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):

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

1 July 2015

Do the minipreps of the 10 liquid cultures.

Do the digestions:

LexABD+KDronpa;α1EcoRI6345, 2296
NDonpa+VP16; α2HindIII6345, 2427
Gal4BD+PIF6; α1EcoRI6345, 1867
LacI+PIF; α1EcoRI6345, 2638
LexABD+PIF6; α1EcoRI6345, 1906

Do the gel:

Gal4+PIF C1Gal4+PIF C2LexA+PIF C1LexA+PIF C2LexA+KDronpa C1
okokokokOk
LexA+Kdronpa C2LacI+PIF C1LacI+PIF C2Ndonpa+VP16 C1Ndronpa+VP16 C2
okokokokok

Prepare liquid culture of:

  • LexA+PIF; Ω1 (C1)
  • LacI+PIF; Ω1 (C1)
  • LexA+K-Dronpa (C1)
  • Gal4+PIF; Ω1 (C1)
  • VP16, pUPD2 (C1)
  • LexABD, pUPD2 (C2)
  • PIF6, pUPD2 (C5)
  • LacIBD, pUPD2 (C1)

EXPERIMENT 1.

  • Luciferase essay.

Pick colonies and make liquid culture of:

  • Gal4+K-Dronpa (C1 and C2)
  • LacI+K-Dronpa (C1 and C2)
  • RepBxbI+GFP (C4-C6)
  • Code:
  • 210.08-249: pUPD2, KDronpa C3
  • 210.08-250: pUPD2, NDronpa C1
  • 210.08-251: pUPD2, NDronpa C3
  • 210.08-252: pUPD2, NDronpa C4

2 July 2015

Minipreps of:

  • Gal4+KDronpa (C1 and C2)
  • LacI+KDronpa (C1 and C2)
  • RepBxbi+GFP (C4-C6)
Gal4+KDronpaEcoRI6345, 3028
LacI+KDronpaEcoRI6345, 2257
RepBxbI+GFPHindIII6300, 2400

Do the gel:

LacI+KDronpa C1LacI+KDronpa C2Gal4+KDronpa C1Gal4+KDronpa C2
okokokok
RepBxb1+GFP C4RepBxb1+GFP C5RepBxb1+GFP C6
okokok

Make ligations:

LexA:Kdonpa+NDronpa; Ω1LacI:Kdronpa+NDronpa; Ω1Gal4:Kdronpa+NDronpa; Ω1
1 µl LexA:Kdronpa1 µl LacI:Kdronpa1 µl Gal4:Kdronpa
1 µl NDronpa1 µl NDronpa1 µl NDronpa
1 µl Ω11 µl Ω11 µl Ω1
4.6 µl H2O4.6 µl H2O4.6 µl H2O
LexA:PIF+PhyB:VP16; Ω1LacI:PIF+PhyB:VP16; Ω1Gal4:PIF+PhyB:VP16; Ω1
1 µl LexA:PIF1 µl LacI:PIF1 µl Gal4:PIF
1 µl PhyB:VP16 (88E)1 µl PhyB:VP161 µl PhyB:VP16
1 µl Ω11 µl Ω11 µl Ω1
4.6 µl H2O4.6 µl H2O4.6 µl H2O
35S:BxbI+RepBxbI:GFP; Ω1E-PIF+phyB+luc+ren; Ω1
1 µl 35s:Bxb1:T35S (alfredo’s)0.5 µl PIF:PhyB:luc (GB0896)
1 µl NoATGProm:RepBxbI:GFP1 µl Renilla GB0160
1 µl Ω11 µl Ω1
4.6 µl H2O4.6 µl H2O

The samples that we sent to sequence have arrived:

  • 210.08-249: pUPD2, KDronpa C3 - ok
  • 210.08-250: pUPD2, NDronpa C1 - ok
  • 210.08-251: pUPD2, NDronpa C3 - ok
  • 210.08-252: pUPD2, NDronpa C4 - ok

The sequences of NDronpa have the desired mutation.

Transformation in E.Coli of the 8 ligations and make petri dish cultures.

Refresh the Agro’s cultures Renilla and PIF:phy:luc.


4 July 2015

Make the 2nd refresh of the culture of Agrobacterium.

Make liquid cultures of the 8 colonies of E.Coli.

  • Red toggle (C1)
  • Gal4:Kdronpa+NDronpa (C1 and C2)
  • LexA:Kdonpa+NDronpa (C1 and C2)
  • LacI:Kdronpa+NDronpa (C1 and C2)
  • LexA:PIF+PhyB:VP16 (C1 and C2)
  • 35S:BxbI+RepBxbI:GFP (C1 and C2)

5 July 2015

Ligations:

LacI:PIF+PhyB:VP16; Ω1Gal4:PIF+PhyB:VP16; Ω1
1 µl LacI:PIF1 µl Gal4:PIF
1 µl PhyB:VP161 µl PhyB:VP16
1 µl Ω11 µl Ω1
4.6µl H2O4.6 µl H2O

Minipreps of the liquid cultures.

