Difference between revisions of "Team:Valencia UPV/Elements"

Latest revision as of 20:50, 29 August 2015

Valencia UPV iGEM 2015

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; ?1	Etr8 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 ligase	1µL α1
1µL Bsmb1	5.8µL 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; ?1	EcoRI	6345, 238
EPIF6 + PhyB-PV16; ?1	BamHI	6686, 1439, 2685, 2237

Agarose gel was made:

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

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-VP16

PvuII (green buffer)

3663, 9472pb

Agarose gel 1%:

EPIF6-PhyB-VP16 (C1)	EPIF6-PhyB-VP16 (C2)	EPIF6-PhyB-VP16 (C3)
no	no	no

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.

Primers	Code	Template	Working temperature? ºC
LacI F	1	LacI (858)	69.7
LacI R	2
Gal4 F	3	We did not take out the glicerynate.	63.2
Gal4	4
LexA F	5	LexA (732)	62.7
LexA R	6
PIF:VP16 F	7	PIF6 (288)	60.1
PIFVP16 R	8
NDronpa F1	9	Kdronpa	67.7
NDronpa R1	10
Dronpa F2	11	58.5
NDronpa R2	12

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

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.

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

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

Transformation of the ligations:

50 µl of electrocompetent cell
1.5 µl of ligation
Put 1 min before the cubettes in ice
Make the transformation (1500V)
Put the transformed cells 1h at 37ºC

We made an stack of Cloranfenicol petri dishes

250ml LB aga
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 + 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.

We make a liquid culture of Agro of Renilla.

5ml of LB agar
5 µl rifampicine
5 µl of kanamicine
5 µl tetracycline
Incubate for 2 days (48h) at 28ºC.

21 June 2015

Pick colonies of and put into a liquid medium of 3 ml of LB agar, 3 µl of each antibiotic (kanamicine, spectomicine, cloranfenicol):

Plates (17/06/15): PIF (C1, C2) (with cloranfenicol), VP16 (C1, C3) (with cloranfenicol), LacI (C1, C2, C3) (with cloranfenicol)
Plates (19/06/15): Bxb1 (C1, C2, C3) (with kanamicine), VP16 (C4, C5) (with cloranfenicol), LacI (C1, C2) (with C1, C2) (with cloranfenicol), PIF (C1, C2, C3, C4, C5) (with cloranfenicol), LexA (C1, C2) (with cloranfenicol).

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, pUPD (C1-C5)
LexABD, pUPD (C1, C2)
Etr8(CMV):Bxb1 (C1-C3)
PIF6,pUPD (C1-C5)
VP16, pUPD (C1, C4, C5)

Make the digestions of all the minipreps:

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

Viral system:……….
We received the reported Bxb1!

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 NDrompa (9-10), the primers of NDrompa are aliquoted

…..

Make the gel with all the digestions writed before.

Lac1	Lac2	Lac3	Lac4	Lac5	Lex1	Lex2	Bxb1,1	Bxb2, 2	Bxb1,3
Ok	ok	ok	ok	ok	no	no	ok	ok	no
PIF1	PIF2	PIF3	PIF4	PIF5	VP16,1	VP16,4	VP16,5
No	no	-	ok	ok	ok	ok	ok
PCRS	…	…	…

We make ligations of:

Etr8(CMV):BxbI in a1 + PhyB:P16 in a2, ?1
LacIBD in pUPD2 + PIFBDless+promoter+terminator, a1
KDrompa in pUPD2 + LacBD+promoter+terminator, a1
Gal4BD, pUPD2
Reporter of BxbI, pUPD2

Tomorrow we have to take out pUPD of constitutive promoters, terminators and GFP (CDS).

23 June 2015

Transformations in E.Coli of the 5 ligations done yesterday and two more transformations of 5+6(1) and 5+6(2) which are the ligations in pUPD of the 18/06.
We put 50 µl of the transformations in the plates. Put in 37ºC.

Etr8(CMV):Bxb1 in a1 + PhyB:P16 in a2, ?1
LacIBD in pUPD2 + PIFBDless+promoter+terminator, a1
KDrompa in pUPD2 + LacBD+promoter+terminator, a1
Gal4BD, pUPD2
Reporter of Bxb2, pUPD
LexABD (5+6(1)), pUPD

We have taken out of the -80ºC fridge the glycerinate of GFP, pUPD/ampicilina/GB0059.
The liquid culture of Renilla (rifampicina/kanamicia/tetraciclina) doesn’t grow before the two days required. So we decide to put two new colonies in une tube with the three antibiotics and another with rifampicina and kanamicine. Asun say 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 nº10 (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 media: 3 µl of LB, 3 µl of antibiotics (K, Spect, Clor).

LacIBD+PIF, a1 (C1, C2)
Gal4BD, pUPD2 (C1)
RepBxb1, pUPD2 (C1, C2, C3)
LacIBD+KDonpa, a1 (C1, C2)
Etr8(CMV)+Bxb1+phyB+VP16, ?1 (C1)
LexABD1, pUPD (C1-C4)
LexABD2, pUPD. No colonies.

The viral systems cultures of Agro for the color mosaics are ready before 2 days at 28ºC. We can make the Agroinfiltration.

Buffers of Agroinfiltration:

First we have to prepare and ajust the pH of the buffer MES and the buffer MgCl.
MES (10x), 100nM; ph=5,6 (adding NaOH). Make 1L.
MgCl (100x), 1M. Make 100ml.
Solution to agroinfiltration: 10ml of MES(10x) + 1ml of MgCl (100x) + 100 µl of DMSO+acetosiningona and finally “enrasar” to 100ml.
19.6mg of acetosiningona for 500 µl of DMSO

Ligation:

ETR8(CMV):Bxb1(a1)+phyB+VP16(a2); ?1	Gal4BD(pcr) + pUPD2
1.5 µl etr8:Bxb1	1 µl Gal4 PCR
1.5 µl 88E (phyB+VP16)	1 µl pUPD2
1 µl ?1	1.2 buffer T4 ligase
1.2 µl buffer T4 ligase	1 µl ligase
1 µl ligase	1.2 µl BSA
1.2 µl BSA	1 µl BsmbI
1 µl BsmbI	5,6 µl H2O
3.6	µl H2O

Quantification of DNA:

pUPD GFF (0059): 249 ng/µl
?2: 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, ?2 (22/06): 158 ng/µl
LacIBD (C1), pUPD (22/06): 129 ng/µl
109k renillaDC: 49 ng/µl
IGEM 534: 13.6 ng/µl
VP16 (C1), pUPD2:102 ng/µl
IGEM 1097: 409 ng/µl
K-donpa (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+phy8 (c1) (08/06): 108 ng/µl
160 renilla, a2 (19/06): 46 ng/µl
159 renilla, ?2 (19/06): 149 ng/µl
Etr8:Bxb1 (C1)(22/06): 149 ng/µl
IGEM 732: 422 ng/µl

Measurement of the OD:

first we have to

25 June 2015

Minipreps of the liquid culture:

We don’t observed growth in LacIBD+PIF and LaciBD+K-donpa.

Digestion of the minipreps and do the gel:

Gal4BD, pUPD2	NotI	2046, 282
RepBxb1, pUPD2	NotI	2046, 460
Etr8(CMV):Bxb1 + phyB,a1	BamHI	6674, 2237, 2806, 1174
LexABD, pUPD2	NotI	2046, 321
9+10, pUPD2	NotI	464

Gel:

Etr8:Bxb1	Lex1	Lex2	Lex3	Lex4	Rep1	Rep2	Rep3	Gal4 C1	PCR 9+10
no	no	no	no	no	ok	ok	ok	no	ok

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 N-donpa (R1).
Refresh the cultures of Agro (28ºC). We pick 7.5 µl of the previous culture. And put into a new liquid media with Rifampicine and Kanamicine for 2 days and 28ºC.
Ligations:

N-dronpa	Rep GFP	Gal4	LexA
1 µl PCR 9+10	1 µl Rep Bxb1	1 µl PCR 3+4	1 µl PCR 5+6
1 µl PCR11+12	1 µl promoter without ATG	1 µl pUPD2	1 µl pUPD2
1 µl pUPD2	1 µl Tnos
1.2 µl buffer ligase	1.2 µl buffer ligase	1.2 µl buffer ligase	1.2 µl buffer ligase
1.2 µl BSA	1.2 µl BSA	1.2 µl BSA	1.2 µl BSA
1 µl BsmbI	1 µl BsaI	1 µl BsmbI	1 µl BsmbI
1 µl T4 ligase	1 µl T4 ligase	1 µl T4 ligase	1 µl T4 ligase
4,6 µl H2O	3,6 µl H2O	5,6 µl H2O	5,6 µl H2O
	1 µl a2
Etr8:Bxb1+phyB
1 µl Etr8:Bxb1
1 µl 88E
1µl ?1
1.2 µl buffer ligase
1.2 µl BSA
1 µl BsmbI
1 µl T4 ligase
3,6 µl H2O

We transform the ligations in E.Coli ant pass them into agar plates with cloranfenicol for all of them except the ligation of Etr8:Bxb1+phy that goes with bhnfnfg

26 June 2015

We do again the two ligations that didn’t gone?

Rep GFP	LacI BD+PIF6
1 µl Rep Bxb1	1 µl LACIBD, pUPD
1 µl promoter without ATG	1 µl PIF6, pUPD
1 µl Tnos	1 µl promoter
1.2 µl buffer ligase	1 µl T35
1.2 µl BSA	1 µl a1
1 µl BsaI	1.2 µl buffer BsaI
1 µl T4 ligase	1.2 µlBSA
2.6 µl H2O	1 µl BsaI
1 µl a2	1 µl ligase
1µl GFP (0059)	2.6 µl H2O

We make a gel of LAcI+PIF6 (C1 and C2). Both of them present the fragment of the vector at 6000 pb but none of them at 2000bp which is the insert one.

LacIBD+PIF; a1

EcoRI

6345, 1997, 641

LacIBD+PIF C1	LacIBD+PIF C2
no	no

Measurement of the ODs of phyB+PIF+luc and renilla+P19.

phyB+PIF+luc: o.35 (1:2)
Ren+P19: 0.34 (1:2)
Final volume= 2
CCo= 0.35*2
CCf= 0.2
Ecuation=> Vo*CCo=Vf*CCf
So we obtain that Vo(LacI+PIF6)=1.429 µl and Vo(ren)= 1.412 µl.

Ligation of:

LacIBD,pUPD + K-donpa, pUPD

1 µl 35S

1 µl LacIBD,pUPD2

1 µl K-dronpa, pUPD

1 µl T35S

1 µl a1

1.2 µl buffer T4 ligase

1 µl BsaI

1 µl T4 ligase

2.6 µl H2O

We make the agorinfiltration of (…..) to start checking if the plant react to the different ..

27 June 2015

Transformation in E.coli of LacIBD+K-Dronpa, a1

We Put into plates LexABD and Etr8(CMV):Bxb1:GFP again and also LAcIBD+K-Dronpa.

We make liquid culture of:

RepBxb1:GFP (C1-C4)
LacIBD+PIF (C1-C5)
N-Dronpa (C1-C4)
Gal4BD (C1-C5)
LexA

28 June 2015

We do the minipreps of the liquid cultures that have grown.

RepBxb1:GFP (C1 and C2)
LacIBD+PIF (C1-C4)
N-Dronpa (C1-C4)
Gal4BD (C1-C5)
LexA: didn’t grow

Do the digestions of the minipreps:

LacIBD+PIF, a1	EcoRI	6345, 1997, 641
RepBxb1:GFP, ?2	HindIII	6345, 2683
Gal4BD; pUPD2	NotI	2681, 644
N-Dronpa; pUPD2	NotI	2046, 744

Make the gel.

RepBxb1:GFP C1	RepBxb1:GFP C2	LacIBD+PIF C1	LacIBD+PIF C2	LacIBD+PIF C3	LacIBD+PIF C4
no	no	no	no	no	No
Gal4BD C1	Gal4BD C2	Gal4BD C3	Gal4BD C4	Gal4BD C5	N-Dronpa C1
ok	ok	ok	ok	ok	ok
N-Dronpa C2	N-Dronpa C3	N-Dronpa C4
no	ok	ok

Take glycerinated:

GB0030: p35S
GB0036: T35S

Make liquid culture of LexABD (C1-C4).
We transform again LacIBD+K-Dronpa 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 4 colonies of LexA and both glycerinates:

LexABD; pPPD2	NotI	2358, 312
35S; pUPD2	NotI	2981, 1074
T35S; pPUD2	NotI	2981, 304

Make the gel:

LexA C1	LexA C2	LexA C3	LexA C4	P35S	T35S
ok	ok	ok	ok	Ok?	Ok?

Make ligations:

LacIBD+K-Dronpa+promoter+termi; a1	Gal4BD+K-Donpa+prom+ter; a1	LexABD+K-Dronpa+prom+term; a1
1 µl LacI, pUPD2	1 µl Gal4, pUPD2	1 µl Gal4, pUPD2
1 µl K-Dronpa, pUPD2	1 µl K-Dronpa, pUPD2	1 µl K-Dronpa, 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)
1.2 µl buffer ligase	1.2 µl buffer ligase	1.2 µl buffer ligase
1.2 µl BSA 10x	1.2 µl BSA 10x	1.2 µl BSA 10x
1 µl BsaI	1 µl BsaI	1 µl BsaI
1 µl ligase T4	1 µl ligase T4	1 µl ligase T4
2.6 µl H2O	2.6 µl H2O	2.6 µl H2O
1 µl a1	1 µl a1	1 µl a1
N-Dronpa+VP16; a2	Gal4BD+PIF6; a1	LacIBD+PIF6; a1
1 µl N-Dronpa, pUPD2	1 µl Gal4BD, pUPD2	1 µl LacIBD, pUPD2
1 µl VP16, pUPD2	1 µl PIF6, pUPD2	1 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)
1.2 µl buffer ligase	1.2 µl buffer ligase	1.2 µl buffer ligase
1.2 µl BSA 10x	1.2 µl BSA 10x	1.2 µl BSA 10x
1 µl BsaI	1 µl BsaI	1 µl BsaI
1 µl ligase T4	1 µl ligase T4	1 µl ligase T4
2.6 µl H2O	2.6 µl H2O	2.6 µl H2O
1 µl a2	1 µl a1	1 µl a1
LexABD+PIF6; a1
1 µl LexABD, pUPD2
1 µl PIF, pUPD2
1 µl 35S (GB0030)
1 µl T35S (GB0036)
1.2 µl buffer ligase
1.2 µl BSA 10x
1 µl BsaI
1 µl ligase T4
2.6 µl H2O
1 µl a2

Transform all the ligations into E.Coli.Gal4BD+K-Dronpa and LacIBD+K-Dronpa goes wrong and we have to do it again.
Put into a plate the colonies before let stay them 1h at 37ºC.

Quantification of DNA:

RepBxb1:GFP (C1): 163.8 ng/µl
N-Dronpa, pUPD2 (C4):113.1 ng/µl
N-Dronpa (C3): 83.2 ng/µl
NDonpa (C1): 116.6 ng/µl
Gal4BD (C1): 95.2 ng/µl
Gal4BD (C2): 120.7 ng/µl
RepBxb1:GFP (C2): 170.6 ng/µl
RepBxb1 (C1): 80.6 ng/µl

30 June 2015

Transform Gal4+K-Dronpa and LacI+K-Dronpa

Miniprep of:

RepBxb1+GFP (C1-C3)

RepBxb1+GFP

HindIII

6345, 2683

Gel:

RepBxb1+GFP C1	RepBxb1+GFP C2	RepBxb1+GFP C3
No	no	no

We pick more colonies and make other liquid cultures.

Save the last samples of the leaves of the plants that were under the first experiment, next to the glycerinates in the freezer (-80ºC).

Put in plates the transformations of Gal4+K-Dronpa and LacI+K-Dronpa (50 µl of bacteria in SOC medium).

Make the gel:

Add to the digestions 2 µl of loading buffer (6x) because for 5 µl of digestion we put 1 µl of the buffer.

Make liquid culture of two colonies for each plates of the transformations done yesterday, 10 tubes in total.

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

1 July 2015

Do the minipreps of the 10 liquid cultures.

Do the digestions:

LexABD+K-Dronpa;a1	EcoRI	6345, 2296
N-Donpa+VP16; a2	HindIII	6345, 2427
Gal4BD+PIF6; a1	EcoRI	6345, 1867
LacI+PIF; a1	EcoRI	6345, 2638
LexABD+PIF6; a1	EcoRI	6345, 1906

Do the gel:

Gal4+PIF C1	Gal4+PIF C2	LexA+PIF C1	LexA+PIF C2	LexA+KDronpa C1
ok	ok	ok	ok	Ok
LexA+Kdronpa C2	LacI+PIF C1	LacI+PIF C2	Ndonpa+VP16 C1	Ndronpa+VP16 C2
ok	ok	ok	ok	ok

Prepare liquid culture of:

LexA+PIF; ?1 (C1)
LacI+PIF; ?1 (C1)
LexA+K-Dronpa (C1)
Gal4+PIF; ?1 (C1)
VP16, pUPD2 (C1)
LexABD, pUPD2 (C2)
PIF6, pUPD2 (C5)
LacIBD, pUPD2 (C1)

Pick colonies and make liquid culture of:

Gal4+K-Dronpa (C1 and C2)
LacI+K-Dronpa (C1 and C2)
RepBxb1+GFP (C4-C6)

We sent to sequence:

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
The solution have: 10µl of miniprep + 5µl (dilution 1:3) of primers.

Data of the luciferase essay with the sample plat RED at 24h (replica2): we obtain a value of 7.4E6.

2 July 2015

Minipreps of:

Gal4+K-Dronpa (C1 and C2)
LacI+K-Dronpa (C1 and C2)
RepBxb1+GFP (C4-C6)

Gal4+K-Dronpa	EcoRI	6345, 3028
LacI+K-Dronpa	EcoRI	6345, 2257
RepBxb1+GFP	HindIII	6300, 2400

Do the gel:

LacI+K-Dronpa C1	LacI+K-Dronpa C2	Gal4+K-Dronpa C1	Gal4+K-Dronpa C2
??
RepBxb1+GFP C4	RepBxb1+GFP C5	RepBxb1+GFP C6

Luciferase essay:

During the whole experiment we lost three samples: T16/FarRed/1; T24/Red/2 and T0/FarRed/1

Results:

Things to keep in mind for the next experiment:

The luminimeter (machine to measure the luminescence) has to be ready before start adding the reactants to the samples because it needs 10min to be ready.
Set the timer (10min) with the first sample of luciferase and add the reactant to the other samples as quick as possible.
We have to let the renilla stay before putting it in the luminimeter the same time as the luciferase, in this case 15min because with the luciferase we didn’t manage well the time. Theoretically we have to wait 10min.

Make ligations:

LexA:Kdonpa+N-Dronpa; ?1	LacI:Kdronpa+N-Dronpa; ?1	Gal4:Kdronpa+N-Dronpa; ?1
1 µl LexA:Kdronpa	1 µl LacI:Kdronpa	1 µl Gal4:Kdronpa
1 µl N-Dronpa	1 µl N-Dronpa	1 µl N-Dronpa
1 µl ?1	1 µl ?1	1 µl ?1
1.2 µl buffer ligase	1.2 µl buffer ligase	1.2 µl buffer ligase
1.2 µl BSA	1.2 µl BSA	1.2 µl BSA
1 µl BsmbI	1 µl BsmbI	1 µl BsmbI
1 µl BsaI	1 µl BsaI	1 µl BsaI
4.6 µl H2O	4.6 µl H2O	4.6 µl H2O
LexA:PIF+PhyB:VP16; ?1	LacI:PIF+PhyB:VP16; ?1	Gal4:PIF+PhyB:VP16; ?1
1 µl LexA:PIF	1 µl LacI:PIF	1 µl Gal4:PIF
1 µl PhyB:VP16 (88E)	1 µl PhyB:VP16	1 µl PhyB:VP16
1 µl ?1	1 µl ?1	1 µl ?1
1.2 µl buffer ligase	1.2 µl buffer ligase	1.2 µl buffer ligase
1.2 µl BSA	1.2 µl BSA	1.2 µl BSA
1 µl BsmbI	1 µl BsmbI	1 µl BsmbI
1 µl BsaI	1 µl BsaI	1 µl BsaI
4.6 µl H2O	4.6 µl H2O	4.6 µl H2O
35S:Bxb1+RepBxb1:GFP; ?1	E-PIF+phyB+luc+ren; ?1
1 µl 35s:Bxb1:T35S (alfredo’s)	0.5 µl 896 (PIF+phy+luc)
1 µl PromsinATG:RepBxb1:GFP	1 µl 160 (renilla)
1 µl ?1	1 µl ?1
1.2 µl buffer ligase	1.2 µl buffer ligase
1.2 µl BSA	1.2 µl BSA
1 µl BsmbI	1 µl BsmbI
1 µl BsaI	1 µl BsaI
4.6 µl H2O	4.6 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 N-Dronpa have the desired mutation.

Transformation in E.Coli of the 8 ligations.
Put the transformation into plates, put at 37ºC.
Refresh the Agro’s cultures (Renilla and PIF+phy+luc):
Add in 5ml of LB, 5 µl of rifampicine, 5 µl kanamicine, 5 µl of culture.

4 July 2015

Make the 2nd refresh of the culture of Agrobacterium. Put 5ml of LB, 5 µl of rifampicine, 5 µl kanamicine, 5 µl of culture.

Make liquid cultures (4ml of LB and 4 µl of spectomicine) of the 8 colonies of E.Coli.

Red toggle (C1)
Gal4:Kdronpa+N-Dronpa (C1 and C2)
LexA:Kdonpa+N-Dronpa (C1 and C2)
LacI:Kdronpa+N-Dronpa (C1 and C2)
LexA:PIF+PhyB:VP16 (C1 and C2)
35S:Bxb1+RepBxb1:GFP (C1 and C2)
This colonies didn’t grow: LacI:PIF+PhyB:VP16; Gal4:PIF+PhyB:VP16 and E-PIF+phyB+luc+ren. Tomorrow we will repeat the ligations.

5 July 2015

Ligations:

LacI:PIF+PhyB:VP16; ?1	Gal4:PIF+PhyB:VP16; ?1
1 µl LacI:PIF	1 µl Gal4:PIF
1 µl PhyB:VP16	1 µl PhyB:VP16
1 µl ?1	1 µl ?1
1.2 µl buffer ligase	1.2 µl buffer ligase
1.2 µl BSA	1.2 µl BSA
1 µl BsmbI	1 µl BsmbI
1 µl T4 ligase	1 µl T4 ligase
4.6	µl H2O	4.6 µl H2O

Minipreps of the liquid cultures.

Digestion of the minipreps.

LacI:Kdronpa+N-Dronpa	BamHI	6674, 5437
35S:Bxb1+RepBxb1:GFP	BamHI	6674, 3859, 1782
Gal4:Kdronpa+N-Dronpa	BamHI	6674, 4666
LexA:Kdonpa+N-Dronpa	BamHI	6674, 4705
LexA:PIF+PhyB:VP16	BamHI	6674, 3513, 2337

Agarose gel (1%):

LacKN C1	LacIKN C2	Bxb1RepGFP C1	Bxb1RepGFP C2	Gal4KN C1	Gal4KN C2
ok	ok	ok	ok	ok	ok
LexA:KN C1	LexA:KN C2	LexAPIFPhy C1	LexAPIFPhy C2	Red toggle
ok	ok	no	no	no

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

Take out the glycerinate 88C (1098): Etr8:luc:Tnos. We will use it like a negative control in the second luciferase essat.
Calculation of the ODs:

Dilution of both samples 1:10.
Renilla:=0.22---182 µl of sample + 1.818 ml MES
PIF+PhyB+Luc=0.26--- 154 µl of sample + 1.646 ml MES

Agroinfiltration of the two samples with renilla and luciferase+PIF+phy in three plants with 4 spots in each leaf and 2 leaf in each plant.

Let the plants 2 days in the darkness till agrobacterium infects the plant. They have to be in the dark because we are trying our red toggle ant it activates with light.

6 July 2015

Transform the negative control into agrobacterium.

Etr8:luc:Tnos
Bxb1:reporterBxb1:GFP

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

7 July 2015

Luciferase essay: Copy the protocol.

Add 150 µl of the lisis buffer and 800µl of MiliQ water, dilution 1:5.
We centrifuge both cultures of agrobacterium at 2900rpm for 10min and remove the supernatant.
We prepare the stock of MES (10ml of MES + 1ml MgCl + 100µl of “Acetosiningona” and level with H2O until 100ml.
Resuspend the pellet of bacteria with 5ml of MES and let grow 2h.

Renilla=0.29 (dilution 1:10): 2.9
PhyB+PIF+luc=0.69 (dilution 1:4):2.76
Solution to agroinflitrate:
Renilla: 0.138ml of sample + 1.862ml of MES
PhyB+PIF+luc: 0.145ml of sample + 1.855ml of MES
We make the infiltration of both samples mix together in 3 different plants, 2 leaf per plant and 4 spots per leaf. Explicacion del experiment (tiempo en oscuridad… luces…)

Digestions:

Red toggle	Enzyme?
LexA+PIF+phy	BamHI	3518, 5855, 6674

Gel:

Red toggle	LexA+PIF+phy C1	LexA+PIF+phy C2
No	Ok	no

It has arrived a new construction: AsLOVpep.

Suspended with 50µl of H2O.

Ligations:

AsLOVpep; pUPD2	Red Toggle	LacI:Kdronpa:Ndronpa:VP16+
35S:renilla:Tnos-35S:P19:Tnos(GB159)	Gal4:Kdronpa:Ndronpa:VP16+GB159
1 µl AsLOVpep	1 µl GB846	1 µl LacI:KNdronpa:VP16	1 µl Gal4:KNdronpa
1 µl pUPD2	1 µl GB160	1 µl GB159	1 µl GB159
1.2 µl buffer	1 µl ?1	1 µl a1	1 µl a1
1.2 µl BSA	1.2 µl buffer	1.2 µl buffer	1.2 µl buffer
1 µl BsmbI	1.2 µl BSA	1.2 µl BSA	1.2 µl BSA
1 µl T4 ligase	1 µl BsmbI	1 µl BsmbI	1 µl BsmbI
5.6 µl H2O	1 µl T4 ligase	1 µl T4 ligase	1 µl T4 ligase
	4.6 µl H2O	4.6 µl H2O	4.6 µl H2O
LexA:Kdronpa:Ndronpa:VP16+GB159	LexA:PIF:Phy:VP16+ GB159
1 µl LexA:Kdronpa:Ndronpa:VP16	1 µl LexA:PIF:Phy:VP16
1 µl GB159	1 µl GB159
1 µl a1	1 µl a1
1.2 µl buffer	1.2 µl buffer
1.2 µl BSA	1.2 µl BSA
1 µl BsmbI	1 µl BsmbI
1 µl T4 ligase	1 µl T4 ligase
4.6 µl H2O	4.6 µl H2O

8 July 2015

Experiment to study the piece PhyB+PIF.

The plants in red are exposed at the light intensity of the leds (we can’t regulate it) and the plants in far red are 100% with far red light and 0% of white light.

How the machines work: (hace falta?)

The machine with red light has the switch …

The 1st sample its at 8:00am and we spend 1h till the machines were working correctly because we didn’t know exactly how they work.

Then, before 12h (21:00), we take the 2nd samples; obtainin 3 discs of each condition (red an far red).

We transform:

AsLOVpep; pUPD2 in DHSa an let incubate 1h at 37ºC.
The last ligations in E.coli. Put in plates. Red toggle with Spectomicine+IPTG+XGal ann the rest (a1) with kanamicine+IPTG+XGal.

9 July 2015

Pick colonies:

AsLOVpep colonies didn’t grow. Repeat.
Red toggle colonies are all blue. Repeat.
The other 4 colonies had grown. we pick them and male liquid culture.
LacI:Kdronpa:Ndronpa:VP16+renilla (C1-C3)
Gal4:Kdronpa:Ndronpa:VP16+renilla (C1-C3)
LexA:Kdronpa:Ndronpa:VP16+GB159 (C1 and C2)
LexA:PIF:Phy:VP16+ GB159 (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 ?1

1.2 µl buffer

1.2 µl BSA

1 µl BsmbI

1 µl T4 ligase

5.1 µl H2O

We do the luciferase essay:

We didn’t obtain goo results, we can’t observed a significative difference between the on(red samples) and off (far red samples). It seems that the red toggle it has been activated. Graphics and tables

Transform the ligations of AsLOVpepe and red toggle.

Add SOC medium and let incubate 1h at 37ºC. Then we make an spin to the red toggle cells to concentrate them and see if we can obtain a colonies in the plates.

10 July 2015

Minipreps of:

LexABD:PIF:phyB:VP16+Renilla; a1 (C1,C2)
LexA:Kdronpa:Ndronpa+renilla; a1C1, C2)
Gal4:Kdronpa:Ndronpa+renilla; a1 (C1-C3)
LacI:Kdronpa:Ndronpa+renilla; a1 (C1,C2)

Digestions of:

LexABD:PIF:phyB:VP16+Renilla; a1 (C1,C2)	EcoRI	6345, 5487, 4891
LexA:Kdronpa:Ndronpa+renilla; a1C1, C2)	EcoRI	9333, 6345
Gal4:Kdronpa:Ndronpa+renilla; a1 (C1-C3)	EcoRI	6345, 9194
LacI:Kdronpa:Ndronpa+renilla; a1 (C1,C2)	EcoRI	6345, 9965
LacIBD+PIF+phyB; Ω1 (C1)	BamHI	6674, 4245, 2337
Gal4BD+PIF+phyB; Ω 1 (C1)	BamHI	6674, 3474, 2337

Make the gel:

LacIBD+PIF+phyB	Gal4BD+PIF+phyB	LexA:PIF:phyB:VP16+Renilla C1	LexA:PIF:phyB:VP16+Renilla C2
Ok	Ok	ok	ok
LexA:KNdronpa+renilla C1	LexA:KNdronpa+renilla C2	Gal4:KNdronpa+renilla C1	Gal4:KNdronpa+renilla C2
Ok	Ok	ok	Ok
Gal4:KNdronpa+renilla C3	LacI:Kdronpa:Ndronpa+renilla C1	LacI:Kdronpa:Ndronpa+renilla C2
Ok	Ok	ok

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). In both liquid cultures we ad YPTG and Xgal to make sure that the colonies are correct, if not the medium will change to blue color.

