid cultures.

Make liquid culture of:

LexABD+KDronpa+prom+term; α1 (C1 and C2)

NDronpa+VP16; α2 (C1 and C2)

Gal4BD+PIF6; α1 (C1 and C2)

LacIBD+PIF6; α1 (C1 and C2)

LexABD+PIF6; α1 (C1 and C2)

Take out a glycerinate 35S:Luciferase:Tnos (GB0227) and do a miniprep.


July

Muy lejos, más allá de las montañas de palabras, alejados de los países de las vocales y las consonantes, viven los textos simulados. Viven aislados en casas de letras, en la costa de la semántica, un gran océano de lenguas. Un riachuelo llamado Pons fluye por su pueblo y los abastece con las normas necesarias. Hablamos de un país paraisomático en el que a uno le caen pedazos de frases asadas en la boca. Ni siquiera los todopoderosos signos de puntuación dominan a los textos simulados; una vida, se puede decir, poco ortográfica.


August and September

Muy lejos, más allá de las montañas de palabras, alejados de los países de las vocales y las consonantes, viven los textos simulados. Viven aislados en casas de letras, en la costa de la semántica, un gran océano de lenguas. Un riachuelo llamado Pons fluye por su pueblo y los abastece con las normas necesarias. Hablamos de un país paraisomático en el que a uno le caen pedazos de frases asadas en la boca. Ni siquiera los todopoderosos signos de puntuación dominan a los textos simulados; una vida, se puede decir, poco ortográfica.