Digestion of the minipreps:

LacI:Kdronpa+NDronpaBamHI6674, 5437
35S:BxbI+RepBxbI:GFPBamHI6674, 3859, 1782
Gal4:Kdronpa+NDronpaBamHI6674, 4666
LexA:Kdonpa+NDronpaBamHI6674, 4705
LexA:PIF+PhyB:VP16BamHI6674, 3513, 2337
E:PIF6:PhyB:VP16BamHI4209, 3756, 6100, 6674
  • Agarose gel (1%):
LacI:KNDronpa C1LacI:KNDronpa C2BxbI:Rep:GFP C1BxbI:Rep:GFP C2Gal4:KNDronpa C1Gal4:KNDronpa C2
okokokokokok
LexA:KNDronpa C1LexA:KNDronpa C2LexA:PIF:Phy C1LexA:PIF:Phy C2Red toggle
okoknonono

We have to repeat the digestion of: LexA+PIF:phy+VP16.

  • Take out the glycerinate 88C (GB1098), Etr8:luc:Tnos. We will use it like a negative control in the second luciferase essay.
  • Calculation of the ODs:
Renilla0.22182 µl of sample + 1.818 ml MES
E:PIF6:PhyB:Luc0.26154 µl of sample + 1.646 ml MES

EXPERIMENT 2.

Agroinfiltration of renilla and PIF6:PhyB:luc. For more info, click here.


6 July 2015

Transform the negative control into agrobacterium and make petri dish culture of:

  • Etr8:luc:Tnos
  • BxbI:ReporterBxbI:GF

We were doing dry lab preparing the power point to present our project to the rector and biotecs companies.


7 July 2015

Prepare this cultures for agroinfiltration:

  • Renilla
  • E:PIF6:PhyB:Luc
Renilla0.290.138ml of sample + 1.862ml of MES
PhyB+PIF+luc0.690.145ml of sample + 1.855ml of MES

EXPERIMENT 3. Start. Agroinfiltrate PhyB+PIF+luc and Renilla

Digestions:

Red toggleBamHI4209, 3756, 6100, 6674
LexA+PIF+phyBamHI3518, 5855, 6674

Gel:

Red toggleLexA+PIF+Phy C1LexA+PIF+Phy C2
NoOkno

It has arrived a new construction: AsLOVpep.

  • Resuspended with 50µl of H2O.

Ligations:

AsLOVpep; pUPD2Red ToggleLacI:Kdronpa:Ndronpa:VP16+renilla; α1Gal4:Kdronpa:Ndronpa:VP16+renilla; α1
1 µl AsLOVpep1 µl GB8461 µl LacI:KNdronpa:VP161 µl Gal4:KNdronpa
1 µl pUPD21 µl GB1601 µl GB1591 µl GB159
5.6 µl H2O1 µl Ω11 µl α11 µl α1
4.6 µl H2O4.6 µl H2O4.6 µl H2O
LexA:Kdronpa:Ndronpa:VP16+renilla; α1LexA:PIF:Phy:VP16+renilla; α1
1 µl LexA:Kdronpa:Ndronpa:VP161 µl LexA:PIF:Phy:VP16
1 µl GB1591 µl GB159
1 µl α11 µl α1
4.6 µl H2O4.6 µl H2O

8 July 2015

EXPERIMENT 2. Change the ligth conditions of the plants.

Transform into E. coli last ligations.


9 July 2015

Pick colonies and make liquid culture of:

  • AsLOVpep; pUPD2 colonies didn’t grow. Repeat.
  • Red toggle colonies are all blue. Repeat.
  • LacI:Kdronpa:Ndronpa:VP16+renilla (C1-C3)
  • Gal4:Kdronpa:Ndronpa:VP16+renilla (C1-C3)
  • LexA:Kdronpa:Ndronpa:VP16+renilla (C1 and C2)
  • LexA:PIF:Phy:VP16+renilla (C1 and C2)

Repeat the ligations.

  • AsLOVpep, as before.
Red toggle (PIF+PhyB+luc+ren)
0.5 µl PIF+phy+luc (896)
1 µl renilla (160)
1 µl Ω1
5.1 µl H2O

EXPERIMENT 2.

Luciferase essay.

Transform the ligations of AsLOVpepe and red toggle. This time we make a spin to concentrate the cells to make petri dish culture.


10 July 2015

Minipreps of:

  • LexABD:PIF:PhyB:VP16+Renilla; α1 (C1,C2)
  • LexA:Kdronpa:Ndronpa+renilla; α1 C1, C2)
  • Gal4:Kdronpa:Ndronpa+renilla; α1 (C1-C3)
  • LacI:Kdronpa:Ndronpa+renilla; α1 (C1,C2)

Digestions of:

LexABD:PIF:PhyB:VP16+Renilla; α1EcoRI6345, 5487, 4891
LexA:Kdronpa:Ndronpa+renilla; α1EcoRI9333, 6345
Gal4:Kdronpa:Ndronpa+renilla; α1EcoRI6345, 9194
LacI:Kdronpa:Ndronpa+renilla; α1EcoRI6345, 9965
LacIBD+PIF+PhyB; Ω1BamHI6674, 4245, 2337
Gal4BD+PIF+PhyB; Ω1BamHI6674, 3474, 2337

Make the gel:

LacIBD+PIF+PhyBGal4BD+PIF+PhyBLexA:PIF:PhyB:VP16+Ren C1LexA:PIF:PhyB:VP16+Ren C2
OkOkokok
LexA:KNdronpa+ren C1LexA:KNdronpa+ren C2Gal4:KNdronpa+ren C1Gal4:KNdronpa+ren C2
OkOkokOk
Gal4:KNdronpa+ren C3LacI:Kdronpa:Ndronpa+ren C1LacI:Kdronpa:Ndronpa+ren C2
OkOkok

We received two constructions:

  • CDS: phiC31. Resuspended with 100µl.
  • Reporter Phi31. Resuspended with 50 µl.