Ligations:

phyC31;pUPD2	Reporter phiC31; pUPD2	LacI:PIF:Phy:VP16+ren; a1
1 µl phiC31	1 µl rep phiC31	1 µl LacI:PIF:Phy:VP16
1 pUPD2	1 pUPD2	1 µl renilla (159)
1.2 µl buffer	1.2 µl buffer	1.2 µl buffer
1.2 µl BSA	1.2 µl BSA	1.2 µl BSA
1 µl T4 ligase	1 µl T4 ligase	1 µl T4 ligase
1 µl BsmbI	1 µl BsmbI	1 µl BSAI
5.6 µl H2O	5.6 µl H2O	4.6 µl H2O
	1 µl a1
Gal4:PIF:phy:VP16+ren; a1	LexA:PIF:phy:ren+opLex:luc; ?1	LexA:KNdronpa:ren+OpLex:Luc; ?1
1 µl Gal4:PIF:phy:VP16	1 µl LexA:PIF:phy:ren	1 µl LexA:KNdronpa:ren
1 µl renilla (159)	1 µl opLex:luc (151)	1 µl OpLex:Luc (151)
1.2 µl buffer	1.2 µl buffer	1.2 µl buffer
1.2 µl BSA	1.2 µl BSA	1.2 µl BSA
1 µl T4 ligase	1 µl T4 ligase	1 µl T4 ligase
1 µl BSAI	1 µl BsmbI	1 µl BsmbI
4.6 µl H2O	4.6 µl H2O	4.6 µl H2O
1 µl a1	1 µl ?1	1 µl ?1
Gal4:KNdronpa:ren+UAS:luc; ?1	LacI:KNdronpa:ren+OpLacI:luc; ?1
1 µl Gal4:KNdronpa:ren	1 µl LacI:KNdronpa:ren
1 µl UAS:luc (227)	1 µl OpLacI:luc (152)
1.2 µl buffer	1.2 µl buffer
1.2 µl BSA	1.2 µl BSA
1 µl T4 ligase	1 µl T4 ligase
1 µl BsmbI	1 µl BsmbI
4.6 µl H2O	4.6 µl H2O
1 µl ?1	1 µl ?1

11 July 2015

Prepare glycerinates of:

Bxb1+Rep:GFP; ?1
N-Dronpa; pUPD2
Bxb1:Etr8; pUPD2
Etr8(CMV):Bxb1:T35S; a1

Pick up the liquid cultures of AsLOVpep and red toggle. We observed that one tube of a red toggle colony is blue, we discard it.

Do miniprep of:

AsLOVpep (C1 and C2)
Red toggle (C2)

Digestions:

Red toggle	BamHI	6674, 6100 / 4209, 3756
AsLOVpep	NotI	2558, 512

Me falta el resultado del gel!!

AsLOVpep C1	AsLOVpep C2	Red toggle C2
¿?

Do transformation of DHSa and yesterday ligations:

phyC31;pUPD2
Reporter phiC31; pUPD2
LacI:PIF:Phy:VP16+ren; a1
Gal4:PIF:phy:VP16+ren; a1
LexA:PIF:phy:ren+opLex:luc; ?1
LexA:KNdronpa:ren+OpLex:Luc; ?1
Gal4:KNdronpa:ren+UAS:luc; ?1
LacI:KNdronpa:ren+OpLacI:luc; ?1
The pUPD2 in plates with cloranfenicol+IPTG+XGal. The a1 in plates with kanamicyn+IPTG+XGal. The ?1 in plates with spectinmicyn+IPTG+XGal.

12 July 2015

Pick colonies and make liquid culture:

phyC31;pUPD2 (C1-C3)
Reporter phiC31; pUPD2 (C1-C3)
LacI:PIF:Phy:VP16+ren; a1 (C1-C3)
Gal4:PIF:phy:VP16+ren; a1 All blue colonies.
LexA:PIF:phy:ren+opLex:luc; ?1 (C1)
LexA:KNdronpa:ren+OpLex:Luc; ?1(C1-C3)
Gal4:KNdronpa:ren+UAS:luc; ?1 (C1)
LacI:KNdronpa:ren+OpLacI:luc; ?1 All blue colonies.
We have to repeat the transformations or do again ligations.

Ligations were repeated:

Gal4:PIF:phy:VP16+ren; a1	LacI:KNdronpa:ren+OpLacI:luc; ?1
1 µl Gal4:PIF:phy:VP16	1 µl LacI:KNdronpa:ren
1 µl renilla (159)	1 µl OpLacI:luc (152)
1.2 µl buffer	1.2 µl buffer
1.2 µl BSA	1.2 µl BSA
1 µl T4 ligase	1 µl T4 ligase
1 µl BSAI	1 µl BsmbI
4.6 µl H2O	4.6 µl H2O
1 µl a1	1 µl ?1

Refresh the liquid cultures of Agrobacterium:

Bxb1:GFP and Etr8:tnos. They were 2 days at 28ºC.
Pnos, it was at the fridge (-4ºC).

13 July 2015

All the liquid cultures have grown, do minipreps.

Do digestions:

phyC31;pUPD2 (C1-C3)	NotI	2046, 1899
Reporter phiC31; pUPD2 (C1-C3)	NotI	2046, 475
LacI:PIF:Phy:VP16+ren; a1 (C1-C3)
	EcoRI	6345, 5623, 5487
LexA:PIF:phy:ren+opLex:luc; ?1 (C1)
	BamHI	9431, 6674, 3531
LexA:KNdronpa:ren+OpLex:Luc; ?1(C1-C3)
	BamHI	1199, 6674
Gal4:KNdronpa:ren+UAS:luc; ?1 (C1)
	BamHI	11582, 6674

Agarose gel:

phyC31 C1	phyC31 C2	phyC31 C3	RepPhiC31 C1
no	no	no	ok
RepPhiC31 C2	LacI:PIF:Phy:VP16+ren C1	LacI:PIF:Phy:VP16+ren C2	LacI:PIF:Phy:VP16+ren C3
no	no	ok	ok
LexA:PIF:phy:ren+opLex:luc	LexA:KNdronpa:ren+OpLex:Luc C1	LexA:KNdronpa:ren+OpLex:Luc C2	LexA:KNdronpa:ren+OpLex:Luc C3
no	ok	ok	ok
Gal4:KNdronpa:ren+UAS:luc C1
no

The Agrobacterium cultures refreshed yesterday were store in the fridge.

It is made another culture of 35S:Bxb1+reporterBxb1:GFP to keep it in the fridge. It had 5ml of LB medium, 5 µl rifampicin and 5 µl of spectinomicyn an 1 µl of the culture.

Mesurement of the OD’s:

35S:Bxb1+reporterBxb1:GFP: 0.28 (dilution 1:10)
143 µl of culture+1857 µl of MES/acetosiningon solution.
With this preparation 2 plants were infiltrated and let in natural light to see the normal activity of the recombinase.

Transformation of the ligation: Gal4:PIF:phy:VP16+ren; a1 and LacI:KNdronpa:ren+OpLacI:luc; ?1.

The liquid cultures of this constructions were repeated:

phyC31;pUPD2 (C4 and C5)
Reporter phiC31; pUPD2 (C4)
LacI:PIF:Phy:VP16+ren; a1 (C4)
LexA:PIF:phy:ren+opLex:luc; ?1 (C1)
LexA:KNdronpa:ren+OpLex:Luc; ?1(C1-C3)
Gal4:KNdronpa:ren+UAS:luc; ?1 (C1)
AsLOVpep; pUPD2 (C3)

14 July 2015

Do minipreps of:

phyC31;pUPD2 (C4 and C5)
Reporter phiC31; pUPD2 (C4)
LacI:PIF:Phy:VP16+ren; a1 (C4)
LexA:PIF:phy:ren+opLex:luc; ?1 (C1)
LexA:KNdronpa:ren+OpLex:Luc; ?1(C1-C3)
Gal4:KNdronpa:ren+UAS:luc; ?1 (C1)
AsLOVpep; pUPD2 (C3)

Do digestions:

phyC31;pUPD2	NotI	2046, 1899
Reporter phiC31; pUPD2	NotI	2046, 475
LacI:PIF:Phy:VP16+ren; a1	EcoRI	6345, 5623, 5487
LexA:PIF:phy:ren+opLex:luc, ?1	BamHI	9431, 6674, 3531
LexA:KNdronpa:ren+OpLex:Luc; ?1	BamHI	1199, 6674
Gal4:KNdronpa:ren+UAS:luc; ?1	BamHI	11582, 6674
AsLOVpep; pUPD2	NotI	2558, 512

Make an agarose gel:

phyC31 (C4)	phyC31 (C5)	AsLOVpep (C4)	LexA:PIF:phy:ren+opLex:luc (C1)
no	no	No	No
LexA:KNdronpa:ren+OpLex:Luc (C3)	Gal4:KNdronpa:ren+UAS:luc (C1)	Gal4:KNdronpa:ren+UAS:luc(C2)	Gal4:KNdronpa:ren+UAS:luc (C3)
Ok	no	no	No
LacI:PIF:Phy:VP16+ren	Reporter phiC31 (C1)
no	Ok

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
PhyC31: 4.6 ng/µl
AsLOVpep: 30.3 ng/µl
Igem151 (op:LexAluc): 40 ng/µl
Gal4:PIF:phyB:ren (C1): 124.4 ng/µl
LacI:PIF:phyB:VP16 (C1): 126 ng/µl
Igem 159 (renilla): 42 ng/µl
Gal4:Kdronpa:Ndronpa:renilla (C3): 172.8 ng/µl
Igem 227 (op:UAS:luc): 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:luc

EcoRI

6345, 9965

Make the gel:

LacI:K:NDronpa:ren:luc C4	LacI:K:NDronpa:ren:luc C5
no	no

Measurement of DNA concentrations:

Gal4BD:PIF:PhyB:VP16: 125.6 ng/µl
LexA:PIF:phyB:VP16:ren: 322.6 ng/µl
LacI:Kdronpa:NDronpa:ran: 209.0 ng/µl

We decided to repeat the ligations due to that we make twice the digestions and we didn’t obtain good results.

phyC31;pUPD2	AsLOVpep; pUPD2	LacI:PIF:Phy:VP16+ren; a1
1 µl phiC31	1 µl AsLOVpep	1.5 µl LacI:PIF:Phy:VP16
1 pUPD2	1 pUPD2	2.5 µl renilla (159)
1.2 µl buffer	1.2 µl buffer	1.2 µl buffer
1.2 µl BSA	1.2 µl BSA	1.2 µl BSA
1 µl T4 ligase	1 µl T4 ligase	1 µl T4 ligase
1 µl BsmbI	1 µl BsmbI	1 µl BSAI
5.6 µl H2O	5.6 µl H2O	4.6 µl H2O
	0.5 µl a1
Gal4:PIF:phy:VP16+ren; a1	LexA:PIF:phy:ren+opLex:luc; ?1	LacI:KNdronpa:ren+OpLex:Luc; ?1
1.5 µl Gal4:PIF:phy:VP16	1 µl LexA:PIF:phy:ren	1 µl LacI:KNdronpa:ren
2 µl renilla (159)	2.5 µl opLex:luc (151)	3 µl OpLac:Luc (152)
1.2 µl buffer	1.2 µl buffer	1.2 µl buffer
1.2 µl BSA	1.2 µl BSA	1.2 µl BSA
1 µl T4 ligase	1 µl T4 ligase	1 µl T4 ligase
1 µl BSAI	1 µl BsmbI	1 µl BsmbI
2.6 µl H2O	2.6 µl H2O	3 µl H2O
1 µl a1	0.5 µl ?1	0.5 µl ?1
Gal4:KNdronpa:ren+OpLex:Luc; ?1	PhyB:VP16+PIF6; ?1
1 µl Gal4:KNdronpa:ren	1 µl PhyB:VP16 (88E)
4 µl OpUAS:Luc (227)	2 µl PIF6 (170)
1.2 µl buffer	1.2 µl buffer
1.2 µl BSA	1.2 µl BSA
1 µl T4 ligase	1 µl T4 ligase
1 µl BsmbI	1 µl BsmbI
3 µl H2O	3.6 µl H2O
0.5 µl ?1	1 µl ?1

These Agrobacterium cultures have been refreshed:

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; a1
Gal4:PIF:phy:VP16+ren; a1
LexA:PIF:phy:ren+opLex:luc; ?1
LacI:KNdronpa:ren+OpLex:Luc; ?1
Gal4:KNdronpa:ren+OpLex:Luc; ?1
PhyB:VP16+PIF6; ?1

The agroinfiltrated leaf with BxbI:rep:GFP has been observed in the magnifying glass with fluorescent lights. The efficiency of the recombinase is lower and it can not been observed a lot of green spots. Tomorrow another leaf will be seen to check again the construction.

Second refresh of the Agrobacterium cultures:

PIF-phyB-luc
Renilla
Pnos
Etr8

Miniprep of these cultures.
Digestion of the minipreps:

PIF-phyB-luc; ?1	EcoRI	??
Renilla; ?2	HindIII	No lo encuentro
Pnos; ?1	EcoRI	2997, 353
Etr8; ?1	EcoRI

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:

No se lo que pone?

Liquid culture of the colonies in the agar plates have been made, 3 colonies for each construction.
OD’s mesurement for the agroinfiltration.

Dilution 1:10 in MES.

PIF:phyB:luc	0.47	41 µl/ml	630 µl
Renilla	0.28	71 µl/ml	1065 µl
Etr8:luc	0.32	52 µl/ml	930 µl
Pnos	0.34	59 µl/ml	885 µl

Red toggle experiement:

The plants were coinfiltrated with the red toggle (PIF+phyB) and renilla. Also with two controls, Pnos (positive control) and Etr8 (negative control).

14 plants were used with 3 infiltrated leafs for each plants and two spots per leaf.

The controls have been infiltrated in leafs of the plants like is shown in the picture. Pnos in the right part and Etr8 in the left part.

Two plants were infiltrated with both controls in the three leafs and they were in natural light during all the experiment.

Another 4 plants were infiltrated with controls following the same pattern in the procedure.

The remaining 8 plants were infiltrated with the red toggle.

Immediately after the infiltration the plants were distributed in three different conditions: natural light, darkness and far red.

So after the agroinfiltration 2 control plants stay in natural light all the experiment; 2 control plants and 4 red toggle went into the far red chamber and the same amount of control and red toggle plan (2+4) went put in darkness.

This conditions were maintained 3 days and then all the infiltrated leafs were cut into small discs and put into a special plates with water.

In this moment was set the time 0 (21/07/2015 at 19:30) and take the first samples.

Then in time 0 some of the dark and far red samples were put in red conditions to activate the red toggle.

This scheme represent the distribution of samples and the times that were taken the samples.

Hacer el esquema!!! Cuando este mas espabilada ;)

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.

18 July 2015

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

Digestions of the minipreps:

phyC31;pUPD2	NotI	2046, 1899
AsLOVpep; pUPD2	NotI
2046, 521
LacI:PIF:Phy:VP16+ren; a1	EcoRI	6345, 5623, 5487
Gal4:PIF:phy:VP16+ren; a1	EcoRI	6345, 5487, 4852
LexA:PIF:phy:ren+opLex:luc; ?1	BamHI	9431, 6674, 3513
LacI:KNdronpa:ren+OpLex:Luc; ?1	BamHI	12632, 6574
Gal4:KNdronpa:ren+OpLex:Luc; ?1	BamHI	11582, 6674
PhyB:VP16+PIF6; ?1	BamHI	6674, 2685, 2337, 1439

Gel has been done:

phyC31 C1	phyC31 C2	phyC31 C3	AsLOVpep C1
no	no	no	ok
AsLOVpep C2	AsLOVpep C3	Gal4:PIF:phy:VP16:ren C1	Gal4:PIF:phy:VP16:ren C2
no	no	no	no
Gal4:PIF:phy:VP16:ren C3	LacI:PIF:phy:ren C1	LacI:PIF:phy:ren C2	LacI:PIF:phy:ren C3
no	ok	no	No
LexA:PIF:phy:ren:luc C1	LexA:PIF:phy:ren:luc C2	Gal4:KNdronpa:ren:luc C1	Gal4:KNdronpa:ren:luc C2
no	no	ok	No
Gal4:KNdronpa:ren:luc C3	LacI:KNdronpa:ren:luc C1	LacI:KNdronpa:ren:luc C2	LacI:KNdronpa:ren:luc C3
no	no	ok	No
PhyB:VP16:PIF6 C1	PhyB:VP16:PIF6 C2	PhyB:VP16:PIF6 C3
ok	no	no

Pick more colonies of:

Gal4:PIF:phy:VP16+ren; a1
LexA:PIF:phy:ren+opLex:luc; ?1

New digestions with new enzymes:

phyC31;pUPD2	XhoI (buffer red)	2119, 934, 894
LacI:PIF:Phy:VP16+ren; a1	NEB4	5949, 5653, 3610, 2246
LacI:PIF:Phy:VP16+ren; a1	HindIII	11568, 5587

After 3 digestions of LacI:PIF:Phy:VP16+ren; a1 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…

19 July 2015

20 July 2015

21 July 2015

Red toggle experiment:

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:luc	BamHI	9431, 6674, 3513
PhyC31	NotI	2046, 1899

Gel with the digestions:

PhyC31 C1	PhyC31 C2	PhyC31 C4	PhyC31 C5
Ok?	ok	ok	ok
LexA:PIF:Phy:ren:luc C1	LexA:PIF:Phy:ren:luc C2
No DNA	No 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
OpLacI:luc (152)

It was made liquid culture of Gal4:PIF:phyB:ren; ?1 (C1-C5)

It was made ligations:

35S:Gal4:AsLOVpep:T35S; ?1	35S:LacI:AsLOVpep:T35S; ?1	35S:LexA:AsLOVpep:T35S; ?1
1 µl 35S (0030)	1 µl 35S (0030)	1 µl 35S (0030)
1 µl Gal4BD	1 µl LacI	1 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 ?1	1 µl ?1	1 µl ?1
2.6 µl H2O	2.6 µl H2O	2.6 µl H2O
PsinATG:RepPhiC31:GFP:T35S; ?2	LacI:PIF:PhyB:ren+luc; ?1	PIF:PhyB+renilla; ?2
1 µl PsinATG (552)	1.5 µl LacI:PIF:PhyB:ren; ?1	1.5 µl PIF:PhyB
1 µl ReporterPhyC31	3 µl OpLacI:luc (152); ?2	2.5 µl renilla (159)
1 µl GFP (0059)	0.5 µl ?1	0.5 µl ?2
1 µl T35S (0036)	2.6 H2O	3 µl H2O
1 µl ?2
2.6 µl H2O

Luciferase essay with all the samples collected in the last three days.

Sampling: 25 samples in total.
Passive lysis 1x (200µl/sample x 25 samples)= 5.000 µl
Crush samples.
Add 150 µl of passive lysis buffer and mix in the vortex.
Centrifuge at 13200rpm for 15’.

E8/li	E8/li	E8/li	P/li	P/li	P/li	TR/Fr	TR/Fr	TR/Fr	TR/D	TR/D	TR/D
TR/Fr
Red	TR/Fr
Red	TR/r
Red	TR/D
Red	TR/D
Red	TR/D
Red

We used 14.4 µl of Stop and Glow. 705.6 destilled H2O

22 July 2015

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

Digestion of the miniprep:

Gal4:PIF:PhyB:ren	BamHI	16684
	EcoRI	4852, 5487, 6345
	EcoRV	1849, 3942, 2475, 381, 8037

Gel was made:

Gal4… (BamHI)	Gal4… (EcoRI)	Gal4… (EcoRV)
no	no	no

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

PhyC31; 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; ?1
35S:LacI:AsLOVpep:T35S; ?1
35S:LexA:AsLOVpep:T35S; ?1
PsinATG:RepPhiC31:GFP:T35S; ?2
LexA:PIF:PhyB:ren+luc; ?1
Add 300 µl of SOC medium and put into a agar plate with the corresponding antibiotics.

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

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

Mesurement of the ODs to agroinfiltrate:

The samples are:

Citoplasm=0.27
DsRed=0.27
GFP=0.36
Recombinase=0.43

Vi= (Vf*Absf)/(Absi*10)

Absi=0.1 (because is a viral system)

Vf=120 µl

Make 16 liquid culture of the white colonies in the agar plates.

24 July 2015

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

Digestion of the minipreps:

Gal4:AsLOVpep	EcoRI	6345, 1972
LacI:AsLOVpep	EcoRI	6345, 2743
LexA:AsLOVpep	EcoRI	6345, 2011
PsinATG:RepPhiC31:GFP	HindIII	6345, 2691
LexA:PIF:PhyB:ren+luc	BamHI	9431, 6674, 3513

PhyC31; pUPD2	NotI	2046, 1899
Gal4:PIF:phyB	BamHI	6674, 3474, 2337
Renilla (GB159)	EcoRV	2909, 2475, 882, 812, 381

It was done 2 gels with ligations:

Gal4:AsLOV C1	Gal4:AsLOV C2	Gal4:AsLOV C3	PsinATG:RepPhi
C31:GFP C1	PsinATG:RepPhi
C31:GFP C2	PsinATG:RepPhi
C31:GFP C3
ok	ok	ok	ok	no	No
Mw/ladder	LacI:AsLOV C1	LacI:AsLOV C2	LexA:AsLOV C1	LexA:AsLOV C2	LexA:AsLOV C3
-	ok	No	no	ok	no
LexA:PIF:PhyB:ren+luc C1	LexA:PIF:PhyB:ren+luc C2
no	Ok

PhyC31 (C1)	PhyC31 (C2)	PhyC31 (C3)	PhyC31 (C4)	PhyC31 (C5)
no	ok	ok	ok	no
Gal4:PIF:phyB	Renilla (GB159)
ok	Ok

26 July 2015

Liquid cultures of Agrobacterium were refreshed:

TsinATG:BxbIreporter:GFP
TsinATG:BxbI:reporterBxbI:GFP
Viral vector??no se cual es

27 July 2015

Sent to sequence:

210.08.256: LacIBD; pUPD2 (C1)
210.08258: 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; ?1	LacI:PIF:phiB:ren + luc; ?2	PIF:PhyB+renilla; ?2
1.5 µl Gal4:PIF:phiB	1.5 µl LacI:PIF:phiB:ren	1.5 µl PIF:phiB
2 µl Renilla (GB159)	2 µl OpLacI:luc	2.5 µl Renilla (GB159)
0.5 µl ?1	0.5 µl ?2	0.5 µl ?2
3.6 µl H2O	2.6 µl H2O	3 µl H2O

OD’s measurements to prepare the agroinfiltrates:

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: 0.41	0.49ml
Reporter: 0.26	0.77ml

3 plants were infiltrated with BxbI and the reporter of BxbI (one leaf with the control an the other with the recombinase)?? No entiendo lo de la libreta

Ligations to join the negative controls with renilla:

Etr8:luc+staffer (SF)	OpLexA:luc+SF	OpLacI:luc+SF	UAS: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; ?2	1 µl SF; ?2	1 µl SF; ?2	1 µl SF; ?2
1 µl ?1	1 µl ?1	1 µl ?1	1 µl ?1
6.1 µl H2O	6.1 µl H2O	6.1 µl H2O	6.1 µl H2O

Transformation of E. coli of the ligations:

Gal4:PIF:phiB + ren; ?1
LacI:PIF:phiB:ren + luc; ?2
PIF:PhyB+renilla; ?2

Make cultures in agar petri dishes of the transformations with the corresponding antibiotics.

Transformation into Agrobacterium with the constructions:

Gal4:KDronpa:NDronpa:ren:luc
LacI:KDronpa:NDronpa:ren:luc
LexA:KDronpa:NDronpa:ren:luc
OpLexA:luc (GB 151)
OpLacI:luc (GB 152)
UAS:luc (GB 227)

Make cultures in agar petri dishes of the transformations with the corresponding antibiotics.

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

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

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:phiB: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:luc	EcoRV	882, 968, 1652, 3942, 2475, 381, 3477, 6674
PIF:PhyB+renilla	HindIII	4316, 5887, 788, 6345

Gel:

LacI:PIF:phiB:ren:luc (C1)	LacI:PIF:phiB:ren:luc (C3)	PIF:PhyB+renilla (C2)
no	no	no

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)
UAS: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)	BamHI	6674, 2766
OpLexA:luc:SF	BamHI	6674, 2746
OpLacI:luc:SF	BamHI	6674, 2847
UAS:luc:SF	BamHI	6674, 2568
Gal4:PIF:phyB:ren	EcoRI	6345, 5487, 4852
LacI:PIF:phy:ren:luc	BamHI	20451
PIF:phyB:ren	HindIII	6345, 5887, 4316, 788

Do the gel:

No se el orden!! Preguntar

Primers have arrived:

ePDZ reverse and forward and phyC31.

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; pUPD2	PIF:phyB+ren; ?2	OpUAS:luc:SF+ren; ?1	OpLexA:luc:SF+ren; ?1
1 µl ePDZ	1 µl PIF:phyB	1 µl OpUAS:luc:SF	1 µl OpLexA:luc:SF
1 µl pUPD2	1 µl ren (159)	1 µl ren	1 µl ren
5.6 µl H2O	1 µl ?2	1 µl ?1	1 µl ?1
	4.6 µl H2O	4.6 µl H2O	4.6 µl H2O
OpEtr8:luc:SF+ren; ?1	Op:LacI:luc:SF+ren; ?1
1 µl OpEtr8:luc:SF	1 µl OpLacI:luc:SF
1 µl ren	1 µl ren
1 µl ?1	1 µl ?1
4.6 µl H2O	4.6 µl H2O

After 3 days till the agroinfiltration we have observed the leaf discs that have BxbI+repBxbI and the repBxbI (negative control).

We could see that the level of GFP expression was higer in the infiltration with the recombinase but there was also expression in the negative control. We think that this can be a contamination due to that we had infiltrated both constructions in he same leaf and we did not change the gloves between agroinfiltrations. FOTOO!

Make liquid culture (E.coli) of:

LTB; pUPD2 (C1-C3)

31 July 2015

Ligation:

LacI:PIF:phyB:ren+OpLacI:luc; ?2

1.5 µl LacI:PIF:phyB:ren

3 µl OpLacI:luc

0.5 µl ?2

2.6 µl H2O

After 4 days till the agroinfiltration we have observed again the samples with BxbI and the negative control but was the same. The negative control expressed GFP but less than the recombinase. Foto!

Minipreps of liquid culture LTB (C1 and C2)

Digestion:

LTB; pUPD2

NotI

2046, 474

Gel:

LTB (C1)	LTB (C2)	PCR ePDZ
??

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; ?2
OpUAS:luc:SF+ren; ?1
OpLexA:luc:SF+ren; ?1
OpEtr8:luc:SF+ren; ?1
Op:LacI:luc:SF+ren; ?1
LacI:PIF:phyB:ren+OpLacI:luc; ?2

Refresh agro cultures to agroinfiltrate tomorrow:

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

New experiment with Nicotiana bentamiana. PROTOCOL

Next constructions were agroinfiltrated, 2 or 3 leafs for each plant:

PhiC31: one leaf for PhiC31+RepPhiC31+P19 (coinfiltration) and the other with RepPhiC31+P19 which is the negative control. 2 plants were infiltrated.

BxbI: one leaf with BxbI:RepBxbI+P19 and the other with RepBxbI+P19 which is negative control. 2 plants were infiltrated.

Red toggle: PIF6:phyB:luc+ren (coinfiltration). They were infiltrated 3 plants with 3 leafs for each.

Blue toggle: Gal4:NDronpa:KDronpa:luc:ren. They were infiltrated 3 plants with 3 leafs for each.

Pnos, the positive control. There are 4 plantas, 3 leafs per plant.

So we will take samples in time 0, 12, 24 and 36h for each construction and control. After 2 days of the agroinfiltration (during this period all the plants were in dark, it is set time 0 we make discs os leaf and put in a plate with water and we change the conditions that were before.

Red toggle plants: 9 discs stay in dark, 9 went to red and 9 went to natural light.

Blue toggle: the same as red but went to ultraviolet instead of red light.

Pnos: one plant were in natural light during all the experiment. The other discs will go, after the 2 days in black, to red, ultraviolet and stay in dark.

Recombinases: they are in natural light during all the experiment.

1 August 2015

Prepare the Agrobacterium cultures for agroinfiltration:

Gal4B:KDronpa:NDronpa:luc:ren, PhiC31 and its control, BxbI and its control and PIF:PhyB and its control were centrifugated 10 min at 3000g. The sobrenadant was discarded and the bacterias were resuspended with the agroinfiltration medium.

Agroinfiltration medium had: 10ml MES, 1ml MgCl2, 89ml H2O, 100ml DMSO+acetosiningone.

Incubate in 2h.

Mesurement oof the ODs:

Ren+P19	0.09	888.9 µl
RepPhiC31	0.69	115.94 µl
BxbI	0.46	174 µl
RepBxbI	0.15	533.3 µl
Gal4:KNDronpa:ren:luc	0.51	156.9 µl
Pnos	0.29	275.9 µl
PIF:phy:luc	0.17	470.6 µl
PhiC31	0.32	250 µl

We went to the greenhouse and infiltrate the 13 plants.

Look the petri dishes culture:

PIF:phyB+ren; ?2
OpLexA:luc:SF+ren; ?1
Op:LacI:luc:SF+ren; ?1

This colonies did not grow, the rest were left in the fridge.

ePDZ; pUPD2
OpUAS:luc:SF+ren; ?1
OpEtr8:luc:SF+ren; ?1
LacI:PIF:phyB:ren+OpLacI:luc; ?2

3 August 2015

Miniprep of the liquid culture:

Sip rotavirus CH2
Sip rotavirus CH2-CH3

Measure of DNA concentration of:

OpLexA:luc:SF=169ng/µl
OpacI:luc:SF=291ng/µl

Pick colonies gb159and make liquid culture of:

ePDZ; pUPD2 (C1-C3)
OpUAS:luc:SF+ren; ?1(C1-C3)
OpEtr8:luc:SF+ren; ?1(C1-C3)
LacI:PIF:phyB:ren+OpLacI:luc; ?2 (C1-C3) Added X-gal and IPTG.

Repeat ligations:

OpLexA:luc:SF+ren; ?1	Op:LacI:luc:SF+ren; ?1	E-PIF:phyB+ren; ?2
1 µl OpLexA:luc:SF	1 µl OpLacI:luc:SF	1 µl E-PIF:phy
3 µl ren	3 µl ren	3 µl ren
1 µl ?1	1 µl ?1	1 µl ?1
2.6 µl H2O	2.6 µl H2O	2.6 µl H2O

Refresh agrobacterium cultures of:

Interferon
SIP-CH2
SIP-CH2-CH3

Take the glycerinate of Renilla; ?2 (GB159) and make liquid culture.

We have made all the leaf discs and put in order in the plates with 300 µl of water, also we put each plate in the conditions light that they have to be. We have taken the samples of time0 (13:00).

So as T0= 13:00, T12=1:00, T24= 13:00 and T36= 1:00.

Imagenes de la colocacion en los platos? Hace falta?

4 August 2015

We do a Western blot with Asun so we can learn how to do it. Lo copio? De verdad!!?

Miniprep of the liquid cultures:

ePDZ; pUPD2 (C1-C3)
OpUAS:luc:SF+ren; ?1(C1-C3)
OpEtr8:luc:SF+ren; ?1(C1-C3)
LacI:PIF:phyB+ren; ?2 (C2 and C3) C1 was blue.

Digestions:

ePDZ; pUPD2	NotI	2046, 642
OpUAS:luc:SF+ren; ?1	EcoRI	6345, 7096
OpEtr8:luc:SF+ren; ?1	EcoRI	6345, 7294
LacI:PIF:phyB:ren+OpLacI:luc; ?2	EcoRV	6674, 3477, 381, 2475, 3942, 1652, 968, 882
Renilla 159	EcoRV	2909, 2475, 882, 812, 381

Gel:

ePDZ C1	ePDZ C2	ePDZ C3	LacI:PIF:phyB+ren
Ok	ok	ok
Renilla 159	OpUAS:luc:SF+ren C1	OpUAS:luc:SF+ren C2	OpUAS:luc:SF+ren C3
Ok	¿?
OpEtr8:luc:SF+ren C1	OpEtr8:luc:SF+ren C2	OpEtr8:luc:SF+ren C3

Transformation into E.coli of yesterday ligations:

OpLexA:luc:SF+ren; ?1
Op:LacI:luc:SF+ren; ?1
E-PIF:phyB+ren; ?2

Make petri dishes culture with the indicate antibiotic.