Pick colonies and make liquid culture of:

  • AsLOVpep; pUPD2 (C1 and C2)
  • Red toggle (C1 and C2).

Ligations:

PhiC31; pUPD2Reporter PhiC31; pUPD2LacI:PIF:PhyB:VP16+ren; α1
1 µl PhiC311 µl RepPhiC311 µl LacI:PIF:PhyB:VP16
1µl pUPD21µl pUPD21 µl renilla (GB159)
5.6 µl H2O5.6 µl H2O1 µl α1
4.6 µl H2O
Gal4:PIF:Phy:VP16+ren; α1LexA:PIF:PhyB:ren+OpLex:luc; Ω1LexA:KNdronpa:ren+OpLex:Luc; Ω1
1 µl Gal4:PIF:phy:VP161 µl LexA:PIF:phy:ren1 µl LexA:KNdronpa:ren
1 µl renilla (GB159)1 µl opLex:luc (GB151)1 µl OpLex:Luc (GB151)
1 µl α11 µl Ω11 µl Ω1
4.6 µl H2O4.6 µl H2O4.6 µl H2O
Gal4:KNdronpa:ren+UAS:luc; Ω1LacI:KNdronpa:ren+OpLacI:luc; Ω1
1 µl Gal4:KNdronpa:ren1 µl LacI:KNdronpa:ren
1 µl OpUAS:luc (GB227)1 µl OpLacI:luc (GB152)
1 µl Ω11 µl Ω1
4.6 µl H2O4.6 µl H2O

11 July 2015

Prepare glycerinates of:

  • BxbI+Rep:GFP; Ω1
  • NDronpa; pUPD2
  • BxbI:Etr8; pUPD2
  • Etr8(CMV):BxbI:T35S; α1

Do miniprep of:

  • AsLOVpep (C1 and C2)
  • Red toggle (C2) (C1 was blue)

Digestions:

E:PIF6:PhyB:luc:renBamHI6674, 6100, 4209, 3756
AsLOVpepNotI2558, 512
AsLOVpep C1AsLOVpep C2Red toggle C2
nonono

Do transformation of DHSa and yesterday ligations:

  • phyC31;pUPD2
  • Reporter PhiC31; pUPD2
  • LacI:PIF:PhyB:VP16+ren; α1
  • Gal4:PIF:phy:VP16+ren; α1
  • LexA:PIF:phy:ren+opLex:luc; Ω1
  • LexA:KNdronpa:ren+OpLex:luc; Ω1
  • Gal4:KNdronpa:ren+OpUAS:luc; Ω1
  • LacI:KNdronpa:ren+OpLacI:luc; Ω1

12 July 2015

Pick colonies and make liquid culture:

  • PhiC31;pUPD2 (C1-C3)
  • Reporter PhiC31; pUPD2 (C1-C3)
  • LacI:PIF:PhyB:VP16+ren; α1 (C1-C3)
  • Gal4:PIF:phy:VP16+ren; α1 All blue colonies.
  • LexA:PIF:phy:ren+OpLex:luc; Ω1 (C1)
  • LexA:KNdronpa:ren+OpLex:Luc; Ω1(C1-C3)
  • Gal4:KNdronpa:ren+UAS:luc; Ω1 (C1)
  • LacI:KNdronpa:ren+OpLacI:luc; Ω1 All blue colonies.

Ligations that were repeated:

Gal4:PIF:phy:VP16+ren; α1LacI:KNdronpa:ren+OpLacI:luc; Ω1
1 µl Gal4:PIF:phy:VP161 µl LacI:KNdronpa:ren
1 µl renilla (GB159)1 µl OpLacI:luc (GB152)
4.6 µl H2O4.6 µl H2O
1 µl α11 µl Ω1

Refresh the liquid cultures of Agrobacterium:

  • BxbI:GFP and Etr8:Tnos.
  • Pnos, it was at the fridge (-4ºC).

13 July 2015

All the liquid cultures have grown, do minipreps.

Do digestions:

phyC31;pUPD2NotI2046, 1899
Reporter phiC31; pUPD2NotI2046, 475
LacI:PIF:Phy:VP16+ren; α1EcoRI6345, 5623, 5487
LexA:PIF:phy:ren+opLex:luc; Ω1BamHI9431, 6674, 3531
LexA:KNdronpa:ren+OpLex:Luc; Ω1BamHI1199, 6674
Gal4:KNdronpa:ren+UAS:luc; Ω1BamHI11582, 6674

Agarose gel:

PhiC31 C1PhiC31 C2PhiC31 C3RepPhiC31 C1
nononook
RepPhiC31 C2 LacI:PIF:PhyB:VP16+ren C1LacI:PIF:PhyB:VP16+ren C2LacI:PIF:PhyB:VP16+ren C3
nonookok
LexA:PIF:phy:ren+OpLex:lucLexA:KNdronpa:ren+OpLex:luc C1LexA:KNdronpa:ren+OpLex:Luc C2LexA:KNdronpa:ren+OpLex:Luc C3
nookokok
Gal4:KNdronpa:ren+OpUAS:luc C1
no

The Agrobacterium cultures refreshed yesterday were store in the fridge.

It is made another culture of 35S:BxbI+reporterBxbI:GFP to keep it in the fridge.

EXPERIMENT 4. Recombinase BxbI.

Mesurement of the OD’s:

35S:BxbI+reporterBxbI:GFP: 0.28143 µl of culture+1857 µl of MES

Transformation of the ligation: Gal4:PIF:phy:VP16+ren; α1 and LacI:KNdronpa:ren+OpLacI:luc; Ω1.

The liquid cultures of this constructions were repeated:

  • phyC31;pUPD2 (C4 and C5)
  • Reporter phiC31; pUPD2 (C4)
  • LacI:PIF:PhyB:VP16+ren; α1 (C4)
  • LexA:PIF:phy:ren+OpLex:luc; Ω1 (C1)
  • LexA:KNdronpa:ren+OpLex:Luc; Ω1(C1-C3)
  • Gal4:KNdronpa:ren+UAS:luc;Ω1 (C1)
  • AsLOVpep; pUPD2 (C3)

14 July 2015

Do minipreps of:

  • phyC31;pUPD2 (C4 and C5)
  • Reporter phiC31; pUPD2 (C4)
  • LacI:PIF:PhyB:VP16+ren; α1 (C4)
  • LexA:PIF:phy:ren+OpLex:luc; Ω1 (C1)
  • LexA:KNdronpa:ren+OpLex:Luc; Ω1(C1-C3)
  • Gal4:KNdronpa:ren+UAS:luc;Ω1 (C1)
  • AsLOVpep; pUPD2 (C3)

Do digestions:

phyC31;pUPD2NotI2046, 1899
Reporter phiC31; pUPD2NotI2046, 475
LacI:PIF:Phy:VP16+ren; α1EcoRI6345, 5623, 5487
LexA:PIF:phy:ren+opLex:luc, Ω1BamHI9431, 6674, 3531
LexA:KNdronpa:ren+OpLex:Luc; Ω1BamHI1199, 6674
Gal4:KNdronpa:ren+UAS:luc; Ω1BamHI11582, 6674
AsLOVpep; pUPD2 NotI2558, 512

Make an agarose gel:

phyC31 (C4)phyC31 (C5)AsLOVpep (C4)LexA:PIF:phy:ren+opLex:luc (C1)
nonoNoNo
LexA:KNdronpa:ren+OpLex:Luc (C3)Gal4:KNdronpa:ren+OpUAS:luc (C1)Gal4:KNdronpa:ren+UAS:luc(C2)Gal4:KNdronpa:ren+UAS:luc (C3)
OknonoNo
LacI:PIF:Phy:VP16+renReporter phiC31 (C1)
noOk

Only LexA:KNdronpa:ren+OpLex:Luc and Reporter phiC31 were correct. Repeat the ligations because is the second digestion of this construction that were made.

  • Measurement of DNA concentration:
    • Reporter:phyC31: 13.6 ng/µl
    • PhiC31: 4.6 ng/µl
    • AsLOVpep: 30.3 ng/µl
    • OpLexA:luc (GB151): 40 ng/µl
    • Gal4:PIF:PhyB:ren (C1): 124.4 ng/µl
    • LacI:PIF:PhyB:VP16 (C1): 126 ng/µl
    • Renilla (GB159): 42 ng/µl
    • Gal4:Kdronpa:Ndronpa:renilla (C3): 172.8 ng/µl
    • OpUAS:luc (GB227): 6.3 ng/µl
  • Pick colonies and make liquid culture of LacI:KDronpa:NDronpa:ren:luc (C4 and C5). The colonies of Gal4:PIF:phy:VP16+ren were all blue.

15 July 2015

  • Miniprep of LacI:KDronpa:NDronpa:ren:luc (C4 and C5).
  • Digestion:
LacI:KDronpa:NDronpa:ren:lucEcoRI6345, 9965
  • Make the gel:
LacI:K:NDronpa:ren:luc C4LacI:K:NDronpa:ren:luc C5
nono
  • Measurement of DNA concentrations:
    • Gal4BD:PIF:PhyB:VP16: 125.6 ng/µl
    • LexA:PIF:PhyB:VP16:ren: 322.6 ng/µl
    • LacI:Kdronpa:NDronpa:ren: 209.0 ng/µl
  • Repeat the ligations:
PhiC31;pUPD2AsLOVpep; pUPD2LacI:PIF:PhyB:VP16+ren; α1
1 µl PhiC311 µl AsLOVpep1.5 µl LacI:PIF:PhyB:VP16
1µl pUPD21µl pUPD22.5 µl renilla (159)
5.6 µl H2O5.6 µl H2O0.5 µl α1
4.6 µl H2O
Gal4:PIF:phy:VP16+ren; α1LexA:PIF:phy:ren+opLex:luc; Ω1LacI:KNdronpa:ren+OpLex:Luc; Ω1
1.5 µl Gal4:PIF:phy:VP161 µl LexA:PIF:phy:ren1 µl LacI:KNdronpa:ren
2 µl renilla (159)2.5 µl opLex:luc (151)3 µl OpLac:Luc (152)
2.6 µl H2O2.6 µl H2O3 µl H2O
1 µl α10.5 µl Ω10.5 µl Ω1
Gal4:KNdronpa:ren+OpLex:Luc; Ω1PhyB:VP16+PIF6; Ω1
1 µl Gal4:KNdronpa:ren1 µl PhyB:VP16 (88E)
4 µl OpUAS:Luc (227)2 µl PIF6 (170)
3 µl H2O3.6 µl H2O
0.5 µl Ω11 µl Ω1
  • These Agrobacterium cultures have been refreshed:
    • E:PIF:PhyB:luc
    • Renilla
    • Pnos
    • Etr8