Ligations:

35S+ePDZ+VP16+T35S;?2	35S+LTB+T35S; ?1
1µl ePDZ	1µl 35S (30)
1 µl VP16	1µl T35S (36)
1 µl 35S (30)	1µl LTB
1 µl T35S (36)	1µl ?1
1 µl ?2	3.6 µl H2O
2.6 µl H2O

Refresh agrobacterium cultures of:

Interferon
SIP-CH2
SIP-CH2-CH3

5 August 2015

Transform ligations:

35S+ePDZ+VP16+T35S; ?2
35S+LTB+T35S; ?1

Pick colonies and make liquid cultures cultures of:

OpLexA:luc:SF+ren; ?1 (C1-C3)
Op:LacI:luc:SF+ren; ?1 (C1-C3)
E-PIF:phyB+ren; ?2 (C1)

35S+ePDZ+VP16+T35S; ?2 (C1 and C2)

35S+LTB+T35S; ?1 (C1 and C2)

Make luciferase essay of red and blue toggle:

Number of samples=75 (a lot)

1. Prepare lisis buffer for 80 samples.

200 µl/sample * 80sample = 16ml

Buffer (5x)—3.2ml of buffer + 12.8ml H2O miliQ: lysis buffer (1x)

2. Crushed samples+ 150 µl of lysis buffer.

3. Centrifuge 15min at 13200rpm in cold.

4. Dilution 2:3 (Add 36 µl lysis buffer + 24 µl sample)

5. Luciferase: 40 µl/sample. Prepare 2.88ml (3 substrate tubes)

6. Renilla:….?

6 August 2015

Miniprep of the liquid cultures.

Digestion:

OpLexA:luc:SF+ren	EcoRI	6345, 7274
Op:LacI:luc:SF+ren	EcoRI	6345, 7375
E-PIF:phyB+ren	HindIII	6345, 788, 5887, 4316
35S+ePDZ+VP16+T35S	HindIII	6345, 2316
35S+LTB+T35S	EcoRI	6345, 1684

Gel:

PIF:phyB+ren	OpLexA:luc:SF+ren C1	OpLexA:luc:SF+ren C2	OpLexA:luc:SF+ren C3
No	Ok, repeat	No	Ok, repeat
Op:LacI:luc:SF+ren C1	Op:LacI:luc:SF+ren C2	Op:LacI:luc:SF+ren C3	35S+LTB+T35S C1
Ok, repeat	Ok	Ok	ok
35S+LTB+T35S C2	35S+ePDZ+VP16+T35S C1	35S+ePDZ+VP16+T35S C2
Ok	ok	ok

Make colony PCR with 6 colonies of PhiC31:

DNA-
JM1: 2 µl (dilution 1:10)
JM2: 2 µl (dilution 1:10)
Buffer Taq (with Mg): 2 µl
dNTPs: 2.5 µl
Taq: 0.5 µl
H2O: 10 µl
Program: 96ºC-2min; 96ºC-30min; 55ºC-30min; 72ºC-30min

Make a gel with the PCR but we did not see the correct bands.

Repeat the colony PCR:

DNA-
JM1: 2.5 µl (dilution 1:10)
JM2: 2.5 µl (dilution 1:10)
Buffer Taq (with Mg): 10 µl
dNTPs: 2 µl
Taq: 0.5 µl
H2O: 7.5 µl
Program: : 96ºC-10min; 96ºC-30min; 55ºC-30min; 72ºC-30min

Ligations:

LexA:AsLOVpep+ePDZ; ?1	LacI:AsLOVpep+ePDZ; ?1	Gal4:AsLOVpep+ePDZ; ?1
1 µl LexA:AsLOVpep; ?1	1 µl LacI:AsLOVpep; ?1	1 µl Gal4:AsLOVpep; ?1
1 µl ePDZ; ?2	1 µl ePDZ; ?2	1 µl ePDZ; ?2
1 µl BsmBI	1 µl BsmBI	1 µl BsmBI
1 µl ?1	1 µl ?1	1 µl ?1
4.6 µl H2O	4.6 µl H2O	4.6 µl H2O

Refresh Agro cultures to agroinfiltrate:

PhiC31:RepPhiC31:GFP
RepPhiC31:GFP
BxbI:RepBxbI:GFP
RepBxbI:GFP
IFN (interferon)
Sip-CH2
Sip-CH2-CH3
Pnos
P19 (this culture ha 10ml of LB + 10 µl antibiotics + 5 µl culture)

7 August 2015

Transformation into Agrobacterium:

35S:LTB:VP16:T35S

Transformation into E.coli and make petri dishes cultures:

LexA:AsLOVpep+ePDZ
LacI:AsLOVpep+ePDZ
Gal4:AsLOVpep+ePDZ

Make a gel with the colonies PCRs:

C7	C8	C9	C10	C11	C12	C13	C14
No	no	no	no	no	no	no	no

There was no DNA, there is a problem with the cells or the procedure, it have to be revised.

Ligation:

PhyC31; pUPD2

3 µl PhiC31

1 µl pUPD2

1 µl BsmBI

3.6 µl H2O

Refresh agrobacterium cultures for tomorrow infiltration:

Sip-CH2
Sip-CH2-CH3
Pnos
Red toggle (PIF6:PhyB:luc )
Renilla
Blue toggle (….:KDronpa:NDronpa:ren:luc)

A new experiment was started:

We are going to check again the recombinase BxbI and its reporter (negative control).

Test the recombinase PhiC31. Due to that we did not obtain yet the complete recombinase, we will coinfiltrate the PhiC31 and the RepPhiC31:GFP following the same scheme as BxbI. 2 plants with 2 leafs, one of them with PhiC31 + RepPhiC31:GFP and the other with only RepPhiC31:GFP.
Infiltration in 2 plants with 2 leafs each of them with interferon.

Measure f the ODs:

Construction	OD	Volume (ml)
IFN	0.13	1.54
RepBxbI:GFP	0.13	1.54
BxbI:RepBxbI:GFP	0.33	0.61
PhiC31	0.19	1.05
RepPhiC31:GFP	0.22	0.91

8 August 2015

Transform the ligation into E.coli and make petri dishes cultures:

PhiC31; pUPD2.

LexA:AsLOVpep+ePDZ (C1-C5)
LacI:AsLOVpep+ePDZ (C1-C4)
Gal4:AsLOVpep+ePDZ (C1-C4)

New experiment:

It will be tested again the red toggle (PIF6:PhyB:luc + ren), the blue toggle (LacI:KDronpa:NDronpa:ren:luc), pnos (positive control to check the ratio renilla luciferasa) and the production of antirotavirus that are SIP rotavirus CH2 and SIP rotavirus CH2-CH3.

2 plants with 3 leafs ache one were infiltrated with pnos.

The same with the red(4) and blue toggle(4) and sip rativurs I DON’T KNOW

Measurement of the ODs:

Construction	OD	Volume (ml)
Sip-CH2	0.04	6.667
Sip-CH2-CH3	0.04	6.667
Red toggle (PIF6:PhyB:luc)	0.09	15
Blue toggle (LacI:KDronpa:NDronpa:ren:luc)	0.09	15.00
Renilla	0.04	6.667
Pnos	0.04	6.667

9 August 2015

Make a colony PCRs with the 10 white colonies of PhiC31 that grow in the petri dishes.

DNA- colony
JM1: 2 µl (dilution 1:10)
JM2: 2 µl (dilution 1:10)
Buffer Taq (with Mg): 2 µl
dNTPs: 2.5 µl
Taq: 0.5 µl
H2O: 1 µl

Minipreps of the liquid cultures:

LexA:AsLOVpep+ePDZ (C1-C4) C5 has turn into blue color.
LacI:AsLOVpep+ePDZ (C1-C4)
Gal4:AsLOVpep+ePDZ (C1-C4)

Digestion of the minipreps:

LexA:AsLOVpep+ePDZ	BamHI	6674, 4309
LacI:AsLOVpep+ePDZ	BamHI	6674, 5041
Gal4:AsLOVpep+ePDZ	BamHI	6674, 4270

Make the gel:

LexA:AsLOVpep+ePDZ (C1)	LexA:AsLOVpep+ePDZ (C2)	LexA:AsLOVpep+ePDZ (C3)	LexA:AsLOVpep+ePDZ (C4)
Ok	ok	ok	ok
oLacI:AsLOVpep+ePDZ (C1)	LacI:AsLOVpep+ePDZ (C2)	LacI:AsLOVpep+ePDZ (C3)	LacI:AsLOVpep+ePDZ (C4)
Ok	ok	ok	ok
Gal4:AsLOVpep+ePDZ (C1)	Gal4:AsLOVpep+ePDZ (C2)	Gal4:AsLOVpep+ePDZ (C3)	Gal4:AsLOVpep+ePDZ (C4)
Ok	ok	ok	Ok

We keep in our inventory the colony C1 of each construction

Make a gel of the colonies PCRs of PhiC31: we did not observed any DNA. Still can’t fix the problem.

Make liquid culture of renilla and PIF6:phyB:luc in Agrobacterium because the last time that we did an experiment they have grown slowly.

Store in the 4ºC fridge an agrobacterium culture with P19.

Ligations:

LexA:AsLOVpep+ePDZ+ren; ?1	LacI:AsLOVpep+ePDZ+ren; ?1	Gal4:AsLOVpep+ePDZ+ren; ?1
1µl LexA:AsLOVpep+ePDZ	1µl LacI:AsLOVpep+ePDZ	1µl Gal4:AsLOVpep+ePDZ
1 µl renilla (159)	1 µl renilla (159)	1 µl renilla (159)
1 µl ?1	1 µl ?1	1 µl ?1
4.6 µl H2O	4.6 µl H2O	4.6 µl H2O

10 August 2015

Transform into E.coli this ligations and make petri dishes cultures:

LexA:AsLOVpep+ePDZ+ren; ?1
LacI:AsLOVpep+ePDZ+ren; ?1
Gal4:AsLOVpep+ePDZ+ren; ?1
(we add into the tubes X-gal and IPTG)

Make liquid culture of 11 colonies of PhiC31 due to that this construction was giving us problems to obtain.

Refresh the agrobacterium cultures of:

BxbI:Rep:BxbI:GFP
Rep:BxbI:GFP
PhiC31
RepPhiC31:GFP
P19
Citoplasm
Integrase
Dsred
GFP

Take the samples of the agroinfiltrated plants that were in darkness for 2 days and make discs of them to change the conditions.

Make liquid culture of:

LexA:AsLOVpep+ePDZ+ren (C1 and C2)
LacI:AsLOVpep+ePDZ+ren (C1-C4)
Gal4:AsLOVpep+ePDZ+ren (C1 and C2)
(we add X-Gal and IPTG because they are small)

11 August 2015

Primers have arrived, they have been resuspended. They were used to eliminate the recognitions sites of the enzymes in BioBricks. This will make our constructions ready to be send to the iGEM

Minipreps of the liquid cultures done yesterday:

PhiC31 (C6-C16)
LacI:AsLOVpep+ePDZ+ren (C1-C4)
Gal4:AsLOVpep+ePDZ+ren (C1 and C2)
(LexA:AsLOVpep+ePDZ+rencolonies were all blue)

Make PCRs with all the primers and constructions:

All of them follow the same composition which is:

10 µl	Buffer HF
26.5 µl	H2O
2 µl	dNTPs
0.5 µl	Taq Phusion
1 µl	DNA (dilution 1:10)
2.5 µl	Primer forward (F) (dilution 1:10)
2.5 µl	Primer reverse (R) (dilution 1:10)

*green ones are the only specify.

IFN (1)	IFN (2)	AsLOVpep (1)	AsLOVpep (2)
IFN	IFN	AsLOVpep	AsLOVpep
Mys int F	IFN domBB R	AsLOVpep F	AsLOVpep Fint
Mys int R	IFN domBB F	AsLOVpep Rint	AsLOVpep R
AsLOVpep (nested)	PhiC31 (1)	PhiC31 (2)	PhiC31 (3)
AsLOVpep	PhiC31	PhiC31	PhiC31
AsLOVpep nested	PhiC31 Fint 1	PhiC31 Fint 2	PhiC31 Fint 3
AsLOVpep	PhiC31 Rint 2	PhiC31 Rint 3	PhiC31 R
PhiC31
PhiC31
PhiC31 F
PhiC31 Rint

Digestions of the minipreps:

PhiC31	NotI	2046, 1899
LacI:AsLOVpep+ePDZ+ren	EcoRI	6345, 8798
Gal4:AsLOVpep+ePDZ+ren	EcoRI	6345, 9569

Make the gel:

PhiC31 (C6)	C7…C16	LacI:AsLOVpep:ePDZ:ren (C1)	C2	C3…C4
no	no	ok	no	ok
Gal4:AsLOVpep+ePDZ+ren (C1)	Gal4:AsLOVpep+ePDZ+ren (C2)
ok	ok

Mesurement of the ODs:

Citoplasm	0.21	7.35 ml
RepPhiC31	0.26	9.1ml
35S:LTB:T35S	0.27	9.45ml
PhiC31	0.27	9.45ml
GFP	0.20	10ml
RepBxbI	0.33	11.55ml

PsinATG:RepBxbI:GFP	0.36	12.6ml
PhiC31	0.34	11.9ml
DsRed	0.26	9.1ml
P19	0.28	9.8ml

12 August 2015

New digestions to check if the negative control constructions are correct and we can transformed it in agro

OpLexA:luc:SF:ren	EcoRI	6345, 7274
Op:UAS:luc:SF:ren	EcoRI	6345, 7096
OpEtr8.luc:SF:ren	EcoRI	6345, 7294

Gel:

Op:UAS:luc:SF:ren C1	Op:UAS:luc:SF:ren C2	Op:UAS:luc:SF:ren C3	OpEtr8.luc:SF:ren
no	no	no	no
OpLexA:luc:SF:ren C1	OpLexA:luc:SF:ren C2	Partner dna	Partner dna
no	no

Transform in Agro and make petri dishes cultures:

LexA:AsLOVpep:ePDZ
Gal4:AsLOVpep:ePDZ
LacI:AsLOVpep:ePDZ
LexA:PIF6:phyB:VP16
Gal4:PIF6:phyB:VP16
LacI:PIF6:phyB:VP16
All of them are in ?1.

Ligations:

LexA:AsLOVpep:ePDZ+ren; ?1	Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc; ?1	LacI:AsLOVpep:ePDZ:ren+OpLacI:luc; ?1
1 µl LexA:AsLOVpep:ePDZ	1 µl Gal4:AsLOVpep:ePDZ:ren	1 µl LacI:AsLOVpep:ePDZ
1 µl renilla (159)	1 µl OpUAS:luc	1 µl OpLacI:luc
1 µl ?1	1 µl ?1	1 µl ?1
4.6 µl H2O	4.6 µl H2O	4.6 µl H2O

@@ Line 17: / Line 17: @@
 	<section id="main" class="container">
-				<section class="box special">
+				<div class="row">
-					<header>
-						<h2>Elements</h2>
-						<p>Just an assorted selection of elements.</p>
-					</header>
-					<div class="row">
 						<div class="12u">
-							<!-- Text -->
 								<section class="box">
-									<h3>Text</h3>
+		<h3 style="color:green">5 June 2015</h3>
-									<p>This is <b>bold</b> and this is <strong>strong</strong>. This is <i>italic</i> and this is <em>emphasized</em>.
-									This is <sup>superscript</sup> text and this is <sub>subscript</sub> text.
-									This is <u>underlined</u> and this is code: <code>for (;;) { ... }</code>. Finally, <a href="#">this is a link</a>.</p>
-									<hr />
+<p>We had 2 cultures from the last day, corresponding to other 2 colonies of ligation. </p>
-									<header>
+<p><i>Agrobacterium</i> culture of promoter less: Luciferase + Renilla </p>
-										<h3>Heading with a Subtitle</h3>
-										<p>Lorem ipsum dolor sit amet nullam id egestas urna aliquam</p>
-									</header>
-									<p>Nunc lacinia ante nunc ac lobortis. Interdum adipiscing gravida odio porttitor sem non mi integer non faucibus ornare mi ut ante amet placerat aliquet. Volutpat eu sed ante lacinia sapien lorem accumsan varius montes viverra nibh in adipiscing blandit tempus accumsan.</p>
-									<header>
-										<h4>Heading with a Subtitle</h4>
-										<p>Lorem ipsum dolor sit amet nullam id egestas urna aliquam</p>
-									</header>
-									<p>Nunc lacinia ante nunc ac lobortis. Interdum adipiscing gravida odio porttitor sem non mi integer non faucibus ornare mi ut ante amet placerat aliquet. Volutpat eu sed ante lacinia sapien lorem accumsan varius montes viverra nibh in adipiscing blandit tempus accumsan.</p>
-									<hr />
+<p>Minipreps</p>
-									<h2>Heading Level 2</h2>
+<p>Digestion with BamHI and EcoRV</p>
-									<h3>Heading Level 3</h3>
-									<h4>Heading Level 4</h4>
-									<h5>Heading Level 5</h5>
-									<h6>Heading Level 6</h6>
-									<hr />
+<p>Agarose gel 1%</p>
-									<h4>Blockquote</h4>
+<p>How to ask and make primers?</p>
-									<blockquote>Fringilla nisl. Donec accumsan interdum nisi, quis tincidunt felis sagittis eget tempus euismod. Vestibulum ante ipsum primis in faucibus vestibulum. Blandit adipiscing eu felis iaculis volutpat ac adipiscing accumsan faucibus. Vestibulum ante ipsum primis in faucibus lorem ipsum dolor sit amet nullam adipiscing eu felis.</blockquote>
-									<h4>Preformatted</h4>
+<ul><li>Select the sequence to amplify and save in FASTA format.</li>
-									<pre>i = 0;
-	while (!deck.isInOrder()) {
+<li>gbCloning, go to Tools-Domesticator-1º Category</li>
-	    print 'Iteration ' + i;
-	    deck.shuffle();
-	    i++;
-	}
-	print 'It took ' + i + ' iterations to sort the deck.';
+<li>Add FASTA and select parts.</li>
-	</pre>
-								</section>
-						</div>
+<li>On the protocol we have the primers </li>
-					</div>
-					<div class="row">
-						<div class="12u">
-							<!-- Lists -->
+<li>The oligos they give us:</li>
-								<section class="box">
-									<h3>Lists</h3>
-									<div class="row">
-										<div class="6u 12u(mobilep)">
-											<h4>Unordered</h4>
+<ul class="ul_2"><li>4 first nucleotides: so the enzyme can recognize without problems</li>
-											<ul>
-												<li>Dolor pulvinar etiam magna etiam.</li>
-												<li>Sagittis adipiscing lorem eleifend.</li>
-												<li>Felis enim feugiat dolore viverra.</li>
-											</ul>
-											<h4>Alternate</h4>
+<li>6 following bingind sites.</li>
-											<ul class="alt">
-												<li>Dolor pulvinar etiam magna etiam.</li>
-												<li>Sagittis adipiscing lorem eleifend.</li>
-												<li>Felis enim feugiat dolore viverra.</li>
-											</ul>
-										</div>
+<li>1 extra nucleotide.</li>
-										<div class="6u 12u(mobilep)">
-											<h4>Ordered</h4>
+<li>4 overhangs. </li>
-											<ol>
-												<li>Dolor pulvinar etiam magna etiam.</li>
-												<li>Etiam vel felis at lorem sed viverra.</li>
-												<li>Felis enim feugiat dolore viverra.</li>
-												<li>Dolor pulvinar etiam magna etiam.</li>
-												<li>Etiam vel felis at lorem sed viverra.</li>
-												<li>Felis enim feugiat dolore viverra.</li>
-											</ol>
-											<h4>Icons</h4>
+</ul></ul>
-											<ul class="icons">
-												<li><a href="#" class="icon fa-twitter"><span class="label">Twitter</span></a></li>
-												<li><a href="#" class="icon fa-facebook"><span class="label">Facebook</span></a></li>
-												<li><a href="#" class="icon fa-instagram"><span class="label">Instagram</span></a></li>
-												<li><a href="#" class="icon fa-github"><span class="label">Github</span></a></li>
-												<li><a href="#" class="icon fa-dribbble"><span class="label">Dribbble</span></a></li>
-												<li><a href="#" class="icon fa-tumblr"><span class="label">Tumblr</span></a></li>
-											</ul>
-										</div>
+<p>Meeting with Daniel Ramón (Biopolis). </p>
-									</div>
-									<h4>Actions</h4>
+<p>Ligation with part 2 and 24 of task sheet.</p>
-									<ul class="actions">
-										<li><a href="#" class="button special">Default</a></li>
-										<li><a href="#" class="button">Default</a></li>
-										<li><a href="#" class="button alt">Default</a></li>
-									</ul>
-									<ul class="actions small">
-										<li><a href="#" class="button special small">Small</a></li>
-										<li><a href="#" class="button small">Small</a></li>
-										<li><a href="#" class="button alt small">Small</a></li>
-									</ul>
-									<div class="row">
-										<div class="3u 6u(narrower) 12u$(mobilep)">
-											<ul class="actions vertical">
-												<li><a href="#" class="button special">Default</a></li>
-												<li><a href="#" class="button">Default</a></li>
-												<li><a href="#" class="button alt">Default</a></li>
-											</ul>
-										</div>
-										<div class="3u 6u$(narrower) 12u$(mobilep)">
-											<ul class="actions vertical small">
-												<li><a href="#" class="button special small">Small</a></li>
-												<li><a href="#" class="button small">Small</a></li>
-												<li><a href="#" class="button alt small">Small</a></li>
-											</ul>
-										</div>
-										<div class="3u 6u(narrower) 12u$(mobilep)">
-											<ul class="actions vertical">
-												<li><a href="#" class="button special fit">Default</a></li>
-												<li><a href="#" class="button fit">Default</a></li>
-												<li><a href="#" class="button alt fit">Default</a></li>
-											</ul>
-										</div>
-										<div class="3u 6u$(narrower) 12u$(mobilep)">
-											<ul class="actions vertical small">
-												<li><a href="#" class="button special small fit">Small</a></li>
-												<li><a href="#" class="button small fit">Small</a></li>
-												<li><a href="#" class="button alt small fit">Small</a></li>
-											</ul>
-										</div>
-									</div>
-								</section>
-						</div>
-					</div>
-					<div class="row">
-						<div class="12u">
-							<!-- Table -->
-								<section class="box">
-									<h3>Table</h3>
-									<h4>Default</h4>
+<div class="table-wrapper"><table class="alt">
-									<div class="table-wrapper">
-										<table>
-											<thead>
-												<tr>
-													<th>Name</th>
-													<th>Description</th>
-													<th>Price</th>
-												</tr>
-											</thead>
-											<tbody>
-												<tr>
-													<td>Something</td>
-													<td>Ante turpis integer aliquet porttitor.</td>
-													<td>29.99</td>
-												</tr>
-												<tr>
-													<td>Nothing</td>
-													<td>Vis ac commodo adipiscing arcu aliquet.</td>
-													<td>19.99</td>
-												</tr>
-												<tr>
-													<td>Something</td>
-													<td> Morbi faucibus arcu accumsan lorem.</td>
-													<td>29.99</td>
-												</tr>
-												<tr>
-													<td>Nothing</td>
-													<td>Vitae integer tempus condimentum.</td>
-													<td>19.99</td>
-												</tr>
-												<tr>
-													<td>Something</td>
-													<td>Ante turpis integer aliquet porttitor.</td>
-													<td>29.99</td>
-												</tr>
-											</tbody>
-											<tfoot>
-												<tr>
-													<td colspan="2"></td>
-													<td>100.00</td>
-												</tr>
-											</tfoot>
-										</table>
-									</div>
-									<h4>Alternate</h4>
+<tr><td>PIF6 + PhyB; ?1</td><td>Etr8 CMV_Bxb1_T35S</td></tr>
-									<div class="table-wrapper">
-										<table class="alt">
-											<thead>
-												<tr>
-													<th>Name</th>
-													<th>Description</th>
-													<th>Price</th>
-												</tr>
-											</thead>
-											<tbody>
-												<tr>
-													<td>Something</td>
-													<td>Ante turpis integer aliquet porttitor.</td>
-													<td>29.99</td>
-												</tr>
-												<tr>
-													<td>Nothing</td>
-													<td>Vis ac commodo adipiscing arcu aliquet.</td>
-													<td>19.99</td>
-												</tr>
-												<tr>
-													<td>Something</td>
-													<td> Morbi faucibus arcu accumsan lorem.</td>
-													<td>29.99</td>
-												</tr>
-												<tr>
-													<td>Nothing</td>
-													<td>Vitae integer tempus condimentum.</td>
-													<td>19.99</td>
-												</tr>
-												<tr>
-													<td>Something</td>
-													<td>Ante turpis integer aliquet porttitor.</td>
-													<td>29.99</td>
-												</tr>
-											</tbody>
-											<tfoot>
-												<tr>
-													<td colspan="2"></td>
-													<td>100.00</td>
-												</tr>
-											</tfoot>
-										</table>
-									</div>
-								</section>
-						</div>
+<tr><td>1µL 892 (PIF &alpha;1)</td><td>1µL 1097 (Etr8 CMV) Pupd2</td></tr>
-					</div>
-					<div class="row">
-						<div class="12u">
-							<!-- Buttons -->
+<tr><td>1µL 88E (Phy &alpha;2)</td><td>1µL Bxb1 (PuPD)</td></tr>
-								<section class="box">
-									<h3>Buttons</h3>
-									<ul class="actions">
-										<li><a href="#" class="button special">Special</a></li>
-										<li><a href="#" class="button">Default</a></li>
-										<li><a href="#" class="button alt">Alternate</a></li>
-									</ul>
-									<ul class="actions">
-										<li><a href="#" class="button special big">Big</a></li>
-										<li><a href="#" class="button">Default</a></li>
-										<li><a href="#" class="button alt small">Small</a></li>
-									</ul>
-									<ul class="actions fit">
-										<li><a href="#" class="button special fit">Fit</a></li>
-										<li><a href="#" class="button fit">Fit</a></li>
-										<li><a href="#" class="button alt fit">Fit</a></li>
-									</ul>
-									<ul class="actions fit small">
-										<li><a href="#" class="button special fit small">Fit + Small</a></li>
-										<li><a href="#" class="button fit small">Fit + Small</a></li>
-										<li><a href="#" class="button alt fit small">Fit + Small</a></li>
-									</ul>
-									<ul class="actions">
-										<li><a href="#" class="button special icon fa-search">Icon</a></li>
-										<li><a href="#" class="button icon fa-download">Icon</a></li>
-										<li><a href="#" class="button alt icon fa-check">Icon</a></li>
-									</ul>
-									<ul class="actions">
-										<li><span class="button special disabled">Special</span></li>
-										<li><span class="button disabled">Default</span></li>
-										<li><span class="button alt disabled">Alternate</span></li>
-									</ul>
-								</section>
-						</div>
+<tr><td>1µL ?1 </td><td>1µL Tnos PuPD</td></tr>
-					</div>
-					<div class="row">
-						<div class="12u">
-							<!-- Form -->
+<tr><td>1.2µL Buffer ligase</td><td>1µL &alpha;1</td></tr>
-								<section class="box">
-									<h3>Form</h3>
-									<form method="post" action="#">
-										<div class="row uniform 50%">
-											<div class="6u 12u(mobilep)">
-												<input type="text" name="name" id="name" value="" placeholder="Name" />
-											</div>
-											<div class="6u 12u(mobilep)">
-												<input type="email" name="email" id="email" value="" placeholder="Email" />
-											</div>
-										</div>
-										<div class="row uniform 50%">
-											<div class="12u">
-												<div class="select-wrapper">
-													<select name="category" id="category">
-														<option value="">- Category -</option>
-														<option value="1">Manufacturing</option>
-														<option value="1">Shipping</option>
-														<option value="1">Administration</option>
-														<option value="1">Human Resources</option>
-													</select>
-												</div>
-											</div>
-										</div>
-										<div class="row uniform 50%">
-											<div class="4u 12u(narrower)">
-												<input type="radio" id="priority-low" name="priority" checked>
-												<label for="priority-low">Low Priority</label>
-											</div>
-											<div class="4u 12u(narrower)">
-												<input type="radio" id="priority-normal" name="priority">
-												<label for="priority-normal">Normal Priority</label>
-											</div>
-											<div class="4u 12u(narrower)">
-												<input type="radio" id="priority-high" name="priority">
-												<label for="priority-high">High Priority</label>
-											</div>
-										</div>
-										<div class="row uniform 50%">
-											<div class="6u 12u(narrower)">
-												<input type="checkbox" id="copy" name="copy">
-												<label for="copy">Email me a copy of this message</label>
-											</div>
-											<div class="6u 12u(narrower)">
-												<input type="checkbox" id="human" name="human" checked>
-												<label for="human">I am a human and not a robot</label>
-											</div>
-										</div>
-										<div class="row uniform 50%">
-											<div class="12u">
-												<textarea name="message" id="message" placeholder="Enter your message" rows="6"></textarea>
-											</div>
-										</div>
-										<div class="row uniform">
-											<div class="12u">
-												<ul class="actions">
-													<li><input type="submit" value="Send Message" /></li>
-													<li><input type="reset" value="Reset" class="alt" /></li>
-												</ul>
-											</div>
-										</div>
-									</form>
-									<hr />
+<tr><td>1µL Bsmb1</td><td>5.8µL H2O</td></tr>
-									<form method="post" action="#">
+<tr><td>6.8µL H2O</td><td></td></tr>
-										<div class="row uniform 50%">
-											<div class="9u 12u(mobilep)">
-												<input type="text" name="query" id="query" value="" placeholder="Query" />
-											</div>
-											<div class="3u 12u(mobilep)">
-												<input type="submit" value="Search" class="fit" />
-											</div>
-										</div>
-									</form>
-								</section>
-						</div>
+</div></table>
-					</div>
-					<div class="row">
-						<div class="12u">
-							<!-- Image -->
-								<section class="box">
-									<h3>Image</h3>
-									<h4>Fit</h4>
-									<span class="image fit"><img src="images/pic04.jpg" alt="" /></span>
-									<div class="box alt">
-										<div class="row no-collapse 50% uniform">
-											<div class="4u"><span class="image fit"><img src="images/pic04.jpg" alt="" /></span></div>
-											<div class="4u"><span class="image fit"><img src="images/pic04.jpg" alt="" /></span></div>
-											<div class="4u"><span class="image fit"><img src="images/pic04.jpg" alt="" /></span></div>
-										</div>
-										<div class="row no-collapse 50% uniform">
-											<div class="4u"><span class="image fit"><img src="images/pic04.jpg" alt="" /></span></div>
-											<div class="4u"><span class="image fit"><img src="images/pic04.jpg" alt="" /></span></div>
-											<div class="4u"><span class="image fit"><img src="images/pic04.jpg" alt="" /></span></div>
-										</div>
-										<div class="row no-collapse 50% uniform">
-											<div class="4u"><span class="image fit"><img src="images/pic04.jpg" alt="" /></span></div>
-											<div class="4u"><span class="image fit"><img src="images/pic04.jpg" alt="" /></span></div>
-											<div class="4u"><span class="image fit"><img src="images/pic04.jpg" alt="" /></span></div>
-										</div>
-									</div>
-									<h4>Left &amp; Right</h4>
-									<p><span class="image left"><img src="images/pic05.jpg" alt="" /></span>Fringilla nisl. Donec accumsan interdum nisi, quis tincidunt felis sagittis eget. tempus euismod. Vestibulum ante ipsum primis in faucibus vestibulum. Blandit adipiscing eu felis iaculis volutpat ac adipiscing accumsan eu faucibus. Integer ac pellentesque praesent tincidunt felis sagittis eget. tempus euismod. Vestibulum ante ipsum primis in faucibus vestibulum. Blandit adipiscing eu felis iaculis volutpat ac adipiscing accumsan eu faucibus. Integer ac pellentesque praesent. Donec accumsan interdum nisi, quis tincidunt felis sagittis eget. tempus euismod. Vestibulum ante ipsum primis in faucibus vestibulum. Blandit adipiscing eu felis iaculis volutpat ac adipiscing accumsan eu faucibus. Integer ac pellentesque praesent tincidunt felis sagittis eget. tempus euismod. Vestibulum ante ipsum primis in faucibus vestibulum. Blandit adipiscing eu felis iaculis volutpat ac adipiscing accumsan eu faucibus. Integer ac pellentesque praesent.</p>
-									<p><span class="image right"><img src="images/pic05.jpg" alt="" /></span>Fringilla nisl. Donec accumsan interdum nisi, quis tincidunt felis sagittis eget. tempus euismod. Vestibulum ante ipsum primis in faucibus vestibulum. Blandit adipiscing eu felis iaculis volutpat ac adipiscing accumsan eu faucibus. Integer ac pellentesque praesent tincidunt felis sagittis eget. tempus euismod. Vestibulum ante ipsum primis in faucibus vestibulum. Blandit adipiscing eu felis iaculis volutpat ac adipiscing accumsan eu faucibus. Integer ac pellentesque praesent. Donec accumsan interdum nisi, quis tincidunt felis sagittis eget. tempus euismod. Vestibulum ante ipsum primis in faucibus vestibulum. Blandit adipiscing eu felis iaculis volutpat ac adipiscing accumsan eu faucibus. Integer ac pellentesque praesent tincidunt felis sagittis eget. tempus euismod. Vestibulum ante ipsum primis in faucibus vestibulum. Blandit adipiscing eu felis iaculis volutpat ac adipiscing accumsan eu faucibus. Integer ac pellentesque praesent.</p>
-								</section>
-						</div>
-					</div>
-				</section>
+<p>If we make a digestion of 160 (35S:Renilla:tNOS-35S:P19:tNOS) with EcoRV, we obtain: 2475, 381, 4601 pb.</p>
+<p>If we make a digestion of 896 (Luc:PIF6:PhyB)with EcoRV, we obtain: 11608, 3942 pb.</p>
+</br><h3 style="color:green">June 2015</h3>
+<p>Transform to E.coli from PIF+Phy and Bxb1</p>
+<ul><li>1.5µL of ligation</li>
+<ul class="ul_2"><li>Cuvette on ice</li>
+<li>Competent cells + 1.5µL of ligation</li>
+<li>Pulse (electroporator) at 1500V</li>
+<li>Add 300µL shock medium and put Eppendorf 1h at 37ºC</li>
+</ul></ul>
+<p>Culture on petri dishes the ligations.</p>
+<p>Digest of 160, 289 and the two ligations, PIF+phy and Etr8+BxbI. </p>
+<p>Agarose gel. </p>
+</br><h3 style="color:green">7 June 2015</h3>
+<p>We’ve got white colonies! (from PIF+Phy and Bxb1)</p>
+<p>Pick two colonies from each construction.</p>
+<p>4 tubes</p>
+<ul><li>3.5µL LB each tube</li>
+</ul>
+<p>2)	2 tubes + 3.5µL Kanamycin (K)</p>
+</br><h3 style="color:green">8 June 2015</h3>
+<p>Minipreps of the 4 liquid cultures and digestion to see the band patterns.</p>
+<p>Digestion:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Etr8(CMV):Bxb1:Tnos; ?1</td><td>EcoRI</td><td>6345, 238</td></tr>
+<tr><td>EPIF6 + PhyB-PV16; ?1</td><td>BamHI</td><td>6686, 1439, 2685, 2237</td></tr>
+</div></table>
+<p>Agarose gel was made:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Bxb1 (C1)</td><td>Bxb1 (C2)</td><td>EPIF6 + PhyB-PV16 (C1)</td><td>EPIF6 + PhyB-PV16 (C2)</td><td></td></tr>
+<tr><td>ok</td><td>ok</td><td></td><td></td></tr>
+<tr><td></td><td></td><td></td></tr>
+<tr><td></td><td></td><td></td></tr>
+</div></table>
+<p>Repeat digestion (errors).</p>
+<p>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.</p>
+<p>Optimized ligation of PIF-Phy-Lac-Renilla-P19</p>
+<ul><li>1 µL vector</li>
+<li>0.8 µL dilution ½ 160</li>
+<li>1.7 µL big part</li>
+<li>1.2 µL BSA</li>
+<li>1.2 µL buffer</li>
+<li>1 µL BsbmI</li>
+<li>1 µL ligase</li>
+<li>4.15 µL H2O</li>
+<li>Ratio 1:2 vector insert</li>
+</ul>
+<p>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.</p>
+<p>We design primers to binding domain (BD) and PIF.</p>
+<ul><li>Problem: domesticator is introduced in an old pUPD. The new one has different bases. </li>
+<li>Change manually the pUPD bases in the program (Benchling).</li>
+</ul></ul>
+</br><h3 style="color:green">9 June 2015</h3>
+<p>Digestion of the ligation of yesterday containing: EPIF6-PhyB-VP16 (C1 y C2)</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>EPIF6-PhyB-VP16</td><td>PvuII (green buffer)</td><td>3663, 9472pb</td></tr>
+</div></table>
+<p>Agarose gel 1%:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>EPIF6-PhyB-VP16 (C1)</td><td>EPIF6-PhyB-VP16 (C2)</td><td>EPIF6-PhyB-VP16 (C3)</td></tr>
+<tr><td>no</td><td>no</td><td>no</td></tr>
+</div></table>
+<p>We see three bands: 7000, 4000, 1900pb</p>
+<p>Transform optimized ligation (yesterday 8/6)</p>
+<ul><li>3.5 µL ligation product (EPIF6-PhyB-VP16 + Luciferase + Renille + P19)</li>
+<li>40 µL electrocompetent cells.</li>
+<li>Pulse of 1500V</li>
+<li>Store 1h at 37ºC</li>
+<li>Plate culture at agar with Spectinomycin: 50 µL of the transformation.</li>
+</ul>
+</br><h3 style="color:green">10 June 2015</h3>
+<ul><li>Check the primers and order LexA, Gal4, PIF6, LacI, Dronpa.</li>
+<li>Check linker VP16 (88E) and make a primer for it.</li>
+<li>Take out glycerinate of &Omega;2.</li>
+</ul>
+<p>Alfredo’s part is not working.</p>
+<ul><li>Pick colonies of transformation and make liquid culture of E:PIF6:PhyB:VP16:luc:ren (C1-C3).</li>
+</ul>
+<ul><li>Minipreps of liquid culture (PIF + Phy), colonies C3, C4, C5, C6</li>
+<li>Digestion:</li>
+</ul></ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>PIF + Phy:VP16</td><td>PvuII (buffer green 10x)</td><td>3663, 9472</td></tr>
+<tr><td>PIF + Phy:VP16</td><td>BamHI</td><td>1939, 2685, 2337, 6674</td></tr>
+</div></table>
+<ul><li>Agarose gel 1% (10 samples, 8 + 2 molecular markers)</li>
+</ul>
+<p>PIF + Phy (PvuII) C3	PIF + Phy (PvuII) C4	PIF + Phy (PvuII) C5	PIF + Phy (PvuII) C6</p>
+<p>no	ok	no	No</p>
+<p>PIF + Phy (BamHI) C3	PIF + Phy (BamHI) C4	PIF + Phy (BamHI) C5	PIF + Phy (BamHI) C6</p>
+<p>no	ok	no	No</p>
+<ul><li>Transformation of <i>Agrobacterium</i> (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).</li>
+</ul>
+</br><h3 style="color:green">11 June 2015</h3>
+<ul><li>Minipreps of the culture:</li>
+</ul>
+<ul><li>Digestion:</li>
+</ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>E:PIF6:PhyB:VP16:luc:ren</td><td>BamHI</td><td>4209, 3756, 6100, 6674</td></tr>
+<tr><td></td><td>EcoRV</td><td>3942, 2989, 2475, 381, 10952</td></tr>
+</div></table>
+<p>Gel:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>PIF6:PhyB:VP16:luc:</td></tr>
+<tr><td>ren C1 (BamHI)</td><td>PIF6:PhyB:VP16:luc:</td></tr>
+<tr><td>ren C3 (BamHI)</td><td>PIF6:PhyB:VP16:luc:ren</td></tr>
+<tr><td>C1 (EcoRV)</td><td>PIF6:PhyB:VP16:luc:ren </td></tr>
+<tr><td>C3 (EcoRV)</td></tr>
+<tr><td>??</td><td></td><td></td></tr>
+</div></table>
+<p>Transformation in <i>Agrobacterium</i> 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.</p>
+</br><h3 style="color:green">12 June 2015</h3>
+<p>The petri dish with PIF:phy:luc was taken out the 37ºC room and put into the fridge to pick  colonies tomorrow.</p>
+</br><h3 style="color:green">13 June 2015</h3>
+<ul><li>Pick colonies to make liquid culture:</li>
+<ul class="ul_2"><li>Renilla in agrobacterium: just one colony, it was made liquid culture but check carefully the gel.</li>
+<li>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).</li>
+</ul><li>Ligation:</li>
+</ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>BxbI; alfa1+phyB; alfa2</td></tr>
+<tr><td>1µl BxbI</td></tr>
+<tr><td>1 µl phyB</td></tr>
+<tr><td>1 µl ?2</td></tr>
+<tr><td>4.6 µl H2O</td></tr>
+</div></table>
+<ul><li>Transform renilla (160) into agrobacterium and make </li>
+</ul></ul>
+</br><h3 style="color:green">15 June 2015</h3>
+<ul><li>Repeat the ligation BxbI+35S:E-PIF6:tnos because PIF was ?2</li>
+</ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>BxbI + 35S:E-PIF6:tnos; ?1</td></tr>
+<tr><td>1µl BxbI</td></tr>
+<tr><td>1 µl phyB</td></tr>
+<tr><td>1 µl ?1</td></tr>
+<tr><td>4.6</td><td>µl H2O</td></tr>
+</div></table>
+<ul><li>KDronpa has arrived:</li>
+<ul class="ul_2"><li>Centrifuge it 2-5sec at max velocity.</li>
+<li>Add 50 µl to have a concentration of 20ng/µl</li>
+<li>Mix it with the vortex and spin.</li>
+</ul><li>Ligation: en la libreta pone que se liga a pUPD2</li>