16 July 2015

  • Yesterday ligations have been transformed into E. coli:
    • phyC31;pUPD2
    • AsLOVpep; pUPD2
    • LacI:PIF:Phy:VP16+ren; α1
    • Gal4:PIF:phy:VP16+ren; α1
    • LexA:PIF:phy:ren+opLex:luc; Ω1
    • LacI:KNdronpa:ren+OpLex:Luc; Ω1
    • Gal4:KNdronpa:ren+OpLex:Luc; Ω1
    • PhyB:VP16+PIF6; Ω1

EXPERIMENT 4.

  • The agroinfiltrated leaf with BxbI:rep:GFP has been observed in the magnifying glass.
  • Second refresh of the Agrobacterium cultures:
    • E:PIF:PhyB:luc
    • Renilla
    • Pnos
    • Etr8
  • Miniprep of these cultures.
  • Digestion of the minipreps:
E:PIF:PhyB:luc; Ω1EcoRI??
Renilla; Ω2HindIII7453
Pnos; α1EcoRI2997, 353
Etr8; Ω1EcoRI2742

The digestions had positive controls that were included in the gel to compare the results obtained.

The digestions are left overnight in the working table.


17 July 2015

Do the gel:

PnosEtr8:luc Etr8:luc C+PIF6:PhyB:luc
nokookok
PIF6:PhyB:luc C+renillarenilla C+
oknook
  • Make liquid cultures of yesterday ligations, three colonies per cultures.
  • OD’s mesurement for the agroinfiltration.

PIF:PhyB:luc0.4741 µl/ml630 µl
Renilla0.2871 µl/ml1065 µl
Etr8:luc0.3252 µl/ml930 µl
Pnos0.3459 µl/ml885 µl

EXPERIMENT 5. Red toggle experiement:

  • PIF:PhyB:luc
  • Renilla
  • Etr8:luc (C-)
  • Pnos (C+)

EXPERIMENT 4.

It was observed another leaf with the recombinase BxbI with GFP in the magnifying glass with fluorescent lights.


18 July 2015

23 minipreps of the liquid cultrures. LexA:PIF:PhyB:ren:luc (C3) has not grown.

Digestions of the minipreps:

PhiC31;pUPD2NotI2046, 1899
AsLOVpep; pUPD2NotI2046, 521
LacI:PIF:PhyB:VP16+ren; α1EcoRI6345, 5623, 5487
Gal4:PIF:phy:VP16+ren; α1EcoRI6345, 5487, 4852
LexA:PIF:phy:ren+OpLex:luc; Ω1BamHI9431, 6674, 3513
LacI:KNdronpa:ren+OpLex:Luc; Ω1BamHI12632, 6574
Gal4:KNdronpa:ren+OpLex:Luc; Ω1BamHI11582, 6674
PhyB:VP16+PIF6; Ω1BamHI6674, 2685, 2337, 1439

Gel has been done:

phiC31 C1phiC31 C2phiC31 C3AsLOVpep C1
nononook
AsLOVpep C2AsLOVpep C3Gal4:PIF:phy:VP16:ren C1Gal4:PIF:phy:VP16:ren C2
nononono
Gal4:PIF:phy:VP16:ren C3LacI:PIF:phy:ren C1LacI:PIF:phy:ren C2LacI:PIF:phy:ren C3
nooknoNo
LexA:PIF:phy:ren:luc C1LexA:PIF:phy:ren:luc C2Gal4:KNdronpa:ren:luc C1Gal4:KNdronpa:ren:luc C2
nonookNo
Gal4:KNdronpa:ren:luc C3LacI:KNdronpa:ren:luc C1LacI:KNdronpa:ren:luc C2LacI:KNdronpa:ren:luc C3
nonookNo
PhyB:VP16:PIF6 C1PhyB:VP16:PIF6 C2PhyB:VP16:PIF6 C3
oknono

Pick more colonies of:

  • Gal4:PIF:phy:VP16+ren; α1
  • LexA:PIF:phy:ren+opLex:luc; Ω1

New digestions with new enzymes:

phiC31;pUPD2XhoI (buffer red)2119, 934, 894
LacI:PIF:Phy:VP16+ren; a1NEB45949, 5653, 3610, 2246
LacI:PIF:Phy:VP16+ren; a1HindIII11568, 5587

After 3 digestions of LacI:PIF:Phy:VP16+ren; α1 is accepted the construction.