+</ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>KDronpa; pUPD2</td></tr>
+<tr><td>1 µl KDronpa</td></tr>
+<tr><td>1 µl pUPD2</td></tr>
+<tr><td>5.6 µl H2O</td></tr>
+</div></table>
+<ul><li>It was not possible to pick colonies of the <i>Agrobacterium</i> transformed because they did not grow. Maybe the problem is that with tetraciclyn bacterias grow slowly. Wait 1 day more.</li>
+<li>Transformation of the ligation, BxbI + 35S:E-PIF6:tnos; ?1, into E.coli.</li>
+<li>Make an agar culture in petri dish and let grow 16h at 37ºC.</li>
+</ul>
+</br><h3 style="color:green">16 June 2015</h3>
+<ul><li>Transformation of the ligation, KDronpa, into E.coli.</li>
+<li>Pick colonies of BxbI:E-PIF6 and make liquid culture (C1-C3).</li>
+<li>Primers had arrived, it has been done the resuspension (dilution 1:10)of all of them.</li>
+</ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Primers</td><td>Code </td><td>Template</td><td>Working temperature? ºC</td></tr>
+<tr><td>LacI F</td><td>1</td><td>LacI (858)</td><td>69.7</td></tr>
+<tr><td>LacI R </td><td>2</td><td></td></tr>
+<tr><td>Gal4 F</td><td>3</td><td>We did not take out the glicerynate.</td><td>63.2</td></tr>
+<tr><td>Gal4 </td><td>4</td><td></td></tr>
+<tr><td>LexA F</td><td>5</td><td>LexA (732)</td><td>62.7</td></tr>
+<tr><td>LexA R</td><td>6</td><td></td></tr>
+<tr><td>PIF:VP16 F</td><td>7</td><td>PIF6 (288)</td><td>60.1</td></tr>
+<tr><td>PIFVP16 R</td><td>8</td><td></td></tr>
+<tr><td>NDronpa F1</td><td>9</td><td>Kdronpa</td><td>67.7</td></tr>
+<tr><td>NDronpa R1</td><td>10</td><td></td></tr>
+<tr><td>Dronpa F2</td><td>11</td><td>58.5</td></tr>
+<tr><td>NDronpa R2</td><td>12</td><td></td></tr>
+</div></table>
+<ul><li>A PCR with all the primers and the fragments was done, the samples were put in order with the temperature.</li>
+<ul class="ul_2"><li>The templates were in dilution 1:50, exception of KDronpa that was dilution 1:5 and the primers 1:10.</li>
+</ul></ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>PCR Fusion Taq (50µl)</td></tr>
+<tr><td>DNA template (10 µg/µl)</td></tr>
+<tr><td>0.5 µl fusion taq</td></tr>
+<tr><td>2.5 µl primer F</td></tr>
+<tr><td>2.5 µl primer R</td></tr>
+<tr><td>2 µl NTPs</td></tr>
+<tr><td>31.5 µl H2O</td></tr>
+<tr><td>17 June 2015</td></tr>
+</div></table>
+<ul><li>Pick colonies and make liquid culture of:</li>
+<ul class="ul_2"><li>KDronpa (C1-C4)</li>
+<li>Ligations with the PCR’s products:</li>
+<li>Templates PCR: 1+2, 5+6, 7+8PIF, 7+8VP16, 9+10, 11+12.</li>
+</ul></ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Template PCR; pUPD2</td></tr>
+<tr><td>0.5µl template</td></tr>
+<tr><td>1µl pUPD2</td></tr>
+<tr><td>6.1µl H2O</td></tr>
+</div></table>
+<ul><li>Minipreps of liquid cultures:</li>
+<ul class="ul_2"><li>BxbI:E-PIF6 (C1-C3)</li>
+</ul><li>Agarose gel with the PCRs:</li>
+</ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Template</td><td>1+2</td><td>5+6</td><td>7+8PIF</td><td>7+8VP16</td><td>9+10</td><td>11+12</td></tr>
+<tr><td>Band pattern</td><td>1017</td><td>284</td><td>391</td><td>478</td><td>464</td><td>290</td></tr>
+<tr><td>Gel result</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>No DNA</td><td>ok</td></tr>
+</div></table>
+<ul><li>Transformation in E.coli of the correct ligations:</li>
+<ul class="ul_2"><li>1+2, 5+6, 7+8PIF, 7+8VP16, 11+12</li>
+<li>Put in cloranfenicol petri dishes. </li>
+</ul></ul>
+</br><h3 style="color:green">18 June 2015</h3>
+<ul><li>Minipreps of the liquid cultrures:</li>
+<ul class="ul_2"><li>KDronpa (C1-C4)	</li>
+</ul><li>Digestions:</li>
+</ul>
+<p>KDronpa	EcoRI	2800</p>
+<ul><li>Gel:</li>
+</ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Kdronpa C1</td><td>Kdronpa C2</td><td>Kdronpa C3</td><td>KdronpaC4</td></tr>
+<tr><td>no</td><td>no</td><td>ok</td><td>no</td></tr>
+<tr><td>Etr8:BxbI:phyB C1</td><td>Etr8:BxbI:phyB C2</td><td>Etr8:BxbI:phyB C3</td><td></td></tr>
+<tr><td>No</td><td>no</td><td>no</td><td></td></tr>
+</div></table>
+<p>We discovered that the construction with BxbI did not go well because our lab college gives us the wrong piece. </p>
+<ul><li>Take glicerynates out:</li>
+<ul class="ul_2"><li>Gal4; pUPD (731)</li>
+<li>?2</li>
+<li>PromotersinATG (GB00552)</li>
+<li>Renilla (160)(159)(109)</li>
+</ul><li>PCR:</li>
+</ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>NDronpa</td></tr>
+<tr><td>2.5 µl (9+10) primer F</td></tr>
+<tr><td>2.5 µl (11+12) primer R</td></tr>
+<tr><td>2 µl NTPs</td></tr>
+<tr><td>0.2 µl Taq</td></tr>
+<tr><td>10 µl Buffer</td></tr>
+<tr><td>31.5</td><td>µl H2O</td></tr>
+</div></table>
+<ul><li>Ligations:</li>
+</ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Etr8:BxbI:T35S; &alpha;1</td><td>Template PCR; pUPD2</td></tr>
+<tr><td>1 µlEtr8</td><td>0.5µl template</td></tr>
+<tr><td>1 µl BxbI</td><td>1µl pUPD2</td></tr>
+<tr><td>1 µl T35S</td><td>6.1µl H2O</td></tr>
+<tr><td>5.8 µl H2O</td><td></td></tr>
+<tr><td></</td></tr>
+<tr><td></td></tr>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Templates PCR: 1+2, 5+6, 7+8PIF, 7+8VP16</td></tr>
+</br><h3 style="color:green">19 June 2015</h3>
+<ul><li>We do a PCR with the normal Taq polymerase.</li>
+<ul class="ul_2"><li>1µl of DNA’s template (9+10, 9+12 and 11+12)</li>
+<li>2µl of specific buffer</li>
+<li>2µl of NTPs</li>
+<li>1µl primer forward</li>
+<li>1µl primer reverse</li>
+<li>0.5 µl of Taq</li>
+<li>12.5 µl H2O</li>
+<li>These quantities multiplied by 3.</li>
+</ul><li>Minipreps of the yesterday’s glycerinated cultures.</li>
+<li>To do the digestions we add in each eppendorf.</li>
+</ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Minipreps:</td><td>Enzime</td><td>Band pattern</td></tr>
+<tr><td>159 pDGB1_?2 renilla</td><td>EcoRV</td><td>2909, 2475,882, 812, 381</td></tr>
+<tr><td>Entry vector, ?2</td><td>EcoRV</td><td>6652, 621</td></tr>
+<tr><td>552 pP35s NoATG, pUPD</td><td>EcoRI</td><td>2997, 1090</td></tr>
+<tr><td>160 renilla pDGB1, a2 </td><td>EcoRV</td><td>4601, 2475, 381</td></tr>
+<tr><td>731 pUPD pGal4BD (CDS)</td><td>EcoRI</td><td>2997, 2493</td></tr>
+<tr><td>109</td><td>GB1_a1 355:renilla:Tnos</td><td>EcoRI</td><td>2580, 2493</td></tr>
+</div></table>
+<ul><li>We make an agarose gel with the digestions made before and the PCR of KDronpa. </li>
+</ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>159</td><td>160</td><td>?2</td><td>552</td><td>731</td><td>109</td><td>9+10</td><td>9+12</td><td>11+12</td></tr>
+<tr><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>no</td><td>ok</td><td>ok</td></tr>
+</div></table>
+<ul><li>Transformation of the ligations:</li>
+<ul class="ul_2"><li>50 µl of electrocompetent cell</li>
+<li>1.5 µl of ligation</li>
+<li>Put 1 min before the cubettes in ice </li>
+<li>Make the transformation (1500V)</li>
+<li>Put the transformed cells 1h at 37ºC</li>
+</ul></ul>
+<ul><li>We made an stack of Cloranfenicol petri dishes</li>
+<ul class="ul_2"><li>250ml  LB aga</li>
+<li>X-gal (1:500): 500 µl</li>
+<li>Iptg (1:1000): 250 µl</li>
+<li>Cloranfenicol (1:2000): 125 µl</li>
+</ul></ul>
+</br><h3 style="color:green">20 june 2015</h3>
+<p>We have White colonies of renilla! Also of Etr8 + Bxb1</p>
+<p>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.</p>
+<ul><li>We make a liquid culture of Agro of Renilla.</li>
+<ul class="ul_2"><li>5ml of LB agar</li>
+<li>5 µl rifampicine</li>
+<li>5 µl of kanamicine</li>
+<li>5 µl tetracycline</li>
+<li>Incubate for 2 days (48h) at 28ºC.</li>
+</ul></ul>
+</br><h3 style="color:green">21 June 2015</h3>
+<ul><li>Pick colonies of and put into a liquid medium of 3 ml of LB agar, 3 µl of each antibiotic (kanamicine, spectomicine, cloranfenicol):</li>
+<ul class="ul_2"><li>Plates (17/06/15): PIF (C1, C2) (with cloranfenicol), VP16 (C1, C3) (with cloranfenicol), LacI (C1, C2, C3) (with cloranfenicol)</li>
+<li>Plates (19/06/15): Bxb1 (C1, C2, C3) (with kanamicine), VP16 (C4, C5) (with cloranfenicol), LacI (C1, C2) (with C1, C2) (with cloranfenicol), PIF (C1, C2, C3, C4, C5) (with cloranfenicol), LexA (C1, C2) (with cloranfenicol).</li>
+</ul><li>We take out two glicerynates of GFP and BFP (of the Alfredo’s box)</li>
+</ul>
+</br><h3 style="color:green">22 June 2015</h3>
+<ul><li>We made minipreps of the liquid culture of the day before:</li>
+<ul class="ul_2"><li>LacIBD, pUPD (C1-C5)</li>
+<li>LexABD, pUPD (C1, C2)</li>
+<li>Etr8(CMV):Bxb1 (C1-C3)</li>
+<li>PIF6,pUPD (C1-C5)</li>
+<li>VP16, pUPD (C1, C4, C5)</li>
+</ul></ul>
+<ul><li>Make the digestions of all the minipreps:</li>
+</ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LacIBD, pUPD</td><td>NotI</td><td>2046, 1053</td></tr>
+<tr><td>LexABD, pUPD </td><td>NotI</td><td>2046, 321</td></tr>
+<tr><td>Etr8(CMV):Bxb1 </td><td>NotI</td><td>1532, 1290, 5896</td></tr>
+<tr><td>PIF6,pUPD </td><td>NotI</td><td>2046, 407</td></tr>
+<tr><td>VP16, pUPD </td><td>NotI</td><td>2046, 500</td></tr>
+</div></table>
+<ul><li>Viral system:……….</li>
+<li>We received the reported Bxb1!</li>
+<ul class="ul_2"><li>500ng of sample</li>
+<li>Centrifuge at 3000rpm for 5 seconds (spin).</li>
+<li>Add 50 µl H2O</li>
+<li>Shake it and let at 50ºC for 20min</li>
+<li>Make a PCR of Gal4 and NDrompa (9-10), the primers of NDrompa are aliquoted</li>
+</ul><li>…..</li>
+</ul>
+<p>Make the gel with all the digestions writed before.</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Lac1</td><td>Lac2</td><td>Lac3</td><td>Lac4</td><td>Lac5</td><td>Lex1</td><td>Lex2</td><td>Bxb1,1</td><td>Bxb2, 2</td><td>Bxb1,3</td></tr>
+<tr><td>Ok</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>no</td><td>no</td><td>ok</td><td>ok</td><td>no</td></tr>
+<tr><td>PIF1</td><td>PIF2</td><td>PIF3</td><td>PIF4</td><td>PIF5</td><td>VP16,1</td><td>VP16,4</td><td>VP16,5</td><td></td></tr>
+<tr><td>No</td><td>no</td><td>-</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td></td></tr>
+<tr><td>PCRS</td><td>…</td><td>…</td><td>…</td><td></td><td></td><td></td></tr>
+</div></table>
+<p>					</p>
+<ul><li>We make ligations of:</li>
+<ul class="ul_2"><li>Etr8(CMV):BxbI in a1 + PhyB:P16 in a2, ?1</li>
+<li>LacIBD in pUPD2 + PIFBDless+promoter+terminator, a1</li>
+<li>KDrompa in pUPD2 + LacBD+promoter+terminator, a1</li>
+<li>Gal4BD, pUPD2</li>
+<li>Reporter of BxbI, pUPD2</li>
+</ul></ul>
+<ul><li>Tomorrow we have to take out pUPD of constitutive promoters, terminators and GFP (CDS).</li>
+</ul>
+</br><h3 style="color:green">23 June 2015</h3>
+<ul><li>Transformations in E.Coli of the 5 ligations done yesterday and two more transformations of 5+6(1) and 5+6(2) which are the ligations in pUPD of the 18/06. </li>
+<li>We put 50 µl of the transformations in the plates. Put in 37ºC.</li>
+<ul class="ul_2"><li>Etr8(CMV):Bxb1 in a1 + PhyB:P16 in a2, ?1</li>
+<li>LacIBD in pUPD2 + PIFBDless+promoter+terminator, a1</li>
+<li>KDrompa in pUPD2 + LacBD+promoter+terminator, a1</li>
+<li>Gal4BD, pUPD2</li>
+<li>Reporter of Bxb2, pUPD</li>
+<li>LexABD (5+6(1)), pUPD</li>
+</ul></ul>
+<ul><li>We have taken out of the -80ºC fridge the glycerinate of GFP, pUPD/ampicilina/GB0059.</li>
+<li>The liquid culture of Renilla (rifampicina/kanamicia/tetraciclina) doesn’t grow before the two days required. So we decide to put two new colonies in une tube with the three antibiotics and another with rifampicina and kanamicine. Asun say that the tetracycline slow down the growth of Agro.</li>
+<li>The 4 liquid cultures of LexA+IPTG/+gal are all blue: throw them.</li>
+<li>We ordered again the primer nº10 (NDronpa R1). Changing one codon in 3’ and delete another in 5’.</li>
+</ul>
+</br><h3 style="color:green">24 June 2015</h3>
+<p>Pick colonies of the plates done yesterday and pass them into a liquid media: 3 µl of LB, 3 µl of antibiotics (K, Spect, Clor).</p>
+<ul><li>LacIBD+PIF, a1 (C1, C2)</li>
+<li>Gal4BD, pUPD2 (C1)</li>
+<li>RepBxb1, pUPD2 (C1, C2, C3)</li>
+<li>LacIBD+KDonpa, a1 (C1, C2)</li>
+<li>Etr8(CMV)+Bxb1+phyB+VP16, ?1 (C1)</li>
+<li>LexABD1, pUPD (C1-C4)</li>
+<li>LexABD2, pUPD. No colonies.</li>
+</ul></ul>
+<p>The viral systems cultures of Agro for the color mosaics are ready before 2 days at 28ºC. We can make the Agroinfiltration.</p>
+<p>Buffers of Agroinfiltration:</p>
+<ul><li>First we have to prepare and ajust the pH of the buffer MES and the buffer MgCl.</li>
+<li>MES (10x), 100nM; ph=5,6 (adding NaOH). Make 1L.</li>
+<li>MgCl (100x), 1M. Make 100ml.</li>
+<li>Solution to agroinfiltration: 10ml of MES(10x) + 1ml of MgCl (100x) + 100 µl of DMSO+acetosiningona and finally “enrasar” to 100ml.</li>
+<li> 19.6mg of acetosiningona for 500 µl of DMSO</li>
+</ul></ul>
+<ul><li>Ligation:</li>
+</ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>ETR8(CMV):Bxb1(a1)+phyB+VP16(a2); ?1 </td><td>Gal4BD(pcr) + pUPD2</td></tr>
+<tr><td>1.5 µl etr8:Bxb1</td><td>1 µl Gal4 PCR</td></tr>
+<tr><td>1.5 µl 88E (phyB+VP16)</td><td>1 µl pUPD2</td></tr>
+<tr><td>1 µl ?1</td><td>1.2 buffer T4 ligase</td></tr>
+<tr><td>1.2 µl buffer T4 ligase</td><td>1 µl ligase</td></tr>
+<tr><td>1 µl ligase</td><td>1.2 µl BSA</td></tr>
+<tr><td>1.2 µl BSA</td><td>1 µl BsmbI</td></tr>
+<tr><td>1 µl BsmbI</td><td>5,6 µl H2O</td></tr>
+<tr><td>3.6</td><td>µl H2O</td><td></td></tr>
+</div></table>
+<p>Quantification of DNA:</p>
+<ul><li>pUPD GFF (0059): 249 ng/µl</li>
+<li>?2: 238 ng/µl</li>
+<li>Alfredo’s pUPD2, domesticator: 102 ng/µl</li>
+<li> iGEM704: 405 ng/µl</li>
+<li>IGEM735: 403 ng/µl</li>
+<li>552 AMP 35S noATG: 45 ng/µl</li>
+<li>PIF (C5), pUPD2: 119 ng/µl</li>
+<li>pD6B3, ?2 (22/06): 158 ng/µl</li>
+<li>LacIBD (C1), pUPD (22/06): 129 ng/µl</li>
+<li>109k renillaDC: 49 ng/µl</li>
+<li>IGEM 534: 13.6 ng/µl</li>
+<li>VP16 (C1), pUPD2:102 ng/µl</li>
+<li>IGEM 1097: 409 ng/µl</li>
+<li>K-donpa (C3), pUPD2 (18/06): 174 ng/µl</li>
+<li>IGEM 858: 487 ng/µl</li>
+<li>731AMP Gal4 (19/06): 81 ng/µl</li>
+<li>IGEM pUPD2 domesticator: 87 ng/µl</li>
+<li>PIF+phy8 (c1) (08/06): 108 ng/µl</li>
+<li>160 renilla, a2 (19/06): 46 ng/µl</li>
+<li>159 renilla, ?2 (19/06): 149 ng/µl</li>
+<li>Etr8:Bxb1 (C1)(22/06): 149 ng/µl</li>
+<li>IGEM 732: 422 ng/µl</li>
+</ul></ul>
+<p>Measurement of the OD:</p>
+<ul><li>first we have to </li>
+</ul></ul>
+</br><h3 style="color:green">25 June 2015</h3>
+<p>Minipreps of the liquid culture:</p>
+<ul><li>We don’t observed growth in LacIBD+PIF and LaciBD+K-donpa.</li>
+</ul></ul>
+<p>Digestion of the minipreps and do the gel:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Gal4BD, pUPD2</td><td>NotI</td><td>2046, 282</td></tr>
+<tr><td>RepBxb1, pUPD2</td><td>NotI</td><td>2046, 460</td></tr>
+<tr><td>Etr8(CMV):Bxb1 + phyB,a1</td><td>BamHI</td><td>6674, 2237, 2806, 1174</td></tr>
+<tr><td>LexABD, pUPD2</td><td>NotI</td><td>2046, 321</td></tr>
+<tr><td>9+10, pUPD2</td><td>NotI</td><td>464</td></tr>
+</div></table>
+<p>Gel:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Etr8:Bxb1</td><td>Lex1</td><td>Lex2</td><td>Lex3</td><td>Lex4</td><td>Rep1</td><td>Rep2</td><td>Rep3</td><td>Gal4 C1</td><td>PCR 9+10</td></tr>
+<tr><td>no</td><td>no</td><td>no</td><td>no</td><td>no</td><td>ok</td><td>ok</td><td>ok</td><td>no</td><td>ok</td></tr>
+</div></table>
+<ul><li>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 N-donpa (R1).</li>
+<li>Refresh the cultures of Agro (28ºC). We pick 7.5 µl of the previous culture. And put into a new liquid media with Rifampicine and Kanamicine for 2 days and 28ºC.</li>
+<li>Ligations:</li>
+</ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>N-dronpa</td><td>Rep GFP</td><td>Gal4</td><td>LexA</td></tr>
+<tr><td>1 µl PCR 9+10</td><td>1 µl Rep Bxb1</td><td>1 µl PCR 3+4</td><td>1 µl PCR 5+6</td></tr>
+<tr><td>1 µl PCR11+12</td><td>1 µl promoter without ATG</td><td>1 µl pUPD2</td><td>1 µl pUPD2</td></tr>
+<tr><td>1 µl pUPD2</td><td>1 µl Tnos</td><td></td></tr>
+<tr><td>1.2 µl buffer ligase</td><td>1.2 µl buffer ligase</td><td>1.2 µl buffer ligase</td><td>1.2 µl buffer ligase</td></tr>
+<tr><td>1.2 µl BSA</td><td>1.2 µl BSA</td><td>1.2 µl BSA</td><td>1.2 µl BSA</td></tr>
+<tr><td>1 µl BsmbI</td><td>1 µl BsaI</td><td>1 µl BsmbI</td><td>1 µl BsmbI</td></tr>
+<tr><td>1 µl T4 ligase</td><td>1 µl T4 ligase</td><td>1 µl T4 ligase</td><td>1 µl T4 ligase</td></tr>
+<tr><td>4,6 µl H2O</td><td>3,6 µl H2O</td><td>5,6 µl H2O</td><td>5,6 µl H2O</td></tr>
+<tr><td></td><td>1 µl a2</td><td></td></tr>
+<tr><td>Etr8:Bxb1+phyB</td><td></td></tr>
+<tr><td>1 µl Etr8:Bxb1</td><td></td></tr>
+<tr><td>1 µl 88E</td><td></td></tr>
+<tr><td>1µl ?1</td><td></td></tr>
+<tr><td>1.2 µl buffer ligase</td><td></td></tr>
+<tr><td>1.2 µl BSA</td><td></td></tr>
+<tr><td>1 µl BsmbI</td><td></td></tr>
+<tr><td>1 µl T4 ligase</td><td></td></tr>
+<tr><td>3,6 µl H2O</td><td></td></tr>
+</div></table>
+<p>We transform the ligations in E.Coli ant pass them into agar plates with cloranfenicol for all of them except the ligation of Etr8:Bxb1+phy that goes with bhnfnfg</p>
+</br><h3 style="color:green">26 June 2015</h3>
+<p>We do again the two ligations that didn’t gone?</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Rep GFP</td><td>LacI BD+PIF6</td></tr>
+<tr><td>1 µl Rep Bxb1</td><td>1 µl LACIBD, pUPD</td></tr>
+<tr><td>1 µl promoter without ATG</td><td>1 µl PIF6, pUPD</td></tr>
+<tr><td>1 µl Tnos</td><td>1 µl promoter</td></tr>
+<tr><td>1.2 µl buffer ligase</td><td>1 µl T35</td></tr>
+<tr><td>1.2 µl BSA</td><td>1 µl a1</td></tr>
+<tr><td>1 µl BsaI</td><td>1.2 µl buffer BsaI</td></tr>
+<tr><td>1 µl T4 ligase</td><td>1.2 µlBSA</td></tr>
+<tr><td>2.6 µl H2O</td><td>1 µl BsaI</td></tr>
+<tr><td>1 µl a2</td><td>1 µl ligase</td></tr>
+<tr><td>1µl GFP (0059)</td><td>2.6 µl H2O</td></tr>
+</div></table>
+<p>We make a gel of LAcI+PIF6 (C1 and C2). Both of them present the fragment of the vector at 6000 pb but none of them at 2000bp which is the insert one.</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LacIBD+PIF; a1</td><td>EcoRI</td><td>6345, 1997, 641</td></tr>
+</div></table>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LacIBD+PIF C1</td><td>LacIBD+PIF C2</td></tr>
+<tr><td>no</td><td>no</td></tr>
+</div></table>
+<p>Measurement of the ODs of phyB+PIF+luc and renilla+P19.</p>
+<ul><li>phyB+PIF+luc: o.35 (1:2)</li>
+<li>Ren+P19: 0.34 (1:2)</li>
+<li>Final volume= 2</li>
+<li>CCo= 0.35*2</li>
+<li>CCf= 0.2</li>
+<li>Ecuation=> Vo*CCo=Vf*CCf</li>
+<li>So we obtain that Vo(LacI+PIF6)=1.429 µl and Vo(ren)= 1.412 µl.</li>
+</ul></ul>
+<ul><li>Ligation of: </li>
+</ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LacIBD,pUPD + K-donpa, pUPD</td></tr>
+<tr><td>1 µl 35S</td></tr>
+<tr><td>1 µl LacIBD,pUPD2</td></tr>
+<tr><td>1 µl K-dronpa, pUPD</td></tr>
+<tr><td>1 µl T35S</td></tr>
+<tr><td>1 µl a1</td></tr>
+<tr><td>1.2 µl buffer T4 ligase</td></tr>
+<tr><td>1 µl BsaI</td></tr>
+<tr><td>1 µl T4 ligase</td></tr>
+<tr><td>2.6 µl H2O</td></tr>
+</div></table>
+<ul><li>We make the agorinfiltration of (…..) to start checking if the plant react to the different ..</li>
+</ul>
+</br><h3 style="color:green">27 June 2015</h3>
+<p>Transformation in E.coli of LacIBD+K-Dronpa, a1</p>
+<p>We Put into plates LexABD and Etr8(CMV):Bxb1:GFP again and also LAcIBD+K-Dronpa.</p>
+<p>We make liquid culture of:</p>
+<ul><li>RepBxb1:GFP (C1-C4)</li>
+<li>LacIBD+PIF (C1-C5)</li>
+<li>N-Dronpa (C1-C4)</li>
+<li>Gal4BD (C1-C5)</li>
+<li>LexA</li>
+</ul></ul>
+</br><h3 style="color:green">28 June 2015</h3>
+<p>We do the minipreps of the liquid cultures that have grown.</p>
+<ul><li>RepBxb1:GFP (C1 and C2)</li>
+<li>LacIBD+PIF (C1-C4)</li>
+<li>N-Dronpa (C1-C4)</li>
+<li>Gal4BD (C1-C5)</li>
+<li>LexA: didn’t grow</li>
+</ul></ul>
+<p>Do the digestions of the minipreps:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LacIBD+PIF, a1</td><td>EcoRI</td><td>6345, 1997, 641</td></tr>
+<tr><td>RepBxb1:GFP, ?2</td><td>HindIII</td><td>6345, 2683</td></tr>
+<tr><td>Gal4BD; pUPD2</td><td>NotI</td><td>2681, 644</td></tr>
+<tr><td>N-Dronpa; pUPD2</td><td>NotI</td><td>2046, 744</td></tr>
+</div></table>
+<p>Make the gel.</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>RepBxb1:GFP C1</td><td>RepBxb1:GFP C2</td><td>LacIBD+PIF C1</td><td>LacIBD+PIF C2</td><td>LacIBD+PIF C3</td><td>LacIBD+PIF C4</td></tr>
+<tr><td>no</td><td>no</td><td>no</td><td>no</td><td>no</td><td>No</td></tr>
+<tr><td>Gal4BD C1</td><td>Gal4BD C2</td><td>Gal4BD C3</td><td>Gal4BD C4</td><td>Gal4BD C5</td><td>N-Dronpa C1</td></tr>
+<tr><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td></tr>
+<tr><td>N-Dronpa C2</td><td>N-Dronpa C3</td><td>N-Dronpa C4</td><td></td><td></td></tr>
+<tr><td>no</td><td>ok</td><td>ok</td><td></td><td></td></tr>
+</div></table>
+<p>Take glycerinated:</p>
+<ul><li>GB0030: p35S</li>
+<li>GB0036: T35S</li>
+</ul></ul>
+<ul><li>Make liquid culture of LexABD (C1-C4).</li>
+<li>We transform again LacIBD+K-Dronpa and RepBxb1:GFP, adding to the agar plates 100 µl of each transformation. </li>
+</ul></ul>
+</br><h3 style="color:green">29 June 2015</h3>
+<p>Do the minipreps of the 4 colonies of LexABD and both glycerinates, 35S and T35S.</p>
+<p>Do the digestion of the 4 colonies of LexA and both glycerinates:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LexABD; pPPD2</td><td>NotI</td><td>2358, 312</td></tr>
+<tr><td>35S; pUPD2</td><td>NotI</td><td>2981, 1074</td></tr>
+<tr><td>T35S; pPUD2</td><td>NotI</td><td>2981, 304</td></tr>
+</div></table>
+<p>Make the gel:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LexA C1</td><td>LexA C2</td><td>LexA C3</td><td>LexA C4</td><td>P35S</td><td>T35S</td></tr>
+<tr><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>Ok?</td><td>Ok?</td></tr>
+</div></table>
+<p>Make ligations:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LacIBD+K-Dronpa+promoter+termi; a1</td><td>Gal4BD+K-Donpa+prom+ter; a1</td><td>LexABD+K-Dronpa+prom+term; a1</td></tr>
+<tr><td>1 µl LacI, pUPD2</td><td>1 µl Gal4, pUPD2</td><td>1 µl Gal4, pUPD2</td></tr>
+<tr><td>1 µl K-Dronpa, pUPD2</td><td>1 µl K-Dronpa, pUPD2</td><td>1 µl K-Dronpa, pUPD2</td></tr>
+<tr><td>1 µl 35S (GB0030)</td><td>1 µl 35S (GB0030)</td><td>1 µl 35S (GB0030)</td></tr>
+<tr><td>1 µl T35S (GB0036)</td><td>1 µl T35S (GB0036)</td><td>1 µl T35S (GB0036)</td></tr>
+<tr><td>1.2 µl  buffer ligase</td><td>1.2 µl  buffer ligase</td><td>1.2 µl  buffer ligase</td></tr>
+<tr><td>1.2 µl BSA 10x</td><td>1.2 µl BSA 10x</td><td>1.2 µl BSA 10x</td></tr>
+<tr><td>1 µl BsaI</td><td>1 µl BsaI</td><td>1 µl BsaI</td></tr>
+<tr><td>1 µl ligase T4</td><td>1 µl ligase T4</td><td>1 µl ligase T4</td></tr>
+<tr><td>2.6 µl H2O</td><td>2.6 µl H2O</td><td>2.6 µl H2O</td></tr>
+<tr><td>1 µl a1</td><td>1 µl a1</td><td>1 µl a1</td></tr>
+<tr><td>N-Dronpa+VP16; a2</td><td>Gal4BD+PIF6; a1</td><td>LacIBD+PIF6; a1</td></tr>
+<tr><td>1 µl N-Dronpa, pUPD2</td><td>1 µl Gal4BD, pUPD2</td><td>1 µl LacIBD, pUPD2</td></tr>
+<tr><td>1 µl VP16, pUPD2</td><td>1 µl PIF6, pUPD2</td><td>1 µl PIF6, pUPD2</td></tr>
+<tr><td>1 µl 35S (GB0030)</td><td>1 µl 35S (GB0030)</td><td>1 µl 35S (GB0030)</td></tr>
+<tr><td>1 µl T35S (GB0036)</td><td>1 µl T35S (GB0036)</td><td>1 µl T35S (GB0036)</td></tr>
+<tr><td>1.2 µl  buffer ligase</td><td>1.2 µl  buffer ligase</td><td>1.2 µl  buffer ligase</td></tr>
+<tr><td>1.2 µl BSA 10x</td><td>1.2 µl BSA 10x</td><td>1.2 µl BSA 10x</td></tr>
+<tr><td>1 µl BsaI</td><td>1 µl BsaI</td><td>1 µl BsaI</td></tr>
+<tr><td>1 µl ligase T4</td><td>1 µl ligase T4</td><td>1 µl ligase T4</td></tr>
+<tr><td>2.6 µl H2O</td><td>2.6 µl H2O</td><td>2.6 µl H2O</td></tr>
+<tr><td>1 µl a2</td><td>1 µl a1</td><td>1 µl a1</td></tr>
+<tr><td>LexABD+PIF6; a1</td></tr>
+<tr><td>1 µl LexABD, pUPD2</td></tr>
+<tr><td>1 µl PIF, pUPD2</td></tr>
+<tr><td>1 µl 35S (GB0030)</td></tr>
+<tr><td>1 µl T35S (GB0036)</td></tr>
+<tr><td>1.2 µl  buffer ligase</td></tr>
+<tr><td>1.2 µl BSA 10x</td></tr>
+<tr><td>1 µl BsaI</td></tr>
+<tr><td>1 µl ligase T4</td></tr>
+<tr><td>2.6 µl H2O</td></tr>
+<tr><td>1 µl a2</td></tr>
+</div></table>
+<ul><li>Transform all the ligations into E.Coli.Gal4BD+K-Dronpa and LacIBD+K-Dronpa goes wrong and we have to do it again. </li>
+<li>Put into a plate the colonies before let stay them 1h at 37ºC.</li>
+</ul>
+<p>Quantification of DNA:</p>
+<ul><li>RepBxb1:GFP (C1): 163.8 ng/µl</li>
+<li>N-Dronpa, pUPD2 (C4):113.1 ng/µl</li>
+<li>N-Dronpa (C3): 83.2 ng/µl</li>
+<li>NDonpa (C1): 116.6 ng/µl</li>
+<li>Gal4BD (C1): 95.2 ng/µl</li>
+<li>Gal4BD (C2): 120.7 ng/µl</li>
+<li>RepBxb1:GFP (C2): 170.6 ng/µl</li>
+<li>RepBxb1 (C1): 80.6 ng/µl</li>
+</ul></ul>
+</br><h3 style="color:green">30 June 2015</h3>
+<p>Transform Gal4+K-Dronpa and LacI+K-Dronpa</p>
+<p>Miniprep of:</p>
+<ul><li>RepBxb1+GFP (C1-C3)</li>
+</ul></ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>RepBxb1+GFP</td><td>HindIII</td><td>6345, 2683</td></tr>
+</div></table>
+<p>Gel:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>RepBxb1+GFP C1</td><td>RepBxb1+GFP C2</td><td>RepBxb1+GFP C3</td></tr>
+<tr><td>No</td><td>no</td><td>no</td></tr>
+</div></table>
+<p>We pick more colonies and make other liquid cultures.</p>
+<p>Save the last samples of the leaves of the plants that were under the first experiment, next to the glycerinates in the freezer (-80ºC). </p>
+<p>Put in plates the transformations of Gal4+K-Dronpa and LacI+K-Dronpa (50 µl of bacteria in SOC medium).</p>
+<p>Make the gel:</p>
+<ul><li>Add to the digestions 2 µl of loading buffer (6x) because for 5 µl of digestion we put 1 µl of the buffer.</li>
+</ul>
+<p>Make liquid culture of two colonies for each plates of the transformations done yesterday, 10 tubes in total.</p>
+<p>Take out a glycerinate 35S:Luciferase:Tnos (GB0227) and do a miniprep.</p>
+</br><h3 style="color:green">1 July 2015</h3>
+<p>Do the minipreps of the 10 liquid cultures.</p>
+<p>Do the digestions:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LexABD+K-Dronpa;a1</td><td>EcoRI</td><td>6345, 2296</td></tr>
+<tr><td>N-Donpa+VP16; a2</td><td>HindIII</td><td>6345, 2427</td></tr>
+<tr><td>Gal4BD+PIF6; a1</td><td>EcoRI</td><td>6345, 1867</td></tr>
+<tr><td>LacI+PIF; a1</td><td>EcoRI</td><td>6345, 2638</td></tr>
+<tr><td>LexABD+PIF6; a1</td><td>EcoRI</td><td>6345, 1906</td></tr>
+</div></table>
+<p>Do the gel:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Gal4+PIF C1</td><td>Gal4+PIF C2</td><td>LexA+PIF C1</td><td>LexA+PIF C2</td><td>LexA+KDronpa C1</td></tr>
+<tr><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>Ok</td></tr>
+<tr><td>LexA+Kdronpa C2</td><td>LacI+PIF C1</td><td>LacI+PIF C2</td><td>Ndonpa+VP16 C1</td><td>Ndronpa+VP16 C2</td></tr>
+<tr><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td></tr>
+</div></table>
+<ul><li>Prepare liquid culture of:</li>
+</ul>
+<ul><li>LexA+PIF; ?1 (C1)</li>
+<li>LacI+PIF; ?1 (C1)</li>
+<li>LexA+K-Dronpa (C1)</li>
+<li>Gal4+PIF; ?1 (C1)</li>
+<li>VP16, pUPD2 (C1)</li>
+<li>LexABD, pUPD2 (C2)</li>
+<li>PIF6, pUPD2 (C5)</li>
+<li>LacIBD, pUPD2 (C1)</li>
+</ul></ul>
+<ul><li>Pick colonies and make liquid culture of:</li>
+<ul class="ul_2"><li>Gal4+K-Dronpa (C1 and C2)</li>
+<li>LacI+K-Dronpa (C1 and C2)</li>
+<li>RepBxb1+GFP (C4-C6)</li>
+</ul><li>We sent to sequence:</li>
+<ul class="ul_2"><li>Code:</li>
+<li>210.08-249: pUPD2, KDronpa C3</li>
+<li>210.08-250: pUPD2, NDronpa C1</li>
+<li>210.08-251: pUPD2, NDronpa C3</li>
+<li>210.08-252: pUPD2, NDronpa C4</li>
+<li>The solution have: 10µl of miniprep + 5µl (dilution 1:3) of primers.</li>
+</ul></ul>
+<ul><li>Data of the luciferase essay with the sample plat RED at 24h (replica2): we obtain a value of 7.4E6.</li>
+</ul>
+</br><h3 style="color:green">2 July 2015</h3>
+<p>Minipreps of:</p>
+<ul><li>Gal4+K-Dronpa (C1 and C2)</li>
+<li>LacI+K-Dronpa (C1 and C2)</li>
+<li>RepBxb1+GFP (C4-C6)</li>
+</ul></ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Gal4+K-Dronpa</td><td>EcoRI</td><td>6345, 3028</td></tr>
+<tr><td>LacI+K-Dronpa</td><td>EcoRI</td><td>6345, 2257</td></tr>
+<tr><td>RepBxb1+GFP</td><td>HindIII</td><td>6300, 2400</td></tr>
+</div></table>
+<p>Do the gel:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LacI+K-Dronpa C1</td><td>LacI+K-Dronpa C2</td><td>Gal4+K-Dronpa C1</td><td>Gal4+K-Dronpa C2</td></tr>
+<tr><td>??</td><td></td><td></td></tr>
+<tr><td>RepBxb1+GFP C4</td><td>RepBxb1+GFP C5</td><td>RepBxb1+GFP C6</td><td></td></tr>
+<tr><td></td><td></td><td></td></tr>
+</div></table>
+<p>Luciferase essay: </p>
+<ul><li>During the whole experiment we lost three samples: T16/FarRed/1; T24/Red/2 and T0/FarRed/1</li>
+</ul>
+<p>Results:</p>
+<p>Things to keep in mind for the next experiment:</p>
+<ul><li>The luminimeter (machine to measure the luminescence) has to be ready before start adding the reactants to the samples because it needs 10min to be ready.</li>
+<li>Set the timer (10min) with the first sample of luciferase and add the reactant to the other samples as quick as possible. </li>
+<li>We have to let the renilla stay before putting it in the luminimeter the same time as the luciferase, in this case 15min because with the luciferase we didn’t manage well the time. Theoretically we have to wait 10min.</li>
+</ul></ul>
+<p>Make ligations:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LexA:Kdonpa+N-Dronpa; ?1</td><td>LacI:Kdronpa+N-Dronpa; ?1</td><td>Gal4:Kdronpa+N-Dronpa; ?1</td></tr>
+<tr><td>1 µl LexA:Kdronpa</td><td>1 µl LacI:Kdronpa</td><td>1 µl Gal4:Kdronpa</td></tr>
+<tr><td>1 µl N-Dronpa</td><td>1 µl N-Dronpa</td><td>1 µl N-Dronpa</td></tr>
+<tr><td>1 µl ?1</td><td>1 µl ?1</td><td>1 µl ?1</td></tr>
+<tr><td>1.2 µl buffer ligase</td><td>1.2 µl buffer ligase</td><td>1.2 µl buffer ligase</td></tr>
+<tr><td>1.2 µl BSA</td><td>1.2 µl BSA</td><td>1.2 µl BSA</td></tr>
+<tr><td>1 µl BsmbI</td><td>1 µl BsmbI</td><td>1 µl BsmbI</td></tr>
+<tr><td>1 µl BsaI</td><td>1 µl BsaI</td><td>1 µl BsaI</td></tr>
+<tr><td>4.6 µl H2O</td><td>4.6 µl H2O</td><td>4.6 µl H2O</td></tr>
+<tr><td>LexA:PIF+PhyB:VP16; ?1</td><td>LacI:PIF+PhyB:VP16; ?1</td><td>Gal4:PIF+PhyB:VP16; ?1</td></tr>
+<tr><td>1 µl LexA:PIF</td><td>1 µl LacI:PIF</td><td>1 µl Gal4:PIF</td></tr>
+<tr><td>1 µl PhyB:VP16 (88E)</td><td>1 µl PhyB:VP16</td><td>1 µl PhyB:VP16</td></tr>
+<tr><td>1 µl ?1</td><td>1 µl ?1</td><td>1 µl ?1</td></tr>
+<tr><td>1.2 µl buffer ligase</td><td>1.2 µl buffer ligase</td><td>1.2 µl buffer ligase</td></tr>
+<tr><td>1.2 µl BSA</td><td>1.2 µl BSA</td><td>1.2 µl BSA</td></tr>
+<tr><td>1 µl BsmbI</td><td>1 µl BsmbI</td><td>1 µl BsmbI</td></tr>
+<tr><td>1 µl BsaI</td><td>1 µl BsaI</td><td>1 µl BsaI</td></tr>
+<tr><td>4.6 µl H2O</td><td>4.6 µl H2O</td><td>4.6 µl H2O</td></tr>
+<tr><td>35S:Bxb1+RepBxb1:GFP; ?1</td><td>E-PIF+phyB+luc+ren; ?1</td></tr>
+<tr><td>1 µl 35s:Bxb1:T35S (alfredo’s)</td><td>0.5 µl 896 (PIF+phy+luc)</td></tr>
+<tr><td>1 µl PromsinATG:RepBxb1:GFP</td><td>1 µl 160 (renilla)</td></tr>
+<tr><td>1 µl ?1</td><td>1 µl ?1</td></tr>
+<tr><td>1.2 µl buffer ligase</td><td>1.2 µl buffer ligase</td></tr>
+<tr><td>1.2 µl BSA</td><td>1.2 µl BSA</td></tr>
+<tr><td>1 µl BsmbI</td><td>1 µl BsmbI</td></tr>
+<tr><td>1 µl BsaI</td><td>1 µl BsaI</td></tr>
+<tr><td>4.6 µl H2O</td><td>4.6 µl H2O</td></tr>
+</div></table>
+<p>The samples that we sent to sequence have arrived:</p>
+<ul><li>210.08-249: pUPD2, KDronpa C3------ok</li>
+<li>210.08-250: pUPD2, NDronpa C1------ok</li>
+<li>210.08-251: pUPD2, NDronpa C3------ok</li>
+<li>210.08-252: pUPD2, NDronpa C4------ok</li>
+</ul></ul>
+<p>The sequences of N-Dronpa have the desired mutation.</p>
+<ul><li>Transformation in E.Coli of the 8 ligations.</li>
+<li>Put the transformation into plates, put at 37ºC.</li>
+<li>Refresh the Agro’s cultures (Renilla and PIF+phy+luc):</li>
+<li>Add in 5ml of LB, 5 µl of rifampicine, 5 µl kanamicine, 5 µl of culture.</li>
+</ul>
+</br><h3 style="color:green">4 July 2015</h3>
+<p>Make the 2nd refresh of the culture of <i>Agrobacterium</i>. Put 5ml of LB, 5 µl of rifampicine, 5 µl kanamicine, 5 µl of culture.</p>
+<p>Make liquid cultures (4ml of LB and 4 µl of spectomicine) of the 8 colonies of E.Coli. </p>
+<ul><li>Red toggle (C1)</li>
+<li>Gal4:Kdronpa+N-Dronpa (C1 and C2)</li>
+<li>LexA:Kdonpa+N-Dronpa (C1 and C2)</li>
+<li>LacI:Kdronpa+N-Dronpa (C1 and C2)</li>
+<li>LexA:PIF+PhyB:VP16 (C1 and C2)</li>
+<li>35S:Bxb1+RepBxb1:GFP (C1 and C2)</li>
+<li>This colonies didn’t grow: LacI:PIF+PhyB:VP16; Gal4:PIF+PhyB:VP16 and E-PIF+phyB+luc+ren. Tomorrow we will repeat the ligations.</li>
+</ul></ul>
+</br><h3 style="color:green">5 July 2015</h3>
+<p>Ligations:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LacI:PIF+PhyB:VP16; ?1</td><td>Gal4:PIF+PhyB:VP16; ?1</td></tr>
+<tr><td>1 µl LacI:PIF</td><td>1 µl Gal4:PIF</td></tr>
+<tr><td>1 µl PhyB:VP16</td><td>1 µl PhyB:VP16</td></tr>
+<tr><td>1 µl ?1</td><td>1 µl ?1</td></tr>
+<tr><td>1.2 µl buffer ligase</td><td>1.2 µl buffer ligase</td></tr>
+<tr><td>1.2 µl BSA</td><td>1.2 µl BSA</td></tr>
+<tr><td>1 µl BsmbI</td><td>1 µl BsmbI</td></tr>
+<tr><td>1 µl T4 ligase</td><td>1 µl T4 ligase</td></tr>
+<tr><td>4.6</td><td>µl H2O</td><td>4.6 µl H2O</td></tr>
+</div></table>
+<p>Minipreps of the liquid cultures.</p>
+<p>Digestion of the minipreps.</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LacI:Kdronpa+N-Dronpa</td><td>BamHI</td><td>6674, 5437</td></tr>
+<tr><td>35S:Bxb1+RepBxb1:GFP</td><td>BamHI</td><td>6674, 3859, 1782</td></tr>
+<tr><td>Gal4:Kdronpa+N-Dronpa</td><td>BamHI</td><td>6674, 4666</td></tr>
+<tr><td>LexA:Kdonpa+N-Dronpa</td><td>BamHI</td><td>6674, 4705</td></tr>
+<tr><td>LexA:PIF+PhyB:VP16</td><td>BamHI</td><td>6674, 3513, 2337</td></tr>
+</div></table>
+<ul><li>Agarose gel (1%): </li>
+</ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LacKN C1</td><td>LacIKN C2</td><td>Bxb1RepGFP C1</td><td>Bxb1RepGFP C2</td><td>Gal4KN C1</td><td>Gal4KN C2</td></tr>
+<tr><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>ok</td></tr>
+<tr><td>LexA:KN C1</td><td>LexA:KN C2</td><td>LexAPIFPhy C1</td><td>LexAPIFPhy C2</td><td>Red toggle</td><td></td></tr>
+<tr><td>ok</td><td>ok</td><td>no</td><td>no</td><td>no</td><td></td></tr>
+</div></table>
+<p>We have to repeat the digestion of: LexA+PIF:phy+VP16.</p>
+<ul><li>Take out the glycerinate 88C (1098): Etr8:luc:Tnos. We will use it like a negative control in the second luciferase essat.</li>
+<li>Calculation of the ODs:</li>
+<ul class="ul_2"><li>Dilution of both samples 1:10.</li>
+<li>Renilla:=0.22---182 µl of sample + 1.818 ml MES</li>
+<li>PIF+PhyB+Luc=0.26--- 154 µl of sample + 1.646 ml MES</li>
+</ul></ul>
+<p>Agroinfiltration of the two samples with renilla and luciferase+PIF+phy in three plants with 4 spots in each leaf and 2 leaf in each plant.</p>
+<p>Let the plants 2 days in the darkness till agrobacterium infects the plant. They have to be in the dark because we are trying our red toggle ant it activates with light.</p>
+</br><h3 style="color:green">6 July 2015</h3>
+<p>Transform the negative control into agrobacterium.</p>
+<ul><li>Etr8:luc:Tnos</li>
+<li>Bxb1:reporterBxb1:GFP</li>
+</ul></ul>
+<p>We were doing dry lab preparing the power point to present our project to the rector and biotecs companies.</p>
+</br><h3 style="color:green">7 July 2015</h3>
+<p>Luciferase essay: Copy the protocol.</p>
+<ul><li>Add 150 µl of the lisis buffer and 800µl of MiliQ water, dilution 1:5.</li>
+<li>We centrifuge both cultures of agrobacterium at 2900rpm for 10min and remove the supernatant. </li>
+<li>We prepare the stock of MES (10ml of MES + 1ml MgCl + 100µl of “Acetosiningona” and level with H2O until 100ml.</li>
+<li>Resuspend the pellet of bacteria with 5ml of MES and let grow 2h.</li>
+</ul></ul>
+<ul><li>Renilla=0.29 (dilution 1:10): 2.9</li>
+<li>PhyB+PIF+luc=0.69 (dilution 1:4):2.76</li>
+<li>Solution to agroinflitrate:</li>
+<li>Renilla: 0.138ml of sample + 1.862ml of MES</li>
+<li>PhyB+PIF+luc: 0.145ml of sample + 1.855ml of MES</li>
+<li>We make the infiltration of both samples mix together in 3 different plants, 2 leaf per plant and 4 spots per leaf. Explicacion del experiment (tiempo en oscuridad… luces…)</li>
+</ul></ul>
+<p>Digestions:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Red toggle</td><td>Enzyme?</td><td></td></tr>
+<tr><td>LexA+PIF+phy</td><td>BamHI</td><td>3518, 5855, 6674</td></tr>
+</div></table>
+<p>Gel:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Red toggle</td><td>LexA+PIF+phy C1</td><td>LexA+PIF+phy C2</td></tr>
+<tr><td>No</td><td>Ok</td><td>no</td></tr>
+</div></table>
+<p>It has arrived a new construction: AsLOVpep.</p>
+<ul><li>Suspended with 50µl of H2O.</li>
+</ul></ul>
+<p>Ligations:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>AsLOVpep; pUPD2</td><td>Red Toggle</td><td>LacI:Kdronpa:Ndronpa:VP16+</td></tr>
+<tr><td>35S:renilla:Tnos-35S:P19:Tnos(GB159)</td><td>Gal4:Kdronpa:Ndronpa:VP16+GB159</td></tr>
+<tr><td>1 µl AsLOVpep</td><td>1 µl GB846</td><td>1 µl LacI:KNdronpa:VP16</td><td>1 µl Gal4:KNdronpa</td></tr>
+<tr><td>1 µl pUPD2</td><td>1 µl GB160</td><td>1 µl GB159</td><td>1 µl GB159</td></tr>
+<tr><td>1.2 µl buffer</td><td>1 µl ?1</td><td>1 µl a1</td><td>1 µl a1</td></tr>
+<tr><td>1.2 µl BSA</td><td>1.2 µl buffer</td><td>1.2 µl buffer</td><td>1.2 µl buffer</td></tr>
+<tr><td>1 µl BsmbI</td><td>1.2 µl BSA</td><td>1.2 µl BSA</td><td>1.2 µl BSA</td></tr>
+<tr><td>1 µl T4 ligase</td><td>1 µl BsmbI</td><td>1 µl BsmbI</td><td>1 µl BsmbI</td></tr>
+<tr><td>5.6 µl H2O</td><td>1 µl T4 ligase</td><td>1 µl T4 ligase</td><td>1 µl T4 ligase</td></tr>
+<tr><td></td><td>4.6 µl H2O</td><td>4.6 µl H2O</td><td>4.6 µl H2O</td></tr>
+<tr><td>LexA:Kdronpa:Ndronpa:VP16+GB159</td><td>LexA:PIF:Phy:VP16+ GB159</td></tr>
+<tr><td>1 µl LexA:Kdronpa:Ndronpa:VP16</td><td>1 µl LexA:PIF:Phy:VP16</td></tr>
+<tr><td>1 µl GB159</td><td>1 µl GB159</td></tr>
+<tr><td>1 µl a1</td><td>1 µl a1</td></tr>
+<tr><td>1.2 µl buffer</td><td>1.2 µl buffer</td></tr>
+<tr><td>1.2 µl BSA</td><td>1.2 µl BSA</td></tr>
+<tr><td>1 µl BsmbI</td><td>1 µl BsmbI</td></tr>
+<tr><td>1 µl T4 ligase</td><td>1 µl T4 ligase</td></tr>
+<tr><td>4.6 µl H2O</td><td>4.6 µl H2O</td></tr>
+</div></table>
+</br><h3 style="color:green">8 July 2015</h3>
+<ul><li>Experiment to study the piece PhyB+PIF.</li>
+</ul>
+<p>	The plants in red are exposed at the light intensity of the leds (we can’t regulate it) and the plants in far red are 100% with far red light and 0% of white light.</p>
+<p>	How the machines work: (hace falta?)</p>
+<p>	The machine with red light has the switch …</p>
+<p>	The 1st sample its at 8:00am and we spend 1h till the machines were working correctly because we didn’t know exactly how they work.</p>
+<p>	Then, before 12h (21:00), we take the 2nd samples; obtainin 3 discs of each condition (red an far red). </p>
+<p>We transform:</p>
+<ul><li>AsLOVpep; pUPD2 in DHSa an let incubate 1h at 37ºC.</li>
+<li>The last ligations in E.coli. Put in plates. Red toggle with Spectomicine+IPTG+XGal ann the rest (a1) with kanamicine+IPTG+XGal.</li>
+</ul></ul>
+</br><h3 style="color:green">9 July 2015</h3>
+<p>Pick colonies:</p>
+<ul><li>AsLOVpep colonies didn’t grow. Repeat.</li>
+<li>Red toggle colonies are all blue. Repeat.</li>
+<li>The other 4 colonies had grown. we pick them and male liquid culture.</li>
+<li>LacI:Kdronpa:Ndronpa:VP16+renilla (C1-C3)</li>
+<li>Gal4:Kdronpa:Ndronpa:VP16+renilla (C1-C3)</li>
+<li>LexA:Kdronpa:Ndronpa:VP16+GB159 (C1 and C2)</li>
+<li>LexA:PIF:Phy:VP16+ GB159 (C1 and C2)</li>
+</ul></ul>
+<p>Repeat the ligations.</p>
+<ul><li>AsLOVpep, as before.</li>
+</ul></ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Red toggle (PIF+PhyB+luc+ren)</td></tr>
+<tr><td>0.5 µl PIF+phy+luc (896)</td></tr>
+<tr><td>1 µl renilla (160)</td></tr>
+<tr><td>1 µl ?1</td></tr>
+<tr><td>1.2 µl buffer</td></tr>
+<tr><td>1.2 µl BSA</td></tr>
+<tr><td>1 µl BsmbI</td></tr>
+<tr><td>1 µl T4 ligase</td></tr>
+<tr><td>5.1 µl H2O</td></tr>
+</div></table>
+<p>We do the luciferase essay:</p>
+<ul><li>We didn’t obtain goo results, we can’t observed a significative difference between the on(red samples) and off (far red samples). It seems that the red toggle it has been activated. Graphics and tables</li>
+</ul></ul>
+<p>Transform the ligations of AsLOVpepe and red toggle. </p>
+<ul><li>Add SOC medium and let incubate 1h at 37ºC. Then we make an spin to the red toggle cells to concentrate them and see if we can obtain a colonies in the plates.</li>
+</ul></ul>
+</br><h3 style="color:green">10 July 2015</h3>
+<p>Minipreps of:</p>
+<ul><li>LexABD:PIF:phyB:VP16+Renilla; a1 (C1,C2)</li>
+<li>LexA:Kdronpa:Ndronpa+renilla; a1C1, C2)</li>
+<li>Gal4:Kdronpa:Ndronpa+renilla; a1 (C1-C3)</li>
+<li>LacI:Kdronpa:Ndronpa+renilla; a1 (C1,C2)</li>
+</ul></ul>
+<p>Digestions of:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LexABD:PIF:phyB:VP16+Renilla; a1 (C1,C2)</td><td>EcoRI</td><td>6345, 5487, 4891</td></tr>
+<tr><td>LexA:Kdronpa:Ndronpa+renilla; a1C1, C2)</td><td>EcoRI</td><td>9333, 6345</td></tr>
+<tr><td>Gal4:Kdronpa:Ndronpa+renilla; a1 (C1-C3)</td><td>EcoRI</td><td>6345, 9194</td></tr>
+<tr><td>LacI:Kdronpa:Ndronpa+renilla; a1 (C1,C2)</td><td>EcoRI</td><td>6345, 9965</td></tr>
+<tr><td>LacIBD+PIF+phyB; &Omega;1 (C1)</td><td>BamHI</td><td>6674, 4245, 2337</td></tr>
+<tr><td>Gal4BD+PIF+phyB; &Omega; 1 (C1)</td><td>BamHI</td><td>6674, 3474, 2337</td></tr>
+</div></table>
+<p>Make the gel:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LacIBD+PIF+phyB</td><td>Gal4BD+PIF+phyB</td><td>LexA:PIF:phyB:VP16+Renilla C1</td><td>LexA:PIF:phyB:VP16+Renilla C2</td></tr>
+<tr><td>Ok</td><td>Ok</td><td>ok</td><td>ok</td></tr>
+<tr><td>LexA:KNdronpa+renilla C1</td><td>LexA:KNdronpa+renilla C2</td><td>Gal4:KNdronpa+renilla C1</td><td>Gal4:KNdronpa+renilla C2</td></tr>
+<tr><td>Ok</td><td>Ok</td><td>ok</td><td>Ok</td></tr>
+<tr><td>Gal4:KNdronpa+renilla C3</td><td>LacI:Kdronpa:Ndronpa+renilla C1</td><td>LacI:Kdronpa:Ndronpa+renilla C2</td><td></td></tr>
+<tr><td>Ok</td><td>Ok</td><td>ok</td><td></td></tr>
+</div></table>
+<p>We received two constructions:</p>
+<ul><li>CDS: phiC31. Resuspended with 100µl.</li>
+<li>Reporter phi31. Resuspended with 50 µl</li>
+</ul></ul>
+<p>Pick colonies and make liquid culture of:</p>
+<ul><li>AsLOVpep; pUPD2 (C1 and C2)</li>
+<li>Red toggle (C1 and C2). In both liquid cultures we ad YPTG and Xgal to make sure that the colonies are correct, if not the medium will change to blue color.</li>
+</ul></ul>
+<p>Ligations:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>phyC31;pUPD2</td><td>Reporter phiC31; pUPD2</td><td>LacI:PIF:Phy:VP16+ren; a1</td></tr>
+<tr><td>1 µl phiC31</td><td>1 µl rep phiC31</td><td>1 µl LacI:PIF:Phy:VP16</td></tr>
+<tr><td>1 pUPD2</td><td>1 pUPD2</td><td>1 µl renilla (159)</td></tr>
+<tr><td>1.2 µl buffer</td><td>1.2 µl buffer</td><td>1.2 µl buffer</td></tr>
+<tr><td>1.2 µl BSA</td><td>1.2 µl BSA</td><td>1.2 µl BSA</td></tr>
+<tr><td>1 µl T4 ligase</td><td>1 µl T4 ligase</td><td>1 µl T4 ligase</td></tr>
+<tr><td>1 µl BsmbI</td><td>1 µl BsmbI</td><td>1 µl BSAI</td></tr>
+<tr><td>5.6 µl H2O</td><td>5.6 µl H2O</td><td>4.6 µl H2O</td></tr>
+<tr><td></td><td>1 µl a1</td></tr>
+<tr><td>Gal4:PIF:phy:VP16+ren; a1</td><td>LexA:PIF:phy:ren+opLex:luc; ?1</td><td>LexA:KNdronpa:ren+OpLex:Luc; ?1</td></tr>
+<tr><td>1 µl Gal4:PIF:phy:VP16</td><td>1 µl LexA:PIF:phy:ren</td><td>1 µl LexA:KNdronpa:ren</td></tr>
+<tr><td>1 µl renilla (159)</td><td>1 µl opLex:luc (151)</td><td>1 µl OpLex:Luc (151)</td></tr>
+<tr><td>1.2 µl buffer</td><td>1.2 µl buffer</td><td>1.2 µl buffer</td></tr>
+<tr><td>1.2 µl BSA </td><td>1.2 µl BSA</td><td>1.2 µl BSA</td></tr>
+<tr><td>1 µl T4 ligase</td><td>1 µl T4 ligase</td><td>1 µl T4 ligase</td></tr>
+<tr><td>1 µl BSAI</td><td>1 µl BsmbI</td><td>1 µl BsmbI</td></tr>
+<tr><td>4.6 µl H2O</td><td>4.6 µl H2O</td><td>4.6 µl H2O</td></tr>
+<tr><td>1 µl a1</td><td>1 µl ?1</td><td>1 µl ?1</td></tr>
+<tr><td>Gal4:KNdronpa:ren+UAS:luc; ?1</td><td>LacI:KNdronpa:ren+OpLacI:luc; ?1</td></tr>
+<tr><td>1 µl Gal4:KNdronpa:ren</td><td>1 µl LacI:KNdronpa:ren</td></tr>
+<tr><td>1 µl UAS:luc (227)</td><td>1 µl OpLacI:luc (152)</td></tr>
+<tr><td>1.2 µl buffer</td><td>1.2 µl buffer</td></tr>
+<tr><td>1.2 µl BSA </td><td>1.2 µl BSA</td></tr>
+<tr><td>1 µl T4 ligase</td><td>1 µl T4 ligase</td></tr>
+<tr><td>1 µl BsmbI</td><td>1 µl BsmbI</td></tr>
+<tr><td>4.6 µl H2O</td><td>4.6 µl H2O</td></tr>
+<tr><td>1 µl ?1</td><td>1 µl ?1</td></tr>
+</div></table>
+</br><h3 style="color:green">11 July 2015</h3>
+<p>Prepare glycerinates of:</p>
+<ul><li>Bxb1+Rep:GFP; ?1</li>
+<li>N-Dronpa; pUPD2</li>
+<li>Bxb1:Etr8; pUPD2</li>
+<li>Etr8(CMV):Bxb1:T35S; a1</li>
+</ul></ul>
+<p>Pick up the liquid cultures of AsLOVpep and red toggle. We observed that one tube of a red toggle colony is blue, we discard it.</p>
+<p>Do miniprep of:</p>
+<ul><li>AsLOVpep (C1 and C2)</li>
+<li>Red toggle (C2)</li>
+</ul></ul>
+<p>Digestions:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Red toggle</td><td>BamHI</td><td>6674, 6100 / 4209, 3756</td></tr>
+<tr><td>AsLOVpep</td><td>NotI</td><td>2558, 512</td></tr>
+</div></table>
+<p>Me falta el resultado del gel!!</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>AsLOVpep C1</td><td>AsLOVpep C2</td><td>Red toggle C2</td></tr>
+<tr><td>¿?</td><td></td></tr>
+</div></table>
+<p>Do transformation of DHSa and yesterday ligations:</p>
+<ul><li>phyC31;pUPD2</li>
+<li>Reporter phiC31; pUPD2</li>
+<li>LacI:PIF:Phy:VP16+ren; a1</li>
+<li>Gal4:PIF:phy:VP16+ren; a1</li>
+<li>LexA:PIF:phy:ren+opLex:luc; ?1</li>
+<li>LexA:KNdronpa:ren+OpLex:Luc; ?1</li>
+<li>Gal4:KNdronpa:ren+UAS:luc; ?1</li>
+<li>LacI:KNdronpa:ren+OpLacI:luc; ?1</li>
+<li>The pUPD2 in plates with cloranfenicol+IPTG+XGal. The a1 in plates with kanamicyn+IPTG+XGal. The ?1 in plates with spectinmicyn+IPTG+XGal.</li>
+</ul></ul>
+</br><h3 style="color:green">12 July 2015</h3>
+<p>Pick colonies and make liquid culture:</p>
+<ul><li>phyC31;pUPD2 (C1-C3)</li>
+<li>Reporter phiC31; pUPD2 (C1-C3)</li>
+<li>LacI:PIF:Phy:VP16+ren; a1 (C1-C3)</li>
+<li>Gal4:PIF:phy:VP16+ren; a1  All blue colonies.</li>
+<li>LexA:PIF:phy:ren+opLex:luc; ?1 (C1)</li>
+<li>LexA:KNdronpa:ren+OpLex:Luc; ?1(C1-C3)</li>
+<li>Gal4:KNdronpa:ren+UAS:luc; ?1 (C1)</li>
+<li>LacI:KNdronpa:ren+OpLacI:luc; ?1 All blue colonies.</li>
+<li>We have to repeat the transformations or do again ligations.</li>
+</ul></ul>
+<p>Ligations were repeated:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Gal4:PIF:phy:VP16+ren; a1</td><td>LacI:KNdronpa:ren+OpLacI:luc; ?1</td></tr>
+<tr><td>1 µl Gal4:PIF:phy:VP16</td><td>1 µl LacI:KNdronpa:ren</td></tr>
+<tr><td>1 µl renilla (159)</td><td>1 µl OpLacI:luc (152)</td></tr>
+<tr><td>1.2 µl buffer</td><td>1.2 µl buffer</td></tr>
+<tr><td>1.2 µl BSA </td><td>1.2 µl BSA</td></tr>
+<tr><td>1 µl T4 ligase</td><td>1 µl T4 ligase</td></tr>
+<tr><td>1 µl BSAI</td><td>1 µl BsmbI</td></tr>
+<tr><td>4.6 µl H2O</td><td>4.6 µl H2O</td></tr>
+<tr><td>1 µl a1</td><td>1 µl ?1</td></tr>
+</div></table>
+<p>Refresh the liquid cultures of <i>Agrobacterium</i>:</p>
+<ul><li>Bxb1:GFP and Etr8:tnos. They were 2 days at 28ºC.</li>
+<li>Pnos, it was at the fridge (-4ºC).</li>
+</ul></ul>
+</br><h3 style="color:green">13 July 2015</h3>
+<p>All the liquid cultures have grown, do minipreps.</p>
+<p>Do digestions:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>phyC31;pUPD2 (C1-C3)</td><td>NotI</td><td>2046, 1899</td></tr>
+<tr><td>Reporter phiC31; pUPD2 (C1-C3)</td><td>NotI</td><td>2046, 475</td></tr>
+<tr><td>LacI:PIF:Phy:VP16+ren; a1 (C1-C3)</td></tr>
+<tr><td></td><td>EcoRI</td><td>6345, 5623, 5487</td></tr>
+<tr><td>LexA:PIF:phy:ren+opLex:luc; ?1 (C1)</td></tr>
+<tr><td></td><td>BamHI</td><td>9431, 6674, 3531</td></tr>
+<tr><td>LexA:KNdronpa:ren+OpLex:Luc; ?1(C1-C3)</td></tr>
+<tr><td></td><td>BamHI</td><td>1199, 6674</td></tr>
+<tr><td>Gal4:KNdronpa:ren+UAS:luc; ?1 (C1)</td></tr>
+<tr><td></td><td>BamHI</td><td>11582, 6674</td></tr>
+</div></table>
+<p>Agarose gel:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>phyC31 C1</td><td>phyC31 C2</td><td>phyC31 C3</td><td>RepPhiC31 C1</td></tr>
+<tr><td>no</td><td>no</td><td>no</td><td>ok</td></tr>
+<tr><td>RepPhiC31 C2 </td><td>LacI:PIF:Phy:VP16+ren C1</td><td>LacI:PIF:Phy:VP16+ren C2</td><td>LacI:PIF:Phy:VP16+ren C3</td></tr>
+<tr><td>no</td><td>no</td><td>ok</td><td>ok</td></tr>
+<tr><td>LexA:PIF:phy:ren+opLex:luc</td><td>LexA:KNdronpa:ren+OpLex:Luc C1</td><td>LexA:KNdronpa:ren+OpLex:Luc C2</td><td>LexA:KNdronpa:ren+OpLex:Luc C3</td></tr>
+<tr><td>no</td><td>ok</td><td>ok</td><td>ok</td></tr>
+<tr><td>Gal4:KNdronpa:ren+UAS:luc C1</td><td></td><td></td></tr>
+<tr><td>no</td><td></td><td></td></tr>
+</div></table>
+<p>The <i>Agrobacterium</i> cultures refreshed yesterday were store in the fridge.</p>
+<p>It is made another culture of 35S:Bxb1+reporterBxb1:GFP to keep it in the fridge. It had 5ml of LB medium, 5 µl rifampicin and 5 µl of spectinomicyn an 1 µl of the culture. </p>
+<p>Mesurement of the OD’s:</p>
+<ul><li>35S:Bxb1+reporterBxb1:GFP: 0.28 (dilution 1:10)</li>
+<li>143 µl of culture+1857 µl of MES/acetosiningon solution.</li>
+<li>With this preparation 2 plants were infiltrated and let in natural light to see the normal activity of the recombinase.</li>
+</ul></ul>
+<p>Transformation of the ligation: Gal4:PIF:phy:VP16+ren; a1 and LacI:KNdronpa:ren+OpLacI:luc; ?1.</p>
+<p>The liquid cultures of this constructions were repeated:</p>
+<ul><li>phyC31;pUPD2 (C4 and C5)</li>
+<li>Reporter phiC31; pUPD2 (C4)</li>
+<li>LacI:PIF:Phy:VP16+ren; a1 (C4)</li>
+<li>LexA:PIF:phy:ren+opLex:luc; ?1 (C1)</li>
+<li>LexA:KNdronpa:ren+OpLex:Luc; ?1(C1-C3)</li>
+<li>Gal4:KNdronpa:ren+UAS:luc; ?1 (C1)</li>
+<li>AsLOVpep; pUPD2 (C3)</li>
+</ul></ul>
+</br><h3 style="color:green">14 July 2015</h3>
+<p>Do minipreps of:</p>
+<ul><li>phyC31;pUPD2 (C4 and C5)</li>
+<li>Reporter phiC31; pUPD2 (C4)</li>
+<li>LacI:PIF:Phy:VP16+ren; a1 (C4)</li>
+<li>LexA:PIF:phy:ren+opLex:luc; ?1 (C1)</li>
+<li>LexA:KNdronpa:ren+OpLex:Luc; ?1(C1-C3)</li>
+<li>Gal4:KNdronpa:ren+UAS:luc; ?1 (C1)</li>
+<li>AsLOVpep; pUPD2 (C3)</li>
+</ul></ul>
+<p>Do digestions:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>phyC31;pUPD2</td><td>NotI</td><td>2046, 1899</td></tr>
+<tr><td>Reporter phiC31; pUPD2</td><td>NotI</td><td>2046, 475</td></tr>
+<tr><td>LacI:PIF:Phy:VP16+ren; a1</td><td>EcoRI</td><td>6345, 5623, 5487</td></tr>
+<tr><td>LexA:PIF:phy:ren+opLex:luc, ?1</td><td>BamHI</td><td>9431, 6674, 3531</td></tr>
+<tr><td>LexA:KNdronpa:ren+OpLex:Luc; ?1</td><td>BamHI</td><td>1199, 6674</td></tr>
+<tr><td>Gal4:KNdronpa:ren+UAS:luc; ?1</td><td>BamHI</td><td>11582, 6674</td></tr>
+<tr><td>AsLOVpep; pUPD2 </td><td>NotI</td><td>2558, 512</td></tr>
+</div></table>
+<p>Make an agarose gel:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>phyC31 (C4)</td><td>phyC31 (C5)</td><td>AsLOVpep (C4)</td><td>LexA:PIF:phy:ren+opLex:luc (C1)</td></tr>
+<tr><td>no</td><td>no</td><td>No</td><td>No</td></tr>