It was observed another leaf with the recombinase BxbI with GFP in the magnifying glass with fluorescent lights. It was not observed a lot of spots, the efficiency is very low. This is the 4th day…

FOTO


19 July 2015

The incredible mistery of the lost notebook papers...


20 July 2015

Another day...


21 July 2015

EXPERIMENT 5. Red toggle.

It was picked disc samples: 3 in far red, 3 in darkness and 6 in red (3 of them were in far red and the other 3 in darkness).

Time lapses:

-19:00=t0

-1:00=t1

-7:00= t2

-19:00= t3 (it was taken the controls in natural light)

  • Minipreps of the colonies that were in 37ºC.
    • LexA:PIF:Phy:ren:luc (C1 and C2)
    • PhyC31 (C1, C2, C4 and C5)
LexA:PIF:Phy:ren:lucBamHI9431, 6674, 3513
PhiC31NotI2046, 1899

Gel with the digestions:

PhiC31 C1PhiC31 C2PhiC31 C4PhiC31 C5
Ok?okokok
LexA:PIF:PhyB:ren:luc C1LexA:PIF:PhyB:ren:luc C2
No DNANo DNA

We had problems with some colonies because in the digestion did not appear DNA. The minipreps will be made with a better kit.

Transform in Agrobacterium this cultures:

  • LexABD:KDronpa:NDronpa:ren:luc
  • Gal4BD:KDronpa:NDronpa:ren:luc
  • LacIBD:KDronpa:NDronpa:ren:luc
  • OpLexA:luc (151)
  • OpUAS:luc (227)
  • OpLacI:luc (152)

It was made liquid culture of Gal4:PIF:phyB:ren; Ω1 (C1-C5)

It was made ligations:

35S:Gal4:AsLOVpep:T35S; α135S:LacI:AsLOVpep:T35S; α135S:LexA:AsLOVpep:T35S; Ω1
1 µl 35S (0030)1 µl 35S (0030)1 µl 35S (0030)
1 µl Gal4BD1 µl LacI1 µl LexA
1 µl AsLOVpep 1 µl AsLOVpep 1 µl AsLOVpep
1 µl T35S (0036)1 µl T35S (0036)1 µl T35S (0036)
1 µl α1 1 µl α1 1 µl α1
2.6 µl H2O2.6 µl H2O2.6 µl H2O
PsinATG:RepPhiC31:GFP:T35S; α2LacI:PIF:PhyB:ren+luc; Ω1PIF:PhyB+renilla; α2
1 µl PsinATG (552)1.5 µl LacI:PIF:PhyB:ren; α11.5 µl PIF:PhyB
1 µl ReporterPhiC313 µl OpLacI:luc (152); α22.5 µl renilla (159)
1 µl GFP (0059)0.5 µl Ω10.5 µl α2
1 µl T35S (0036)2.6 H2O3 µl H2O
1 µl α2
2.6 µl H2O

EXPERIMENT 5.

Luciferase essay.


22 July 2015

Miniprep of Gal4:PIF:PhyB:ren (C1-C3) C4 and C5 did not grow.

Digestion of the miniprep:

Gal4:PIF:PhyB:renBamHI16684
EcoRI4852, 5487, 6345
EcoRV1849, 3942, 2475, 381, 8037

Gel was made:

Gal4:PIF:PhyB:ren (BamHI)Gal4:PIF:PhyB:ren (EcoRI)Gal4:PIF:PhyB:ren (EcoRV)
nonono

After doing several digestions with different enzyme all with wrong band patterns, it was decided to revise each part making digestions. The parts are:

  • PhiC31; pUPD2
  • Gal4:PIF:PhyB
  • Renilla (GB159)

Made liquid culture of the ReporterBxbI:GFP in Agrobacterium.

Transform the ligations into E. coli:

  • 35S:Gal4:AsLOVpep:T35S; α1
  • 35S:LacI:AsLOVpep:T35S; α1
  • 35S:LexA:AsLOVpep:T35S; α1
  • PsinATG:RepPhiC31:GFP:T35S; α2
  • LacI:PIF:PhyB:ren+luc; Ω1

EXPERIMENT 6.

Luciferase essay:

  • Sampling: samples that have been in red and natural light 48h, 3 samples.
  • 200 µl/sample x 3 sample= 600 µl of passive lissis buffer (5x)
  • Passive lissis buffer 1x= 120 µl+480 µl water.
  • 2.4 µl of Stop and glow
  • 118 µl of buffer.

23 July 2015

EXPERIMENT 7.

We decided to infiltrate soybean sprouts. First we decided to infiltrate with dye to observe the characteristics and the capacity of absorption.

FOTO

Make liquid cultures of yesterday transformations.


24 July 2015

Minipreps of the 13 liquid culture. LacI:PIF:phyB:ren cultures did not grow.