+<tr><td>LexA:KNdronpa:ren+OpLex:Luc (C3)</td><td>Gal4:KNdronpa:ren+UAS:luc (C1)</td><td>Gal4:KNdronpa:ren+UAS:luc(C2)</td><td>Gal4:KNdronpa:ren+UAS:luc (C3)</td></tr>
+<tr><td>Ok</td><td>no</td><td>no</td><td>No</td></tr>
+<tr><td>LacI:PIF:Phy:VP16+ren</td><td>Reporter phiC31 (C1)</td><td></td></tr>
+<tr><td>no</td><td>Ok</td><td></td></tr>
+</div></table>
+<p>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.</p>
+<ul><li>Measurement of DNA concentration:</li>
+<ul class="ul_2"><li>Reporter:phyC31: 13.6 ng/µl</li>
+<li>PhyC31: 4.6 ng/µl </li>
+<li>AsLOVpep: 30.3 ng/µl</li>
+<li>Igem151 (op:LexAluc): 40 ng/µl</li>
+<li>Gal4:PIF:phyB:ren (C1): 124.4 ng/µl</li>
+<li>LacI:PIF:phyB:VP16 (C1): 126 ng/µl</li>
+<li>Igem 159 (renilla): 42 ng/µl</li>
+<li>Gal4:Kdronpa:Ndronpa:renilla (C3): 172.8 ng/µl</li>
+<li>Igem 227 (op:UAS:luc): 6.3 ng/µl</li>
+</ul></ul>
+<ul><li>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.</li>
+</ul>
+</br><h3 style="color:green">15 July 2015</h3>
+<ul><li>Miniprep of LacI:KDronpa:NDronpa:ren:luc (C4 and C5). </li>
+<li>Digestion:</li>
+</ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LacI:KDronpa:NDronpa:ren:luc</td><td>EcoRI</td><td>6345, 9965</td></tr>
+</div></table>
+<ul><li>Make the gel:</li>
+</ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LacI:K:NDronpa:ren:luc C4</td><td>LacI:K:NDronpa:ren:luc C5</td></tr>
+<tr><td>no</td><td>no</td></tr>
+</div></table>
+<ul><li>Measurement of DNA concentrations:</li>
+<ul class="ul_2"><li>Gal4BD:PIF:PhyB:VP16: 125.6 ng/µl</li>
+<li>LexA:PIF:phyB:VP16:ren: 322.6 ng/µl</li>
+<li>LacI:Kdronpa:NDronpa:ran: 209.0 ng/µl</li>
+</ul></ul>
+<ul><li>We decided to repeat the ligations due to that we make twice the digestions and we didn’t obtain good results. </li>
+</ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>phyC31;pUPD2</td><td>AsLOVpep; pUPD2</td><td>LacI:PIF:Phy:VP16+ren; a1</td></tr>
+<tr><td>1 µl phiC31</td><td>1 µl AsLOVpep</td><td>1.5 µl LacI:PIF:Phy:VP16</td></tr>
+<tr><td>1 pUPD2</td><td>1 pUPD2</td><td>2.5 µl renilla (159)</td></tr>
+<tr><td>1.2 µl buffer</td><td>1.2 µl buffer</td><td>1.2 µl buffer</td></tr>
+<tr><td>1.2 µl BSA</td><td>1.2 µl BSA</td><td>1.2 µl BSA</td></tr>
+<tr><td>1 µl T4 ligase</td><td>1 µl T4 ligase</td><td>1 µl T4 ligase</td></tr>
+<tr><td>1 µl BsmbI</td><td>1 µl BsmbI</td><td>1 µl BSAI</td></tr>
+<tr><td>5.6 µl H2O</td><td>5.6 µl H2O</td><td>4.6 µl H2O</td></tr>
+<tr><td></td><td>0.5 µl a1</td></tr>
+<tr><td>Gal4:PIF:phy:VP16+ren; a1</td><td>LexA:PIF:phy:ren+opLex:luc; ?1</td><td>LacI:KNdronpa:ren+OpLex:Luc; ?1</td></tr>
+<tr><td>1.5 µl Gal4:PIF:phy:VP16</td><td>1 µl LexA:PIF:phy:ren</td><td>1 µl LacI:KNdronpa:ren</td></tr>
+<tr><td>2 µl renilla (159)</td><td>2.5 µl opLex:luc (151)</td><td>3 µl OpLac:Luc (152)</td></tr>
+<tr><td>1.2 µl buffer</td><td>1.2 µl buffer</td><td>1.2 µl buffer</td></tr>
+<tr><td>1.2 µl BSA </td><td>1.2 µl BSA</td><td>1.2 µl BSA</td></tr>
+<tr><td>1 µl T4 ligase</td><td>1 µl T4 ligase</td><td>1 µl T4 ligase</td></tr>
+<tr><td>1 µl BSAI</td><td>1 µl BsmbI</td><td>1 µl BsmbI</td></tr>
+<tr><td>2.6 µl H2O</td><td>2.6 µl H2O</td><td>3 µl H2O</td></tr>
+<tr><td>1 µl a1</td><td>0.5 µl ?1</td><td>0.5 µl ?1</td></tr>
+<tr><td>Gal4:KNdronpa:ren+OpLex:Luc; ?1</td><td>PhyB:VP16+PIF6; ?1</td></tr>
+<tr><td>1 µl Gal4:KNdronpa:ren</td><td>1 µl PhyB:VP16 (88E)</td></tr>
+<tr><td>4 µl OpUAS:Luc (227)</td><td>2 µl PIF6 (170)</td></tr>
+<tr><td>1.2 µl buffer</td><td>1.2 µl buffer</td></tr>
+<tr><td>1.2 µl BSA</td><td>1.2 µl BSA</td></tr>
+<tr><td>1 µl T4 ligase</td><td>1 µl T4 ligase</td></tr>
+<tr><td>1 µl BsmbI</td><td>1 µl BsmbI</td></tr>
+<tr><td>3 µl H2O</td><td>3.6 µl H2O</td></tr>
+<tr><td>0.5 µl ?1</td><td>1 µl ?1</td></tr>
+</div></table>
+<ul><li>These <i>Agrobacterium</i> cultures have been refreshed:</li>
+<ul class="ul_2"><li>PIF-phyB-luc</li>
+<li>Renilla</li>
+<li>Pnos</li>
+<li>Etr8</li>
+</ul></ul>
+</br><h3 style="color:green">16 July 2015</h3>
+<ul><li>Yesterday ligations have been transformed into <i>E. coli</i>:</li>
+<ul class="ul_2"><li>phyC31;pUPD2</li>
+<li>AsLOVpep; pUPD2</li>
+<li>LacI:PIF:Phy:VP16+ren; a1</li>
+<li>Gal4:PIF:phy:VP16+ren; a1</li>
+<li>LexA:PIF:phy:ren+opLex:luc; ?1</li>
+<li>LacI:KNdronpa:ren+OpLex:Luc; ?1</li>
+<li>Gal4:KNdronpa:ren+OpLex:Luc; ?1</li>
+<li>PhyB:VP16+PIF6; ?1</li>
+</ul></ul>
+<ul><li>The agroinfiltrated leaf with BxbI:rep:GFP has been observed in the magnifying glass with fluorescent lights. The efficiency of the recombinase is lower and it can not been observed a lot of green spots. Tomorrow another leaf will be seen to check again the construction.</li>
+</ul>
+<ul><li>Second refresh of the <i>Agrobacterium</i> cultures:</li>
+<ul class="ul_2"><li>PIF-phyB-luc</li>
+<li>Renilla</li>
+<li>Pnos</li>
+<li>Etr8</li>
+</ul></ul>
+<ul><li>Miniprep of these cultures.</li>
+<li>Digestion of the minipreps:</li>
+</ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>PIF-phyB-luc; ?1</td><td>EcoRI</td><td>??</td></tr>
+<tr><td>Renilla; ?2</td><td>HindIII</td><td>No lo encuentro</td></tr>
+<tr><td>Pnos; ?1</td><td>EcoRI</td><td>2997, 353</td></tr>
+<tr><td>Etr8; ?1</td><td>EcoRI</td><td></td></tr>
+</div></table>
+<p>The digestions had positive controls that were included in the gel to compare the results obtained.</p>
+<p>The digestions are left overnight in the working table.</p>
+</br><h3 style="color:green">17 July 2015</h3>
+<p>Do the gel:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>No se lo que pone?</td><td></td><td></td></tr>
+<tr><td></td><td></td><td></td></tr>
+<tr><td></td><td></td><td></td></tr>
+<tr><td></td><td></td><td></td></tr>
+</div></table>
+<ul><li>Liquid culture of the colonies in the agar plates have been made, 3 colonies for each construction.</li>
+<li>OD’s mesurement for the agroinfiltration.</li>
+<ul class="ul_2"><li>Dilution 1:10 in MES.</li>
+</ul></ul>
+<div class="table-wrapper"><table class="alt">
+</br><h3 style="color:green"> </h3>
+<tr><td>PIF:phyB:luc</td><td>0.47</td><td>41 µl/ml</td><td>630 µl</td></tr>
+<tr><td>Renilla</td><td>0.28</td><td>71 µl/ml</td><td>1065 µl</td></tr>
+<tr><td>Etr8:luc</td><td>0.32</td><td>52 µl/ml</td><td>930 µl</td></tr>
+<tr><td>Pnos</td><td>0.34</td><td>59 µl/ml</td><td>885 µl</td></tr>
+</div></table>
+<ul><li>Red toggle experiement:</li>
+</ul>
+<p>The plants were coinfiltrated with the red toggle (PIF+phyB) and renilla. Also with two controls, Pnos (positive control) and Etr8 (negative control).</p>
+<p>14 plants were used with 3 infiltrated leafs for each plants and two spots per leaf.</p>
+<p>The controls have been infiltrated in leafs of the plants like is shown in the picture. Pnos in the right part and Etr8 in the left part.</p>
+<p> </p>
+<p>Two plants were infiltrated with both controls in the three leafs and they were in natural light during all the experiment. </p>
+<p>Another 4 plants were infiltrated with controls following the same pattern in the procedure. </p>
+<p>The remaining 8 plants were infiltrated with the red toggle.</p>
+<p>Immediately after the infiltration the plants were distributed in three different conditions: natural light, darkness and far red.</p>
+<p>So after the agroinfiltration 2 control plants stay in natural light all the experiment; 2 control plants and 4 red toggle went into the far red chamber and the same amount of control and red toggle plan (2+4) went put in darkness.</p>
+<p>This conditions were maintained 3 days and then all the infiltrated leafs were cut into small discs and put into a special plates with water. </p>
+<p>In this moment was set the time 0 (21/07/2015 at 19:30) and take the first samples.</p>
+<p>Then in time 0 some of the dark and far red samples were put in red conditions to activate the red toggle.</p>
+<p>This scheme represent the distribution of samples and the times that were taken the samples.</p>
+<p>Hacer el esquema!!! Cuando este mas espabilada ;)</p>
+<p>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.</p>
+</br><h3 style="color:green">18 July 2015</h3>
+<p>23 minipreps of the liquid cultrures. LexA:PIF:phtB:ren:luc (C3) has not grown.</p>
+<p>Digestions of the minipreps:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>phyC31;pUPD2</td><td>NotI</td><td>2046, 1899</td></tr>
+<tr><td>AsLOVpep; pUPD2</td><td>NotI</td><td></td></tr>
+<tr><td>2046, 521</td></tr>
+<tr><td>LacI:PIF:Phy:VP16+ren; a1</td><td>EcoRI</td><td>6345, 5623, 5487</td></tr>
+<tr><td>Gal4:PIF:phy:VP16+ren; a1</td><td>EcoRI</td><td>6345, 5487, 4852</td></tr>
+<tr><td>LexA:PIF:phy:ren+opLex:luc; ?1</td><td>BamHI</td><td>9431, 6674, 3513</td></tr>
+<tr><td>LacI:KNdronpa:ren+OpLex:Luc; ?1</td><td>BamHI</td><td>12632, 6574</td></tr>
+<tr><td>Gal4:KNdronpa:ren+OpLex:Luc; ?1</td><td>BamHI</td><td>11582, 6674</td></tr>
+<tr><td>PhyB:VP16+PIF6; ?1</td><td>BamHI</td><td>6674, 2685, 2337, 1439</td></tr>
+</div></table>
+<p>Gel has been done:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>phyC31 C1</td><td>phyC31 C2</td><td>phyC31 C3</td><td>AsLOVpep C1</td></tr>
+<tr><td>no</td><td>no</td><td>no</td><td>ok</td></tr>
+<tr><td>AsLOVpep C2</td><td>AsLOVpep C3</td><td>Gal4:PIF:phy:VP16:ren C1</td><td>Gal4:PIF:phy:VP16:ren C2</td></tr>
+<tr><td>no</td><td>no</td><td>no</td><td>no</td></tr>
+<tr><td>Gal4:PIF:phy:VP16:ren C3</td><td>LacI:PIF:phy:ren C1</td><td>LacI:PIF:phy:ren C2</td><td>LacI:PIF:phy:ren C3</td></tr>
+<tr><td>no</td><td>ok</td><td>no</td><td>No</td></tr>
+<tr><td>LexA:PIF:phy:ren:luc C1</td><td>LexA:PIF:phy:ren:luc C2</td><td>Gal4:KNdronpa:ren:luc C1</td><td>Gal4:KNdronpa:ren:luc C2</td></tr>
+<tr><td>no</td><td>no</td><td>ok</td><td>No</td></tr>
+<tr><td>Gal4:KNdronpa:ren:luc C3</td><td>LacI:KNdronpa:ren:luc C1</td><td>LacI:KNdronpa:ren:luc C2</td><td>LacI:KNdronpa:ren:luc C3</td></tr>
+<tr><td>no</td><td>no</td><td>ok</td><td>No</td></tr>
+<tr><td>PhyB:VP16:PIF6 C1</td><td>PhyB:VP16:PIF6 C2</td><td>PhyB:VP16:PIF6 C3</td><td></td></tr>
+<tr><td>ok</td><td>no</td><td>no</td><td></td></tr>
+</div></table>
+<p>Pick more colonies of:</p>
+<ul><li>Gal4:PIF:phy:VP16+ren; a1</li>
+<li>LexA:PIF:phy:ren+opLex:luc; ?1</li>
+</ul></ul>
+<p>New digestions with new enzymes:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>phyC31;pUPD2</td><td>XhoI (buffer red)</td><td>2119, 934, 894</td></tr>
+<tr><td>LacI:PIF:Phy:VP16+ren; a1</td><td>NEB4</td><td>5949, 5653, 3610, 2246</td></tr>
+<tr><td>LacI:PIF:Phy:VP16+ren; a1</td><td>HindIII</td><td>11568, 5587</td></tr>
+</div></table>
+<p>After 3 digestions of LacI:PIF:Phy:VP16+ren; a1 is accepted the construction.</p>
+<p>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…</p>
+</br><h3 style="color:green">19 July 2015</h3>
+</br><h3 style="color:green">20 July 2015</h3>
+</br><h3 style="color:green">21 July 2015</h3>
+<ul><li>Red toggle experiment:</li>
+</ul>
+<p>Time lapses: </p>
+<p>-19:00=t0</p>
+<p>-1:00=t1</p>
+<p>-7:00= t2</p>
+<p>-19:00= t3 (it was taken the controls in natural light)</p>
+<ul><li>Minipreps of the colonies that were in 37ºC.</li>
+<ul class="ul_2"><li>LexA:PIF:Phy:ren:luc (C1 and C2)</li>
+<li>PhyC31 (C1, C2, C4 and C5)</li>
+</ul></ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LexA:PIF:Phy:ren:luc</td><td>BamHI</td><td>9431, 6674, 3513</td></tr>
+<tr><td>PhyC31</td><td>NotI</td><td>2046, 1899</td></tr>
+</div></table>
+<p>Gel with the digestions:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>PhyC31 C1</td><td>PhyC31 C2</td><td>PhyC31 C4</td><td>PhyC31 C5</td></tr>
+<tr><td>Ok?</td><td>ok</td><td>ok</td><td>ok</td></tr>
+<tr><td>LexA:PIF:Phy:ren:luc C1</td><td>LexA:PIF:Phy:ren:luc C2</td><td></td></tr>
+<tr><td>No DNA</td><td>No DNA</td><td></td></tr>
+</div></table>
+<p>We had problems with some colonies because in the digestion did not appear DNA. The minipreps will be made with a better kit.</p>
+<p>Transform in <i>Agrobacterium</i> this cultures:</p>
+<ul><li>LexABD:KDronpa:NDronpa:ren:luc</li>
+<li>Gal4BD:KDronpa:NDronpa:ren:luc</li>
+<li>LacIBD:KDronpa:NDronpa:ren:luc</li>
+<li>OpLexA:luc (151)</li>
+<li>OpUAS:luc</li>
+<li>OpLacI:luc (152)</li>
+</ul></ul>
+<p>It was made liquid culture of Gal4:PIF:phyB:ren; ?1 (C1-C5)</p>
+<p>It was made ligations:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>35S:Gal4:AsLOVpep:T35S; ?1</td><td>35S:LacI:AsLOVpep:T35S; ?1</td><td>35S:LexA:AsLOVpep:T35S; ?1</td></tr>
+<tr><td>1 µl 35S (0030)</td><td>1 µl 35S (0030)</td><td>1 µl 35S (0030)</td></tr>
+<tr><td>1 µl Gal4BD</td><td>1 µl LacI</td><td>1 µl LexA</td></tr>
+<tr><td>1 µl AsLOVpep </td><td>1 µl AsLOVpep </td><td>1 µl AsLOVpep </td></tr>
+<tr><td>1 µl T35S (0036)</td><td>1 µl T35S (0036)</td><td>1 µl T35S (0036)</td></tr>
+<tr><td>1 µl ?1 </td><td>1 µl ?1 </td><td>1 µl ?1 </td></tr>
+<tr><td>2.6 µl H2O</td><td>2.6 µl H2O</td><td>2.6 µl H2O</td></tr>
+<tr><td>PsinATG:RepPhiC31:GFP:T35S; ?2</td><td>LacI:PIF:PhyB:ren+luc; ?1</td><td>PIF:PhyB+renilla; ?2</td></tr>
+<tr><td>1 µl PsinATG (552)</td><td>1.5 µl LacI:PIF:PhyB:ren; ?1</td><td>1.5 µl PIF:PhyB</td></tr>
+<tr><td>1 µl ReporterPhyC31</td><td>3 µl OpLacI:luc (152); ?2</td><td>2.5 µl renilla (159)</td></tr>
+<tr><td>1 µl GFP (0059)</td><td>0.5 µl ?1</td><td>0.5 µl ?2</td></tr>
+<tr><td>1 µl T35S (0036)</td><td>2.6 H2O</td><td>3 µl H2O</td></tr>
+<tr><td>1 µl ?2 </td><td></td></tr>
+<tr><td>2.6 µl H2O</td><td></td></tr>
+</div></table>
+<p>Luciferase essay with all the samples collected in the last three days.</p>
+<ul><li>Sampling: 25 samples in total.</li>
+<li>Passive lysis 1x (200µl/sample x 25 samples)= 5.000 µl</li>
+<li>Crush samples.</li>
+<li>Add 150 µl of passive lysis buffer and mix in the vortex.</li>
+<li>Centrifuge at 13200rpm for 15’.</li>
+</ul></ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>E8/li</td><td>E8/li</td><td>E8/li</td><td>P/li</td><td>P/li</td><td>P/li</td><td>TR/Fr</td><td>TR/Fr</td><td>TR/Fr</td><td>TR/D</td><td>TR/D</td><td>TR/D</td></tr>
+<tr><td>TR/Fr</td></tr>
+<tr><td>Red</td><td>TR/Fr</td></tr>
+<tr><td>Red</td><td>TR/r</td></tr>
+<tr><td>Red</td><td>TR/D</td></tr>
+<tr><td>Red</td><td>TR/D</td></tr>
+<tr><td>Red</td><td>TR/D</td></tr>
+<tr><td>Red</td><td></td><td></td><td></td></tr>
+</div></table>
+<p>We used 14.4 µl of Stop and Glow. 705.6 destilled H2O</p>
+</br><h3 style="color:green">22 July 2015</h3>
+<p>Miniprep of Gal4:PIF:PhyB:ren (C1-C3) C4 and C5 did not grow.</p>
+<p>Digestion of the miniprep:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Gal4:PIF:PhyB:ren</td><td>BamHI</td><td>16684</td></tr>
+<tr><td></td><td>EcoRI</td><td>4852, 5487, 6345</td></tr>
+<tr><td></td><td>EcoRV</td><td>1849, 3942, 2475, 381, 8037</td></tr>
+</div></table>
+<p>Gel was made:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Gal4… (BamHI)</td><td>Gal4… (EcoRI)</td><td>Gal4… (EcoRV)</td></tr>
+<tr><td>no</td><td>no</td><td>no</td></tr>
+</div></table>
+<p>After doing several digestions with different enzyme all with wrong band patterns, it was decided to revise each part making digestions. The parts are: </p>
+<ul><li>PhyC31; pUPD2</li>
+<li>Gal4:PIF:phyB; </li>
+<li>Renilla (GB159)</li>
+</ul></ul>
+<p>Made liquid culture of the ReporterBxbI:GFP in <i>Agrobacterium</i>.</p>
+<p>Transform the ligations into <i>E. coli</i>:</p>
+<ul><li>35S:Gal4:AsLOVpep:T35S; ?1</li>
+<li>35S:LacI:AsLOVpep:T35S; ?1</li>
+<li>35S:LexA:AsLOVpep:T35S; ?1</li>
+<li>PsinATG:RepPhiC31:GFP:T35S; ?2</li>
+<li>LexA:PIF:PhyB:ren+luc; ?1</li>
+<li>Add 300 µl of SOC medium and put into a agar plate with the corresponding antibiotics.</li>
+</ul></ul>
+<p>Luciferase essay:</p>
+<ul><li>Sampling: samples that have been in red and natural light 48h, 3 samples.</li>
+<li>200 µl/sample x 3 sample= 600 µl of passive lissis buffer (5x)</li>
+<li>Passive lissis buffer 1x= 120 µl+480 µl water.</li>
+<li>2.4 µl of Stop and glow</li>
+<li>118 µl of  buffer.</li>
+</ul></ul>
+</br><h3 style="color:green">23 July 2015</h3>
+<p>We decided to infiltrate soybean sprouts. First we decided to infiltrate with dye to observe the characteristics and the capacity of absorption.</p>
+<p> </p>
+<p>Mesurement of the ODs to agroinfiltrate:</p>
+<p>The samples are:</p>
+<ul><li>Citoplasm=0.27</li>
+<li>DsRed=0.27</li>
+<li>GFP=0.36</li>
+<li>Recombinase=0.43</li>
+</ul></ul>
+<p>Vi= (Vf*Absf)/(Absi*10)</p>
+<p>Absi=0.1 (because is a viral system)</p>
+<p>Vf=120 µl</p>
+<p>Make 16 liquid culture of the white colonies in the agar plates.</p>
+</br><h3 style="color:green">24 July 2015</h3>
+<p>Minipreps of the 13 liquid culture. LacI:PIF:phyB:ren cultures did not grow.</p>
+<p>Digestion of the minipreps:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Gal4:AsLOVpep</td><td>EcoRI</td><td>6345, 1972</td></tr>
+<tr><td>LacI:AsLOVpep</td><td>EcoRI</td><td>6345, 2743</td></tr>
+<tr><td>LexA:AsLOVpep</td><td>EcoRI</td><td>6345, 2011</td></tr>
+<tr><td>PsinATG:RepPhiC31:GFP</td><td>HindIII</td><td>6345, 2691</td></tr>
+<tr><td>LexA:PIF:PhyB:ren+luc</td><td>BamHI</td><td>9431, 6674, 3513</td></tr>
+<tr><td></td><td></td></tr>
+<tr><td>PhyC31; pUPD2</td><td>NotI</td><td>2046, 1899</td></tr>
+<tr><td>Gal4:PIF:phyB</td><td>BamHI</td><td>6674, 3474, 2337</td></tr>
+<tr><td>Renilla (GB159)</td><td>EcoRV</td><td>2909, 2475, 882, 812, 381</td></tr>
+</div></table>
+<p>It was done 2 gels with ligations:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Gal4:AsLOV C1</td><td>Gal4:AsLOV C2</td><td>Gal4:AsLOV C3</td><td>PsinATG:RepPhi</td></tr>
+<tr><td>C31:GFP C1</td><td>PsinATG:RepPhi</td></tr>
+<tr><td>C31:GFP C2</td><td>PsinATG:RepPhi</td></tr>
+<tr><td>C31:GFP C3</td></tr>
+<tr><td>ok</td><td>ok</td><td>ok</td><td>ok</td><td>no</td><td>No</td></tr>
+<tr><td>Mw/ladder</td><td>LacI:AsLOV C1</td><td>LacI:AsLOV C2</td><td>LexA:AsLOV C1</td><td>LexA:AsLOV C2</td><td>LexA:AsLOV C3</td></tr>
+<tr><td>-</td><td>ok</td><td>No</td><td>no</td><td>ok</td><td>no</td></tr>
+<tr><td>LexA:PIF:PhyB:ren+luc C1</td><td>LexA:PIF:PhyB:ren+luc C2</td><td></td><td></td></tr>
+<tr><td>no</td><td>Ok</td><td></td><td></td></tr>
+</div></table>
+<div class="table-wrapper"><table class="alt">
+<tr><td>PhyC31 (C1)</td><td>PhyC31 (C2)</td><td>PhyC31 (C3)</td><td>PhyC31 (C4)</td><td>PhyC31 (C5)</td></tr>
+<tr><td>no</td><td>ok</td><td>ok</td><td>ok</td><td>no</td></tr>
+<tr><td>Gal4:PIF:phyB</td><td>Renilla (GB159)</td><td></td><td></td></tr>
+<tr><td>ok</td><td>Ok</td><td></td><td></td></tr>
+</div></table>
+</br><h3 style="color:green">26 July 2015</h3>
+<p>Liquid cultures of <i>Agrobacterium</i> were refreshed:</p>
+<ul><li>TsinATG:BxbIreporter:GFP</li>
+<li>TsinATG:BxbI:reporterBxbI:GFP</li>
+<li>Viral vector??no se cual es</li>
+</ul></ul>
+</br><h3 style="color:green">27 July 2015</h3>
+<p>Sent to sequence:</p>
+<ul><li>210.08.256: LacIBD; pUPD2 (C1)</li>
+<li>210.08258: Gal4BD; pUPD2 (C2)</li>
+<li>210.08.259:LexABD; pUPD2 (C1) </li>
+<li>210.08.260: PIF6; pUPD2 (C5)</li>
+<li>210.08.261: VP16; pUPD2 (C1)</li>
+<li>210.08.262: PhiC31; pUPD2 (C2)</li>
+<li>210.08.264: PhiC31; pUPD2 (C3)</li>
+<li>210.08.266: PhiC31; pUPD2 (C4)</li>
+<li>210.08.268: ReporterBxbI; pUPD2 (C1)</li>
+<li>210.08.269: ReporterPhiC31; pUPD2 (C1)</li>
+<li>210.08.270: AsLOVpep; pUPD2 (C1)</li>
+</ul></ul>
+<p>The sample had to have 10µl of miniprep (200ng/µl aprox) + 5 µl of primer (dilution 1:3)</p>
+<p>Ligations:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Gal4:PIF:phiB + ren; ?1</td><td>LacI:PIF:phiB:ren + luc; ?2</td><td>PIF:PhyB+renilla; ?2</td></tr>
+<tr><td>1.5 µl Gal4:PIF:phiB</td><td>1.5 µl LacI:PIF:phiB:ren</td><td>1.5 µl PIF:phiB</td></tr>
+<tr><td>2 µl Renilla (GB159)</td><td>2 µl OpLacI:luc</td><td>2.5 µl Renilla (GB159)</td></tr>
+<tr><td>0.5 µl ?1</td><td>0.5 µl ?2</td><td>0.5 µl ?2</td></tr>
+<tr><td>3.6 µl H2O</td><td>2.6 µl H2O</td><td>3 µl H2O</td></tr>
+</div></table>
+<p>OD’s measurements to prepare the agroinfiltrates:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Cytoplasm: 0.39 (viral)</td><td>0.25ml</td></tr>
+<tr><td>Integrase: 0.35 (viral)</td><td>0.28ml</td></tr>
+<tr><td>GFP: 0.32 (viral)</td><td>0.31ml</td></tr>
+<tr><td>Dsred: 0.31 (viral)</td><td>0.32ml</td></tr>
+<tr><td>BxbI:reporter: 0.41</td><td>0.49ml</td></tr>
+<tr><td>Reporter: 0.26</td><td>0.77ml</td></tr>
+</div></table>
+<p>3 plants were infiltrated with BxbI and the reporter of BxbI (one leaf with the control an the other with the recombinase)?? No entiendo lo de la libreta </p>
+<p>Ligations to join the negative controls with renilla:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Etr8:luc+staffer (SF)</td><td>OpLexA:luc+SF</td><td>OpLacI:luc+SF</td><td>UAS:luc+SF</td></tr>
+<tr><td>1.5 µl Etr8:luc (88C o 1098)</td><td>1.5 µl OpLexA:luc (151)</td><td>1.5 µl OpLacI:luc (152)</td><td>1.5 µl UAS:luc (227)</td></tr>
+<tr><td>1 µl SF; ?2</td><td>1 µl SF; ?2</td><td>1 µl SF; ?2</td><td>1 µl SF; ?2</td></tr>
+<tr><td>1 µl ?1</td><td>1 µl ?1</td><td>1 µl ?1</td><td>1 µl ?1</td></tr>
+<tr><td>6.1 µl H2O</td><td>6.1 µl H2O</td><td>6.1 µl H2O</td><td>6.1 µl H2O</td></tr>
+</div></table>
+<p>Transformation of <i>E. coli</i> of the ligations:</p>
+<ul><li>Gal4:PIF:phiB + ren; ?1</li>
+<li>LacI:PIF:phiB:ren + luc; ?2</li>
+<li>PIF:PhyB+renilla; ?2</li>
+</ul></ul>
+<p>Make cultures in agar petri dishes of the transformations with the corresponding antibiotics.</p>
+<p>Transformation into <i>Agrobacterium</i> with the constructions:</p>
+<ul><li>Gal4:KDronpa:NDronpa:ren:luc</li>
+<li>LacI:KDronpa:NDronpa:ren:luc</li>
+<li>LexA:KDronpa:NDronpa:ren:luc</li>
+<li>OpLexA:luc (GB 151)</li>
+<li>OpLacI:luc (GB 152)</li>
+<li>UAS:luc (GB 227)</li>
+</ul></ul>
+<p>Make cultures in agar petri dishes of the transformations with the corresponding antibiotics.</p>
+<p>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.</p>
+</br><h3 style="color:green">28 July 2015</h3>
+<p>Miniprep of the liquid culture: ePDZ.</p>
+<p>Transformation into E.coli of the ligations:</p>
+<ul><li>Etr8:luc+staffer (SF)</li>
+<li>OpLexA:luc+SF</li>
+<li>OpLacI:luc+SF</li>
+<li>UAS:luc+SF</li>
+<li>Gal4:PIF:phyB:ren</li>
+</ul></ul>
+<p>It was observed the soybean sprouts that were infiltrated with a dye with green light and red filter. It was not observed nothing significant, moreover, the damage is evident. </p>
+<p>Transformation in <i>Agrobacterium</i> the ReporterPhiC31:GFP.</p>
+<p>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.</p>
+<ul><li>Gal4:PIF:phiB + ren. Did not grow any colony.</li>
+<li>LacI:PIF:phiB:ren + luc (C1-C3)</li>
+<li>PIF:PhyB+renilla (C1- C3)</li>
+</ul></ul>
+<p>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. </p>
+<p>We add 50µl to have a final concentration of 10ng/µl.</p>
+<p>Ligation:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LTB; pUPD2</td></tr>
+<tr><td>1 µl LTB</td></tr>
+<tr><td>1 µl pUPD2</td></tr>
+<tr><td>5.6 µl H2O</td></tr>
+</div></table>
+</br><h3 style="color:green">29 July 2015</h3>
+<p>Miniprep of yesterday liquid culture:</p>
+<ul><li>LacI:PIF:phiB:ren + luc (C1 and C3) C2 turn into blue.</li>
+<li>PIF:PhyB+renilla (C2) C1 and C3 did not grow.</li>
+</ul></ul>
+<p>Digestion:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LacI:PIF:phiB:ren:luc</td><td>EcoRV</td><td>882, 968, 1652, 3942, 2475, 381, 3477, 6674</td></tr>
+<tr><td>PIF:PhyB+renilla</td><td>HindIII</td><td>4316, 5887, 788, 6345</td></tr>
+</div></table>
+<p>Gel:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LacI:PIF:phiB:ren:luc (C1)</td><td>LacI:PIF:phiB:ren:luc (C3)</td><td>PIF:PhyB+renilla (C2)</td></tr>
+<tr><td>no</td><td>no</td><td>no</td></tr>
+</div></table>
+<p>Pick colonies and make liquid culture of:</p>
+<ul><li>Etr8:luc:staffer(SF) (C1-C3)</li>
+<li>OpLexA:luc:SF (C1-C3)</li>
+<li>OpLacI:luc:SF (C1-C3)</li>
+<li>UAS:luc:SF (C1-C3)</li>
+<li>Gal4:PIF:phyB:ren (C1-C3)</li>
+</ul></ul>
+<p>Transformation in E.coli of:</p>
+<ul><li>LTB; pUPD2</li>
+</ul></ul>
+</br><h3 style="color:green">30 July 2015</h3>
+<p>Minipreps have been done:</p>
+<ul><li>Etr8:luc:staffer(SF) (C1-C3)</li>
+<li>OpLexA:luc:SF (C1 and C3) C2 did not grow.	</li>
+<li>OpLacI:luc:SF (C1-C3)</li>
+<li>UAS:luc:SF (C1-C3)</li>
+<li>Gal4:PIF:phyB:ren (C1-C3)</li>
+</ul></ul>
+<ul><li>LacI:PIF:phy:ren:luc (C1-C3)</li>
+<li>PIF:phyB:ren (C1-C3)</li>
+</ul></ul>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Etr8:luc:staffer(SF)</td><td>BamHI</td><td>6674, 2766</td></tr>
+<tr><td>OpLexA:luc:SF</td><td>BamHI</td><td>6674, 2746</td></tr>
+<tr><td>OpLacI:luc:SF</td><td>BamHI</td><td>6674, 2847</td></tr>
+<tr><td>UAS:luc:SF</td><td>BamHI</td><td>6674, 2568</td></tr>
+<tr><td>Gal4:PIF:phyB:ren</td><td>EcoRI</td><td>6345, 5487, 4852</td></tr>
+<tr><td>LacI:PIF:phy:ren:luc</td><td>BamHI</td><td>20451</td></tr>
+<tr><td>PIF:phyB:ren</td><td>HindIII</td><td>6345, 5887, 4316, 788</td></tr>
+</div></table>
+<p>Do the gel:</p>
+<p>No se el orden!! Preguntar </p>
+<p>Primers have arrived:</p>
+<ul><li>ePDZ reverse and forward and phyC31.</li>
+</ul></ul>
+<p>Make a PCR with ePDZ and its primers to obtain the desired fragment and put it in pUPD2.</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>ePDZ PCR</td></tr>
+<tr><td>10µl Buffer HF</td></tr>
+<tr><td>31.5 µl H2O</td></tr>
+<tr><td>2 µl dNTPs</td></tr>
+<tr><td>2.5 µl  Primer forward</td></tr>
+<tr><td>2.5 µl primer reverse</td></tr>
+<tr><td>1 µl ePDZ (dilution 1:50)</td></tr>
+<tr><td>0.5 µl Taq phunion</td></tr>
+</div></table>
+<p>Pick 2 colonies of phiC31:GFP in <i>Agrobacterium</i> and make liquid culture.</p>