Digestion of the minipreps:

Gal4:AsLOVpepEcoRI6345, 1972
LacI:AsLOVpepEcoRI6345, 2743
LexA:AsLOVpepEcoRI6345, 2011
PsinATG:RepPhiC31:GFPHindIII6345, 2691
LexA:PIF:PhyB:ren+lucBamHI9431, 6674, 3513
PhyC31; pUPD2NotI2046, 1899
Gal4:PIF:phyBBamHI6674, 3474, 2337
Renilla (GB159)EcoRV2909, 2475, 882, 812, 381

It was done 2 gels with ligations:

Gal4:AsLOV C1Gal4:AsLOV C2Gal4:AsLOV C3PsinATG:RepPhiC31:GFP C1PsinATG:RepPhiC31:GFP C2
okokokokno
PsinATG:RepPhiC31:GFP C3LacI:AsLOV C1LacI:AsLOV C2LexA:AsLOV C1LexA:AsLOV C2
nookNonook
LexA:AsLOV C3LexA:PIF:PhyB:ren+luc C1LexA:PIF:PhyB:ren+luc C2
nonoOk
PhiC31 (C1)PhiC31 (C2)PhiC31 (C3)PhiC31 (C4)PhiC31 (C5)
nookokokno
Gal4:PIF:phyBRenilla (GB159)
okOk

26 July 2015

Liquid cultures of Agrobacterium were refreshed:

  • TsinATG:BxbIreporter:GFP
  • TsinATG:BxbI:reporterBxbI:GFP
  • Viral vectors (citoplasm, integrase)
  • GFP
  • Dsred

27 July 2015

Sent to sequence:

  • 210.08.256: LacIBD; pUPD2 (C1)
  • 210.08.258: Gal4BD; pUPD2 (C2)
  • 210.08.259:LexABD; pUPD2 (C1)
  • 210.08.260: PIF6; pUPD2 (C5)
  • 210.08.261: VP16; pUPD2 (C1)
  • 210.08.262: PhiC31; pUPD2 (C2)
  • 210.08.264: PhiC31; pUPD2 (C3)
  • 210.08.266: PhiC31; pUPD2 (C4)
  • 210.08.268: ReporterBxbI; pUPD2 (C1)
  • 210.08.269: ReporterPhiC31; pUPD2 (C1)
  • 210.08.270: AsLOVpep; pUPD2 (C1)

The sample had to have 10µl of miniprep (200ng/µl aprox) + 5 µl of primer (dilution 1:3)

Ligations:

Gal4:PIF:phiB + ren; α1LacI:PIF:phiB:ren + luc; Ω2PIF:PhyB+renilla; α2
1.5 µl Gal4:PIF:PhyB1.5 µl LacI:PIF:PhyB:ren1.5 µl PIF:PhyB
2 µl Renilla (GB159)2 µl OpLacI:luc2.5 µl Renilla (GB159)
0.5 µl α10.5 µl Ω20.5 µl α2
3.6 µl H2O2.6 µl H2O3 µl H2O

OD’s measurements to agroinfiltrate:

Cytoplasm: 0.39 (viral)0.25ml
Integrase: 0.35 (viral)0.28ml
GFP: 0.32 (viral)0.31ml
Dsred: 0.31 (viral)0.32ml
BxbI:reporter: 0.410.49ml
Reporter: 0.260.77ml

EXPERIMENT 8.

  • BxbI:RepBxbI:GFP
  • RepBxbI:GFP

EXPERIMENT 9.

Infiltrate at vaccum the soyabeans sprouts with viral system and GFP.

Ligations to join the negative controls with renilla:

Etr8:luc+staffer(SF)OpLexA:luc+SFOpLacI:luc+SFOpUAS:luc+SF
1.5 µl Etr8:luc (88C o 1098)1.5 µl OpLexA:luc (151)1.5 µl OpLacI:luc (152)1.5 µl UAS:luc (227)
1 µl SF; α21 µl SF; α21 µl SF; α21 µl SF;α2
1 µl Ω11 µl Ω11 µl Ω11 µl Ω1
6.1 µl H2O6.1 µl H2O6.1 µl H2O6.1 µl H2O

Transformation of E. coli of the ligations and make petri dish cultures:

  • Gal4:PIF:PhyB + ren; α1
  • LacI:PIF:PhyB:ren + luc; Ω2
  • PIF:PhyB+renilla; α2

Transformation into Agrobacterium with the constructions and make petri dish cultures:

  • Gal4:KDronpa:NDronpa:ren:luc
  • LacI:KDronpa:NDronpa:ren:luc
  • LexA:KDronpa:NDronpa:ren:luc
  • OpLexA:luc (GB151)
  • OpLacI:luc (GB152)
  • OpUAS:luc (GB227)

It was received a new piece (ePDZ) which is part of the blue toggle (plan A). It arrived in E. coli so it was made a lquid culture and let it grow at 37ºC overnight.


28 July 2015

Miniprep of the liquid culture: ePDZ.

Transformation into E. coli of the ligations:

  • Etr8:luc+staffer(SF)
  • OpLexA:luc+SF
  • OpLacI:luc+SF
  • UAS:luc+SF
  • Gal4:PIF:PhyB:ren

EXPERIMENT 9.

It was observed the soybean sprouts that were infiltrated with Agrobacteriumwith green light and red filter. It was not observed nothing significant, moreover, the damage is evident.

FOTO

Transformation in Agrobacterium the ReporterPhiC31:GFP.

Pick colonies and make liquid cultures adding X-Gal and IPTG because the colonies were little and we can not observe clearly if they were white or blue.

  • Gal4:PIF:phiB+ren. Did not grow any colony.
  • LacI:PIF:PhyB:ren+luc (C1-C3)
  • PIF:PhyB+renilla (C1- C3)

The medicine LTB (heat labile toxin B subunit) which is part of an enterotoxin of Echerichia coli homologous to the same toxin in Vibrio cholerae that causes diarrhea.

We add 50µl to have a final concentration of 10ng/µl.

Ligation:

LTB; pUPD2
1 µl LTB
1 µl pUPD2
5.6 µl H2O

29 July 2015

Miniprep of yesterday liquid culture:

  • LacI:PIF:phiB:ren+luc (C1 and C3) C2 turn into blue.
  • PIF:PhyB+renilla (C2) C1 and C3 did not grow.

Digestion:

LacI:PIF:phiB:ren:lucEcoRV882, 968, 1652, 3942, 2475, 381, 3477, 6674
PIF:PhyB+renillaHindIII4316, 5887, 788, 6345

Gel:

LacI:PIF:phiB:ren:luc (C1)LacI:PIF:phiB:ren:luc (C3)PIF:PhyB+renilla (C2)
nonono

Pick colonies and make liquid culture of:

  • Etr8:luc:staffer(SF) (C1-C3)
  • OpLexA:luc:SF (C1-C3)
  • OpLacI:luc:SF (C1-C3)
  • UAS:luc:SF (C1-C3)
  • Gal4:PIF:phyB:ren (C1-C3)

Transformation in E. coli of:

  • LTB; pUPD2

30 July 2015

Minipreps have been done:

  • Etr8:luc:staffer(SF) (C1-C3)
  • OpLexA:luc:SF (C1 and C3) C2 did not grow.
  • OpLacI:luc:SF (C1-C3)
  • OpUAS:luc:SF (C1-C3)
  • Gal4:PIF:phyB:ren (C1-C3)
  • LacI:PIF:phy:ren:luc (C1-C3)
  • PIF:PhyB:ren (C1-C3)
Etr8:luc:staffer(SF)BamHI6674, 2766
OpLexA:luc:SFBamHI6674, 2746
OpLacI:luc:SFBamHI6674, 2847
OpUAS:luc:SFBamHI6674, 2568
Gal4:PIF:phyB:renEcoRI6345, 5487, 4852
LacI:PIF:phy:ren:lucBamHI20451
PIF:PhyB:renHindIII6345, 5887, 4316, 788

Do the gel:

Primers have arrived:

  • ePDZ reverse and forward and phiC31.

Make a PCR with ePDZ and its primers to obtain the desired fragment and put it in pUPD2.

ePDZ PCR
10µl Buffer HF
31.5 µl H2O
2 µl dNTPs
2.5 µl Primer forward
2.5 µl primer reverse
1 µl ePDZ (dilution 1:50)
0.5 µl Taq phunion

Pick 2 colonies of PhiC31:GFP in Agrobacterium and make liquid culture.

Ligations:

ePDZ; pUPD2PIF:phyB+ren; α2OpUAS:luc:SF+ren; α1OpLexA:luc:SF+ren; α1
1 µl ePDZ1 µl PIF:phyB1 µl OpUAS:luc:SF1 µl OpLexA:luc:SF
1 µl pUPD21 µl ren (159)1 µl ren1 µl ren
5.6 µl H2O1 µl α21 µl α11 µl α1
4.6 µl H2O4.6 µl H2O4.6 µl H2O
OpEtr8:luc:SF+ren; α1Op:LacI:luc:SF+ren; α1
1 µl OpEtr8:luc:SF1 µl OpLacI:luc:SF
1 µl ren1 µl ren
1 µl α11 µl aplha1
4.6 µl H2O4.6 µl H2O

Make liquid culture (E. coli) of:

  • LTB; pUPD2 (C1-C3)

31 July 2015

Ligation:

LacI:PIF:PhyB:ren+OpLacI:luc; Ω2
1.5 µl LacI:PIF:phyB:ren
3 µl OpLacI:luc
0.5 µl Ω2
2.6 µl H2O

EXPERIMENT 8.

After 4 days till the agroinfiltration we have observed again the samples with BxbI and the negative control.

Foto!

Minipreps of liquid culture LTB (C1 and C2)

Digestion:

LTB; pUPD2NotI2046, 474

Gel:

LTB (C1)LTB (C2)PCR ePDZ
okokok

Take out glicerynates of Paloma, lab mate:

  • Sip rotavirus CH2
  • Sip rotavirus CH2-CH3

For E. coli and Agrobacterium.

Pick a colony of Asun interferon (IFN) in Agro, make liquid culture.

We had miniprep of IFN in pUPD. Make ligations.

Transform in E. coli the ligations and make petri dishes cultures:

  • ePDZ; pUPD2
  • PIF:phyB+ren; α2
  • OpUAS:luc:SF+ren; α1
  • OpLexA:luc:SF+ren; α1
  • OpEtr8:luc:SF+ren; α1
  • Op:LacI:luc:SF+ren; α1
  • LacI:PIF:phyB:ren+OpLacI:luc; Ω2

Refresh agro cultures to agroinfiltrate tomorrow:

  • PhiC31 (viral system)
  • ReporterPhiC31
  • BxbI+reporterBxbI
  • ReporterBxbI
  • Gal4:KDronpa:NDronpa:luc:ren (blue toggle)
  • EPIF:phi:luc
  • Renilla
  • P19
  • Pnos


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.