+<p>Ligations:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>ePDZ; pUPD2</td><td>PIF:phyB+ren; ?2</td><td>OpUAS:luc:SF+ren; ?1</td><td>OpLexA:luc:SF+ren; ?1</td></tr>
+<tr><td>1 µl ePDZ</td><td>1 µl PIF:phyB</td><td>1 µl OpUAS:luc:SF</td><td>1 µl OpLexA:luc:SF</td></tr>
+<tr><td>1 µl pUPD2</td><td>1 µl ren (159)</td><td>1 µl ren</td><td>1 µl ren</td></tr>
+<tr><td>5.6 µl H2O</td><td>1 µl ?2</td><td>1 µl ?1</td><td>1 µl ?1</td></tr>
+<tr><td></td><td>4.6 µl H2O</td><td>4.6 µl H2O</td><td>4.6 µl H2O</td></tr>
+<tr><td>OpEtr8:luc:SF+ren; ?1</td><td>Op:LacI:luc:SF+ren; ?1</td></tr>
+<tr><td>1 µl OpEtr8:luc:SF</td><td>1 µl OpLacI:luc:SF</td></tr>
+<tr><td>1 µl ren</td><td>1 µl ren</td></tr>
+<tr><td>1 µl ?1</td><td>1 µl ?1</td></tr>
+<tr><td>4.6 µl H2O</td><td>4.6 µl H2O</td></tr>
+</div></table>
+<p>After 3 days till the agroinfiltration we have observed the leaf discs that have BxbI+repBxbI and the repBxbI (negative control). </p>
+<p>We could see that the level of GFP expression was higer in the infiltration with the recombinase but there was also expression in the negative control. We think that this can be a contamination due to that we had infiltrated both constructions in he same leaf and we did not change the gloves between agroinfiltrations. FOTOO!</p>
+<p>Make liquid culture (E.coli) of:</p>
+<ul><li>LTB; pUPD2 (C1-C3)</li>
+</ul></ul>
+</br><h3 style="color:green">31 July 2015</h3>
+<p>Ligation:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LacI:PIF:phyB:ren+OpLacI:luc; ?2</td></tr>
+<tr><td>1.5 µl LacI:PIF:phyB:ren</td></tr>
+<tr><td>3 µl OpLacI:luc</td></tr>
+<tr><td>0.5 µl ?2</td></tr>
+<tr><td>2.6 µl H2O</td></tr>
+</div></table>
+<p>After 4 days till the agroinfiltration we have observed again the samples with BxbI and the negative control but was the same. The negative control expressed GFP but less than the recombinase. Foto!</p>
+<p>Minipreps of liquid culture LTB (C1 and C2)</p>
+<p>Digestion:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LTB; pUPD2</td><td>NotI</td><td>2046, 474</td></tr>
+</div></table>
+<p>Gel:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LTB (C1)</td><td>LTB (C2)</td><td>PCR ePDZ</td></tr>
+<tr><td>??</td><td></td></tr>
+</div></table>
+<p>Take out glicerynates of Paloma, lab mate:</p>
+<ul><li>Sip rotavirus CH2</li>
+<li>Sip rotavirus CH2-CH3</li>
+</ul></ul>
+<p>For E.coli and <i>Agrobacterium</i>.</p>
+<p>Pick a colony of Asun interferon (IFN) in Agro, make liquid culture.</p>
+<p>We had miniprep of IFN in pUPD. Make ligations.</p>
+<p>Transform in E.coli the ligations and make petri dishes cultures:</p>
+<ul><li>ePDZ; pUPD2</li>
+<li>PIF:phyB+ren; ?2</li>
+<li>OpUAS:luc:SF+ren; ?1</li>
+<li>OpLexA:luc:SF+ren; ?1</li>
+<li>OpEtr8:luc:SF+ren; ?1</li>
+<li>Op:LacI:luc:SF+ren; ?1</li>
+<li>LacI:PIF:phyB:ren+OpLacI:luc; ?2</li>
+</ul></ul>
+<p>Refresh agro cultures to agroinfiltrate tomorrow:</p>
+<ul><li>PhiC31 (viral system)</li>
+<li>ReporterPhiC31</li>
+<li>BxbI+reporterBxbI</li>
+<li>ReporterBxbI</li>
+<li>Gal4:KDronpa:NDronpa:luc:ren (brue toggle)</li>
+<li>PIF:phi:luc</li>
+<li>Renilla</li>
+<li>P19</li>
+<li>Pnos</li>
+</ul></ul>
+<p>New experiment with Nicotiana bentamiana. PROTOCOL</p>
+<p>Next constructions were agroinfiltrated, 2 or 3 leafs for each plant:</p>
+<p>PhiC31: one leaf for PhiC31+RepPhiC31+P19 (coinfiltration) and the other with RepPhiC31+P19 which is the negative control. 2 plants were infiltrated.</p>
+<p>BxbI: one leaf with BxbI:RepBxbI+P19 and the other with RepBxbI+P19 which is negative control. 2 plants were infiltrated.</p>
+<p>Red toggle: PIF6:phyB:luc+ren (coinfiltration). They were infiltrated 3 plants with 3 leafs for each.</p>
+<p>Blue toggle: Gal4:NDronpa:KDronpa:luc:ren. They were infiltrated 3 plants with 3 leafs for each.</p>
+<p>Pnos, the positive control. There are 4 plantas, 3 leafs per plant. </p>
+<p>So we will take samples in time 0, 12, 24 and 36h for each construction and control. After 2 days of the agroinfiltration (during this period all the plants were in dark, it is set time 0 we make discs os leaf and put in a plate with water and we change the conditions that were before. </p>
+<p>Red toggle plants: 9 discs stay in dark, 9 went to red and 9 went to natural light.</p>
+<p>Blue toggle: the same as red but went to ultraviolet instead of red light.</p>
+<p>Pnos: one plant were in natural light during all the experiment. The other discs will go, after the 2 days in black, to red, ultraviolet and stay in dark.</p>
+<p>Recombinases: they are in natural light during all the experiment.</p>
+</br><h3 style="color:green">1 August 2015</h3>
+<p>Prepare the <i>Agrobacterium</i> cultures for agroinfiltration:</p>
+<p>Gal4B:KDronpa:NDronpa:luc:ren, PhiC31 and its control, BxbI and its control and PIF:PhyB and its control were centrifugated 10 min at 3000g. The sobrenadant was discarded and the bacterias were resuspended with the agroinfiltration medium.</p>
+<p>Agroinfiltration medium had: 10ml MES, 1ml MgCl2, 89ml H2O, 100ml DMSO+acetosiningone.</p>
+<p>Incubate in 2h.</p>
+<p>Mesurement oof the ODs:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Ren+P19</td><td>0.09</td><td>888.9 µl</td></tr>
+<tr><td>RepPhiC31</td><td>0.69</td><td>115.94 µl</td></tr>
+<tr><td>BxbI</td><td>0.46</td><td>174 µl</td></tr>
+<tr><td>RepBxbI</td><td>0.15</td><td>533.3 µl</td></tr>
+<tr><td>Gal4:KNDronpa:ren:luc</td><td>0.51</td><td>156.9 µl</td></tr>
+<tr><td>Pnos</td><td>0.29</td><td>275.9 µl</td></tr>
+<tr><td>PIF:phy:luc</td><td>0.17</td><td>470.6 µl</td></tr>
+<tr><td>PhiC31</td><td>0.32</td><td>250 µl</td></tr>
+</div></table>
+<p>We went to the greenhouse and infiltrate the 13 plants.</p>
+<p>Look the petri dishes culture:</p>
+<ul><li>PIF:phyB+ren; ?2</li>
+<li>OpLexA:luc:SF+ren; ?1</li>
+<li>Op:LacI:luc:SF+ren; ?1</li>
+</ul></ul>
+<p>This colonies did not grow, the rest were left in the fridge.</p>
+<ul><li>ePDZ; pUPD2</li>
+<li>OpUAS:luc:SF+ren; ?1</li>
+<li>OpEtr8:luc:SF+ren; ?1</li>
+<li>LacI:PIF:phyB:ren+OpLacI:luc; ?2</li>
+</ul></ul>
+</br><h3 style="color:green">3 August 2015</h3>
+<p>Miniprep of the liquid culture:</p>
+<ul><li>Sip rotavirus CH2</li>
+<li>Sip rotavirus CH2-CH3</li>
+</ul></ul>
+<p>Measure of DNA concentration of:</p>
+<ul><li>OpLexA:luc:SF=169ng/µl</li>
+<li>OpacI:luc:SF=291ng/µl</li>
+</ul></ul>
+<p>Pick colonies gb159and make liquid culture of:</p>
+<ul><li>ePDZ; pUPD2 (C1-C3)</li>
+<li>OpUAS:luc:SF+ren; ?1(C1-C3)</li>
+<li>OpEtr8:luc:SF+ren; ?1(C1-C3)</li>
+<li>LacI:PIF:phyB:ren+OpLacI:luc; ?2 (C1-C3) Added X-gal and IPTG.</li>
+</ul></ul>
+<p>Repeat ligations:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>OpLexA:luc:SF+ren; ?1</td><td>Op:LacI:luc:SF+ren; ?1</td><td>E-PIF:phyB+ren; ?2</td></tr>
+<tr><td>1 µl OpLexA:luc:SF</td><td>1 µl OpLacI:luc:SF</td><td>1 µl E-PIF:phy</td></tr>
+<tr><td>3 µl ren</td><td>3 µl ren</td><td>3 µl ren</td></tr>
+<tr><td>1 µl ?1</td><td>1 µl ?1</td><td>1 µl ?1</td></tr>
+<tr><td>2.6 µl H2O</td><td>2.6 µl H2O</td><td>2.6 µl H2O</td></tr>
+</div></table>
+<p>Refresh agrobacterium cultures of:</p>
+<ul><li>Interferon</li>
+<li>SIP-CH2</li>
+<li>SIP-CH2-CH3</li>
+</ul></ul>
+<p>Take the glycerinate of Renilla; ?2 (GB159) and make liquid culture.</p>
+<p>We have made all the leaf discs and put in order in the plates with 300 µl of water, also we put each plate in the conditions light that they have to be. We have taken the samples of time0 (13:00).</p>
+<p>So as T0= 13:00, T12=1:00, T24= 13:00 and T36= 1:00.</p>
+<p>Imagenes de la colocacion en los platos? Hace falta?</p>
+</br><h3 style="color:green">4 August 2015</h3>
+<p>We do a Western blot with Asun so we can learn how to do it. Lo copio? De verdad!!?</p>
+<p>Miniprep of the liquid cultures:</p>
+<ul><li>ePDZ; pUPD2 (C1-C3)</li>
+<li>OpUAS:luc:SF+ren; ?1(C1-C3)</li>
+<li>OpEtr8:luc:SF+ren; ?1(C1-C3)</li>
+<li>LacI:PIF:phyB+ren; ?2 (C2 and C3) C1 was blue.</li>
+</ul></ul>
+<p>Digestions:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>ePDZ; pUPD2</td><td>NotI</td><td>2046, 642</td></tr>
+<tr><td>OpUAS:luc:SF+ren; ?1</td><td>EcoRI</td><td>6345, 7096</td></tr>
+<tr><td>OpEtr8:luc:SF+ren; ?1</td><td>EcoRI</td><td>6345, 7294</td></tr>
+<tr><td>LacI:PIF:phyB:ren+OpLacI:luc; ?2</td><td>EcoRV</td><td>6674, 3477, 381, 2475, 3942, 1652, 968, 882</td></tr>
+<tr><td>Renilla 159</td><td>EcoRV</td><td>2909, 2475, 882, 812, 381</td></tr>
+</div></table>
+<p>Gel:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>ePDZ C1</td><td>ePDZ C2</td><td>ePDZ C3</td><td>LacI:PIF:phyB+ren </td></tr>
+<tr><td>Ok</td><td>ok</td><td>ok</td><td></td></tr>
+<tr><td>Renilla 159</td><td>OpUAS:luc:SF+ren C1</td><td>OpUAS:luc:SF+ren C2</td><td>OpUAS:luc:SF+ren C3</td></tr>
+<tr><td>Ok</td><td>¿?</td><td></td></tr>
+<tr><td>OpEtr8:luc:SF+ren C1</td><td>OpEtr8:luc:SF+ren C2</td><td>OpEtr8:luc:SF+ren C3</td><td></td></tr>
+<tr><td></td><td></td><td></td></tr>
+</div></table>
+<p>Transformation into E.coli of yesterday ligations:</p>
+<ul><li>OpLexA:luc:SF+ren; ?1</li>
+<li>Op:LacI:luc:SF+ren; ?1</li>
+<li>E-PIF:phyB+ren; ?2</li>
+</ul></ul>
+<p>Make petri dishes culture with the indicate antibiotic.</p>
+<p>Ligations:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>35S+ePDZ+VP16+T35S;?2</td><td>35S+LTB+T35S; ?1</td></tr>
+<tr><td>1µl ePDZ</td><td>1µl 35S (30)</td></tr>
+<tr><td>1 µl VP16</td><td>1µl T35S (36)</td></tr>
+<tr><td>1 µl 35S (30)</td><td>1µl LTB</td></tr>
+<tr><td>1 µl T35S (36)</td><td>1µl ?1</td></tr>
+<tr><td>1 µl ?2</td><td>3.6 µl H2O</td></tr>
+<tr><td>2.6 µl H2O</td><td></td></tr>
+</div></table>
+<p>Refresh agrobacterium cultures of:</p>
+<ul><li>Interferon</li>
+<li>SIP-CH2</li>
+<li>SIP-CH2-CH3</li>
+</ul></ul>
+</br><h3 style="color:green">5 August 2015</h3>
+<p>Transform ligations:</p>
+<ul><li>35S+ePDZ+VP16+T35S; ?2</li>
+<li>35S+LTB+T35S; ?1</li>
+</ul></ul>
+<p>Pick colonies and make liquid cultures cultures of:</p>
+<ul><li>OpLexA:luc:SF+ren; ?1 (C1-C3)</li>
+<li>Op:LacI:luc:SF+ren; ?1 (C1-C3)</li>
+<li>E-PIF:phyB+ren; ?2 (C1)</li>
+<ul class="ul_ul_ul_notebook"><li>35S+ePDZ+VP16+T35S; ?2 (C1 and C2)</li>
+</ul><li>35S+LTB+T35S; ?1 (C1 and C2)</li>
+</ul></ul>
+<p>Make luciferase essay of red and blue toggle:</p>
+<p>Number of samples=75 (a lot)</p>
+<p>1.	Prepare lisis buffer for 80 samples.</p>
+<p>200 µl/sample * 80sample = 16ml</p>
+<p>Buffer (5x)—3.2ml of buffer + 12.8ml H2O miliQ: lysis buffer (1x)</p>
+<p>2.	Crushed samples+ 150 µl of lysis buffer.</p>
+<p>3.	Centrifuge 15min at 13200rpm in cold.</p>
+<p>4.	Dilution 2:3 (Add 36 µl lysis buffer + 24 µl sample)</p>
+<p>5.	Luciferase: 40 µl/sample. Prepare 2.88ml (3 substrate tubes)</p>
+<p>6.	Renilla:….?</p>
+</br><h3 style="color:green">6 August 2015</h3>
+<p>Miniprep of the liquid cultures.</p>
+<p>Digestion:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>OpLexA:luc:SF+ren</td><td>EcoRI</td><td>6345, 7274</td></tr>
+<tr><td>Op:LacI:luc:SF+ren</td><td>EcoRI</td><td>6345, 7375</td></tr>
+<tr><td>E-PIF:phyB+ren</td><td>HindIII</td><td>6345, 788, 5887, 4316</td></tr>
+<tr><td>35S+ePDZ+VP16+T35S</td><td>HindIII</td><td>6345, 2316</td></tr>
+<tr><td>35S+LTB+T35S</td><td>EcoRI</td><td>6345, 1684</td></tr>
+</div></table>
+<p>Gel:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>PIF:phyB+ren</td><td>OpLexA:luc:SF+ren C1</td><td>OpLexA:luc:SF+ren C2</td><td>OpLexA:luc:SF+ren C3</td></tr>
+<tr><td>No</td><td>Ok, repeat</td><td>No</td><td>Ok, repeat</td></tr>
+<tr><td>Op:LacI:luc:SF+ren C1</td><td>Op:LacI:luc:SF+ren C2</td><td>Op:LacI:luc:SF+ren C3</td><td>35S+LTB+T35S C1</td></tr>
+<tr><td>Ok, repeat</td><td>Ok</td><td>Ok</td><td>ok</td></tr>
+<tr><td>35S+LTB+T35S C2</td><td>35S+ePDZ+VP16+T35S C1</td><td>35S+ePDZ+VP16+T35S C2</td><td></td></tr>
+<tr><td>Ok</td><td>ok</td><td>ok</td><td></td></tr>
+</div></table>
+<p>Make colony PCR with 6 colonies of PhiC31:</p>
+<ul><li>DNA- </li>
+<li>JM1: 2 µl (dilution 1:10)</li>
+<li>JM2: 2 µl (dilution 1:10)</li>
+<li>Buffer Taq (with Mg): 2 µl</li>
+<li>dNTPs: 2.5 µl</li>
+<li>Taq: 0.5 µl</li>
+<li>H2O: 10 µl</li>
+<li>Program: 96ºC-2min; 96ºC-30min; 55ºC-30min; 72ºC-30min</li>
+</ul></ul>
+<p>Make a gel with the PCR but we did not see the correct bands.</p>
+<p>Repeat the colony PCR:</p>
+<ul><li>DNA- </li>
+<li>JM1: 2.5 µl (dilution 1:10)</li>
+<li>JM2: 2.5 µl (dilution 1:10)</li>
+<li>Buffer Taq (with Mg): 10 µl</li>
+<li>dNTPs: 2 µl</li>
+<li>Taq: 0.5 µl</li>
+<li>H2O: 7.5 µl</li>
+<li>Program: : 96ºC-10min; 96ºC-30min; 55ºC-30min; 72ºC-30min</li>
+</ul></ul>
+<p>Ligations:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LexA:AsLOVpep+ePDZ; ?1</td><td>LacI:AsLOVpep+ePDZ; ?1</td><td>Gal4:AsLOVpep+ePDZ; ?1</td></tr>
+<tr><td>1 µl LexA:AsLOVpep; ?1</td><td>1 µl LacI:AsLOVpep; ?1</td><td>1 µl Gal4:AsLOVpep; ?1</td></tr>
+<tr><td>1 µl ePDZ; ?2</td><td>1 µl ePDZ; ?2</td><td>1 µl ePDZ; ?2</td></tr>
+<tr><td>1 µl BsmBI</td><td>1 µl BsmBI</td><td>1 µl BsmBI</td></tr>
+<tr><td>1 µl ?1</td><td>1 µl ?1</td><td>1 µl ?1</td></tr>
+<tr><td>4.6 µl H2O</td><td>4.6 µl H2O</td><td>4.6 µl H2O</td></tr>
+</div></table>
+<p>Refresh Agro cultures to agroinfiltrate:</p>
+<ul><li>PhiC31:RepPhiC31:GFP</li>
+<li>RepPhiC31:GFP</li>
+<li>BxbI:RepBxbI:GFP</li>
+<li>RepBxbI:GFP</li>
+<li>IFN (interferon)</li>
+<li>Sip-CH2</li>
+<li>Sip-CH2-CH3</li>
+<li>Pnos</li>
+<li>P19 (this culture ha 10ml of LB + 10 µl antibiotics + 5 µl culture)</li>
+</ul></ul>
+</br><h3 style="color:green">7 August 2015</h3>
+<p>Transformation into <i>Agrobacterium</i>:</p>
+<ul><li>35S:LTB:VP16:T35S</li>
+</ul></ul>
+<p>Transformation into E.coli and make petri dishes cultures:</p>
+<ul><li>LexA:AsLOVpep+ePDZ</li>
+<li>LacI:AsLOVpep+ePDZ</li>
+<li>Gal4:AsLOVpep+ePDZ</li>
+</ul></ul>
+<p>Make a gel with the colonies PCRs:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>C7</td><td>C8</td><td>C9</td><td>C10</td><td>C11</td><td>C12</td><td>C13</td><td>C14</td></tr>
+<tr><td>No</td><td>no</td><td>no</td><td>no</td><td>no</td><td>no</td><td>no</td><td>no</td></tr>
+</div></table>
+<p>There was no DNA, there is a problem with the cells or the procedure, it have to be revised.</p>
+<p>Ligation:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>PhyC31; pUPD2</td></tr>
+<tr><td>3 µl PhiC31</td></tr>
+<tr><td>1 µl pUPD2</td></tr>
+<tr><td>1 µl BsmBI</td></tr>
+<tr><td>3.6 µl H2O</td></tr>
+</div></table>
+<p>Refresh agrobacterium cultures for tomorrow infiltration:</p>
+<ul><li>Sip-CH2</li>
+<li>Sip-CH2-CH3</li>
+<li>Pnos</li>
+<li>Red toggle (PIF6:PhyB:luc )</li>
+<li>Renilla</li>
+<li>Blue toggle (….:KDronpa:NDronpa:ren:luc)</li>
+</ul></ul>
+<ul><li>A new experiment was started:</li>
+<ul class="ul_2"><li>We are going to check again the recombinase BxbI and its reporter (negative control). </li>
+</ul></ul>
+<ul><li>Test the recombinase PhiC31. Due to that we did not obtain yet the complete recombinase, we will coinfiltrate the PhiC31 and the RepPhiC31:GFP following the same scheme as BxbI. 2 plants with 2 leafs, one of them with PhiC31 + RepPhiC31:GFP and the other with only RepPhiC31:GFP.</li>
+<li>Infiltration in 2 plants with 2 leafs each of them with interferon.</li>
+</ul></ul>
+<p>Measure f the ODs:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Construction</td><td>OD</td><td>Volume (ml)</td></tr>
+<tr><td>IFN</td><td>0.13</td><td>1.54</td></tr>
+<tr><td>RepBxbI:GFP</td><td>0.13</td><td>1.54</td></tr>
+<tr><td>BxbI:RepBxbI:GFP</td><td>0.33</td><td>0.61</td></tr>
+<tr><td>PhiC31</td><td>0.19</td><td>1.05</td></tr>
+<tr><td>RepPhiC31:GFP</td><td>0.22</td><td>0.91</td></tr>
+</div></table>
+</br><h3 style="color:green">8 August 2015</h3>
+<p>Transform the ligation into E.coli and make petri dishes cultures:</p>
+<ul><li>PhiC31; pUPD2.</li>
+</ul></ul>
+<ul><li>LexA:AsLOVpep+ePDZ (C1-C5)</li>
+<li>LacI:AsLOVpep+ePDZ (C1-C4)</li>
+<li>Gal4:AsLOVpep+ePDZ (C1-C4)</li>
+</ul></ul>
+<p>New experiment:</p>
+<p>It will be tested again the red toggle (PIF6:PhyB:luc + ren), the blue toggle (LacI:KDronpa:NDronpa:ren:luc), pnos  (positive control to check the ratio renilla luciferasa) and the production of antirotavirus that are SIP rotavirus CH2 and SIP rotavirus CH2-CH3.</p>
+<p>2 plants with 3 leafs ache one were infiltrated with pnos.</p>
+<p>The same with the red(4) and blue toggle(4) and sip rativurs I DON’T KNOW</p>
+<p>Measurement of the ODs:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Construction</td><td>OD</td><td>Volume (ml)</td></tr>
+<tr><td>Sip-CH2</td><td>0.04</td><td>6.667</td></tr>
+<tr><td>Sip-CH2-CH3</td><td>0.04</td><td>6.667</td></tr>
+<tr><td>Red toggle (PIF6:PhyB:luc)</td><td>0.09</td><td>15</td></tr>
+<tr><td>Blue toggle (LacI:KDronpa:NDronpa:ren:luc)</td><td>0.09</td><td>15.00</td></tr>
+<tr><td>Renilla</td><td>0.04</td><td>6.667</td></tr>
+<tr><td>Pnos</td><td>0.04</td><td>6.667</td></tr>
+</div></table>
+</br><h3 style="color:green">9 August 2015</h3>
+<p>Make a colony PCRs with the 10 white colonies of PhiC31 that grow in the petri dishes.</p>
+<ul><li>DNA- colony</li>
+<li>JM1: 2 µl (dilution 1:10)</li>
+<li>JM2: 2 µl (dilution 1:10)</li>
+<li>Buffer Taq (with Mg): 2 µl</li>
+<li>dNTPs: 2.5 µl</li>
+<li>Taq: 0.5 µl</li>
+<li>H2O: 1 µl</li>
+</ul></ul>
+<p>Minipreps of the liquid cultures:</p>
+<ul><li>LexA:AsLOVpep+ePDZ (C1-C4) C5 has turn into blue color.</li>
+<li>LacI:AsLOVpep+ePDZ (C1-C4)</li>
+<li>Gal4:AsLOVpep+ePDZ (C1-C4)</li>
+</ul></ul>
+<p>Digestion of the minipreps:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LexA:AsLOVpep+ePDZ</td><td>BamHI</td><td>6674, 4309</td></tr>
+<tr><td>LacI:AsLOVpep+ePDZ</td><td>BamHI</td><td>6674, 5041</td></tr>
+<tr><td>Gal4:AsLOVpep+ePDZ</td><td>BamHI</td><td>6674, 4270</td></tr>
+</div></table>
+<p>Make the gel:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LexA:AsLOVpep+ePDZ (C1)</td><td>LexA:AsLOVpep+ePDZ (C2)</td><td>LexA:AsLOVpep+ePDZ (C3)</td><td>LexA:AsLOVpep+ePDZ (C4)</td></tr>
+<tr><td>Ok</td><td>ok</td><td>ok</td><td>ok</td></tr>
+<tr><td>oLacI:AsLOVpep+ePDZ (C1)</td><td>LacI:AsLOVpep+ePDZ (C2)</td><td>LacI:AsLOVpep+ePDZ (C3)</td><td>LacI:AsLOVpep+ePDZ (C4)</td></tr>
+<tr><td>Ok</td><td>ok</td><td>ok</td><td>ok</td></tr>
+<tr><td>Gal4:AsLOVpep+ePDZ (C1)</td><td>Gal4:AsLOVpep+ePDZ (C2)</td><td>Gal4:AsLOVpep+ePDZ (C3)</td><td>Gal4:AsLOVpep+ePDZ (C4)</td></tr>
+<tr><td>Ok</td><td>ok</td><td>ok</td><td>Ok</td></tr>
+</div></table>
+<p>We keep in our inventory the colony C1 of each construction</p>
+<p>Make a gel of the colonies PCRs of PhiC31: we did not observed any DNA. Still can’t fix the problem.	</p>
+<p>Make liquid culture of renilla and PIF6:phyB:luc in <i>Agrobacterium</i> because the last time that we did an experiment they have grown slowly.</p>
+<p>Store in the 4ºC fridge an agrobacterium culture with P19.</p>
+<p>Ligations:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LexA:AsLOVpep+ePDZ+ren; ?1</td><td>LacI:AsLOVpep+ePDZ+ren; ?1</td><td>Gal4:AsLOVpep+ePDZ+ren; ?1</td></tr>
+<tr><td>1µl LexA:AsLOVpep+ePDZ</td><td>1µl LacI:AsLOVpep+ePDZ</td><td>1µl Gal4:AsLOVpep+ePDZ</td></tr>
+<tr><td>1 µl renilla (159)</td><td>1 µl renilla (159)</td><td>1 µl renilla (159)</td></tr>
+<tr><td>1 µl ?1</td><td>1 µl ?1</td><td>1 µl ?1</td></tr>
+<tr><td>4.6 µl H2O</td><td>4.6 µl H2O</td><td>4.6 µl H2O</td></tr>
+</div></table>
+</br><h3 style="color:green">10 August 2015</h3>
+<p>Transform into E.coli this ligations and make petri dishes cultures:</p>
+<ul><li>LexA:AsLOVpep+ePDZ+ren; ?1</li>
+<li>LacI:AsLOVpep+ePDZ+ren; ?1</li>
+<li>Gal4:AsLOVpep+ePDZ+ren; ?1</li>
+<li>(we add into the tubes X-gal and IPTG)</li>
+</ul></ul>
+<p>Make liquid culture of 11 colonies of PhiC31 due to that this construction was giving us problems to obtain.</p>
+<p>Refresh the agrobacterium cultures of:</p>
+<ul><li>BxbI:Rep:BxbI:GFP</li>
+<li>Rep:BxbI:GFP</li>
+<li>PhiC31</li>
+<li>RepPhiC31:GFP</li>
+<li>P19</li>
+<li>Citoplasm</li>
+<li>Integrase</li>
+<li>Dsred</li>
+<li>GFP</li>
+</ul></ul>
+<p>Take the samples of the agroinfiltrated plants that were in darkness for 2 days and make discs of them to change the conditions.</p>
+<p>Make liquid culture of:</p>
+<ul><li>LexA:AsLOVpep+ePDZ+ren (C1 and C2)</li>
+<li>LacI:AsLOVpep+ePDZ+ren (C1-C4)</li>
+<li>Gal4:AsLOVpep+ePDZ+ren (C1 and C2)</li>
+<li>(we add X-Gal and IPTG because they  are small)</li>
+</ul></ul>
+</br><h3 style="color:green">11 August 2015</h3>
+<p>Primers have arrived, they have been resuspended. They were used to eliminate the recognitions sites of the enzymes in BioBricks. This will make our constructions ready to be send to the iGEM</p>
+<p>Minipreps of the liquid cultures done yesterday:</p>
+<ul><li>PhiC31 (C6-C16)</li>
+<li>LacI:AsLOVpep+ePDZ+ren (C1-C4)</li>
+<li>Gal4:AsLOVpep+ePDZ+ren (C1 and C2)</li>
+<li>(LexA:AsLOVpep+ePDZ+rencolonies were all blue)</li>
+</ul></ul>
+<p>Make PCRs with all the primers and constructions:</p>
+<p>All of them follow the same composition which is:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>10 µl </td><td>Buffer HF</td></tr>
+<tr><td>26.5 µl</td><td>H2O</td></tr>
+<tr><td>2 µl</td><td>dNTPs</td></tr>
+<tr><td>0.5 µl</td><td>Taq Phusion</td></tr>
+<tr><td>1 µl </td><td>DNA (dilution 1:10)</td></tr>
+<tr><td>2.5 µl</td><td>Primer forward (F) (dilution 1:10)</td></tr>
+<tr><td>2.5 µl</td><td>Primer reverse (R) (dilution 1:10)</td></tr>
+</div></table>
+<p>*green ones are the only specify.</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>IFN (1)</td><td>IFN (2)</td><td>AsLOVpep (1)</td><td>AsLOVpep (2)</td></tr>
+<tr><td>IFN</td><td>IFN</td><td>AsLOVpep</td><td>AsLOVpep</td></tr>
+<tr><td>Mys int F</td><td>IFN domBB R</td><td>AsLOVpep F</td><td>AsLOVpep Fint</td></tr>
+<tr><td>Mys int R</td><td>IFN domBB F</td><td>AsLOVpep Rint</td><td>AsLOVpep R</td></tr>
+<tr><td>AsLOVpep (nested)</td><td>PhiC31 (1)</td><td>PhiC31 (2)</td><td>PhiC31 (3)</td></tr>
+<tr><td>AsLOVpep</td><td>PhiC31</td><td>PhiC31</td><td>PhiC31</td></tr>
+<tr><td>AsLOVpep nested</td><td>PhiC31 Fint 1</td><td>PhiC31 Fint 2</td><td>PhiC31 Fint 3</td></tr>
+<tr><td>AsLOVpep</td><td>PhiC31 Rint 2</td><td>PhiC31 Rint 3</td><td>PhiC31 R</td></tr>
+<tr><td>PhiC31</td></tr>
+<tr><td>PhiC31</td></tr>
+<tr><td>PhiC31 F</td></tr>
+<tr><td>PhiC31 Rint</td></tr>
+</div></table>
+<p>Digestions of the minipreps:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>PhiC31</td><td>NotI</td><td>2046, 1899</td></tr>
+<tr><td>LacI:AsLOVpep+ePDZ+ren </td><td>EcoRI</td><td>6345, 8798</td></tr>
+<tr><td>Gal4:AsLOVpep+ePDZ+ren </td><td>EcoRI</td><td>6345, 9569</td></tr>
+</div></table>
+<p>Make the gel:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>PhiC31 (C6)</td><td>C7…C16</td><td>LacI:AsLOVpep:ePDZ:ren (C1)</td><td>C2</td><td>C3…C4</td></tr>
+<tr><td>no</td><td>no</td><td>ok</td><td>no</td><td>ok</td></tr>
+<tr><td>Gal4:AsLOVpep+ePDZ+ren (C1)</td><td>Gal4:AsLOVpep+ePDZ+ren (C2)</td><td></td><td></td></tr>
+<tr><td>ok</td><td>ok</td><td></td><td></td></tr>
+</div></table>
+<p>Mesurement of the ODs:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Citoplasm</td><td>0.21</td><td>7.35 ml</td></tr>
+<tr><td>RepPhiC31</td><td>0.26</td><td>9.1ml</td></tr>
+<tr><td>35S:LTB:T35S</td><td>0.27</td><td>9.45ml</td></tr>
+<tr><td>PhiC31</td><td>0.27</td><td>9.45ml</td></tr>
+<tr><td>GFP</td><td>0.20</td><td>10ml</td></tr>
+<tr><td>RepBxbI</td><td>0.33</td><td>11.55ml</td></tr>
+<tr><td></td><td></td></tr>
+<tr><td>PsinATG:RepBxbI:GFP</td><td>0.36</td><td>12.6ml</td></tr>
+<tr><td>PhiC31</td><td>0.34</td><td>11.9ml</td></tr>
+<tr><td>DsRed</td><td>0.26</td><td>9.1ml</td></tr>
+<tr><td>P19</td><td>0.28</td><td>9.8ml</td></tr>
+<tr><td></td><td></td></tr>
+</div></table>
+</br><h3 style="color:green">12 August 2015</h3>
+<p>New digestions to check if the negative control constructions are correct and we can transformed it in agro</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>OpLexA:luc:SF:ren</td><td>EcoRI</td><td>6345, 7274</td></tr>
+<tr><td>Op:UAS:luc:SF:ren</td><td>EcoRI</td><td>6345, 7096</td></tr>
+<tr><td>OpEtr8.luc:SF:ren</td><td>EcoRI</td><td>6345, 7294</td></tr>
+</div></table>
+<p>Gel:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>Op:UAS:luc:SF:ren C1</td><td>Op:UAS:luc:SF:ren C2</td><td>Op:UAS:luc:SF:ren C3</td><td>OpEtr8.luc:SF:ren</td></tr>
+<tr><td>no</td><td>no</td><td>no</td><td>no</td></tr>
+<tr><td>OpLexA:luc:SF:ren C1</td><td>OpLexA:luc:SF:ren C2</td><td>Partner dna</td><td>Partner dna</td></tr>
+<tr><td>no</td><td>no</td><td></td></tr>
+</div></table>
+<p>Transform in Agro and make petri dishes cultures:</p>
+<ul><li>LexA:AsLOVpep:ePDZ</li>
+<li>Gal4:AsLOVpep:ePDZ</li>
+<li>LacI:AsLOVpep:ePDZ</li>
+<li>LexA:PIF6:phyB:VP16</li>
+<li>Gal4:PIF6:phyB:VP16</li>
+<li>LacI:PIF6:phyB:VP16</li>
+<li>All of them are in ?1.</li>
+</ul></ul>
+<p>Ligations:</p>
+<div class="table-wrapper"><table class="alt">
+<tr><td>LexA:AsLOVpep:ePDZ+ren; ?1</td><td>Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc; ?1</td><td>LacI:AsLOVpep:ePDZ:ren+OpLacI:luc; ?1</td></tr>
+<tr><td>1 µl LexA:AsLOVpep:ePDZ</td><td>1 µl Gal4:AsLOVpep:ePDZ:ren</td><td>1 µl LacI:AsLOVpep:ePDZ</td></tr>
+<tr><td>1 µl renilla (159)</td><td>1 µl OpUAS:luc</td><td>1 µl OpLacI:luc</td></tr>
+<tr><td>1 µl ?1</td><td>1 µl ?1</td><td>1 µl ?1</td></tr>
+<tr><td>4.6 µl H2O</td><td>4.6 µl H2O</td><td>4.6 µl H2O</td></tr>
+</div></table>
+</section>
+</div>
+</div>
 </section>
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