Difference between revisions of "Team:Valencia UPV/Notebook/Content"

Revision as of 15:57, 18 September 2015

Valencia UPV iGEM 2015

Daily notebook

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

June

27 May 2015

Starts our work in the lab!

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

Make the ligation (step 2 in the protocol):

E:PIF6:PhyB:VP16:Etr8:luc+ren; Ω1

1µL E:PIF6:PhyB:VP16:Etr8:luc; α1

1µL renilla; apha2

1µL Ω1

6,8µL H₂O

28 May 2015

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

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

29 May 2015

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

30 May 2015

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

E:PIF6:PhyB:VP16:Etr8:luc+ren; Ω1

0.5µL E:PIF6:PhyB:VP16:Etr8:luc; α1

1µL renilla; apha2

1µL Ω1

7.2µL H₂O

1 June 2015

Electroporation of the new ligation.

2 June 2015

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

Make liquid culture of just some glycerinates:

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

3 June 2015

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

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

α1	-	-
α2	-	-
Ω1	-	-
pUPD2	-	-
E:PIF6:NLS; pUPD2 (GB0288)	EcoRI	3000, 1000
E:PIF6:NLS; α1 (GB892)	EcoRI	6300, 2500
E:PIF6:NLS; Ω2 (GB893)	EcoRV	1800, 6600, 900
E:PIF6:NLS:luc:PhyB; α1 (GB896)	EcoRI	3600, 6300, 5600
Luc:PhyB; Ω1 (GB890)	BamHI	2300, 6300, 4200
PhyB:VP16; pUPD2 (GB289)	EcoRI	3000, 2000, 500
PhyB:VP16; α2 (GB88E)	HindIII	2100, 6300, 1800
Etr8:CMVmini; pUPD2 (GB1097)	EcoRI	3000, 480
OpLexA:mini35S; pUPD2 (GB733)	EcoRI	3000, 460
OpLexA:mini35S:luc:Tnos; α2 (GB151)	HindIII	2500
LexABD; pUPD2 (GB0732)	EcoRI	3000, 300
LacI for N-Tfusion; pUPD2 (GB858)	EcoRI	3000, 1000
Linker:LacIBD; pUPD2 (GB704)	EcoRI	3000, 1000
OpLacI:mini35S:luc:Tnos; α2 (GB152)	HindIII	2500, 2600
OpLacI:mini35S; pPUD2 (GB534)	EcoRI	3000, 560
E:PIF6:PhyB:VP16:Etr8:luc+ren; Ω1	BamHI	3700, 6100, 6600, 4200
E:PIF6:PhyB:VP16:Etr8:luc+ren; Ω1	EcoRV	11000, 400, 2500, 3000, 4000

Make the gel:

pUPD2	Alf	Alpha1	288	289	534
ok	ok	ok	ok	ok	?
704	732	733	858	892	896
ok	ok	ok	ok	ok	ok
1097	Alpha2	88E	151	152	Omega1
ok	ok	ok	ok	ok	ok
890	893	Red toggle (C1) (EcoRV)	Red toggle (C1) (BamHI)	Red toggle (C2) (EcoRV)	Red toggle (C2) (BamHI)
ok	ok	ok	no	no	no

4 June 2015

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

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

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

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

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

5 June 2015

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

Agrobacterium culture of promoter less: Luciferase + Renilla

Minipreps

Digestion with BamHI and EcoRV

Agarose gel 1%

How to ask and make primers?

Select the sequence to amplify and save in FASTA format.
gbCloning, go to Tools-Domesticator-1?Category
Add FASTA and select parts.
On the protocol we have the primers
The oligos they give us:

4 first nucleotides: so the enzyme can recognize without problems
6 following bingind sites.
1 extra nucleotide.
4 overhangs.

Meeting with Daniel Ramón (Biopolis).

Ligations:

PIF6 + PhyB; Ω1	Etr8(CMV)+BxbI:T35S; α1
1µL (GB892) PIF; α1	1µL (GB1097) Etr8(CMV); pUPD2
1µL (GB88E) PhyB; α2	1µL BxbI; pUPD2
1µL Ω1	1µL Tnos pUPD2
6.8µL H₂O	1µL α1
	5.8µL H₂O

Digestions:

(GB160) 35S:Renilla:tNOS-35S:P19:tNOS	EcoRV	2475, 381, 4601
(GB896) Luc:PIF6:PhyB	EcoRV	11608, 3942

6 June 2015

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

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

Agarose gel.

GB160	289	PIF+PhyB	BxbI
ok	no	?	?

7 June 2015

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

Pick two colonies from each construction.

8 June 2015

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

Digestion:

Etr8(CMV):Bxb1:Tnos; α1	EcoRI	6345, 238
EPIF6 + PhyB-PV16; Ω1	BamHI	6686, 1439, 2685, 2237

Agarose gel was made:

BxbI (C1)	BxbI (C2)	E:PIF6+PhyB-VP16 (C1)	E:PIF6+PhyB-PV16 (C2)
ok	ok	no	no

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

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

Optimized ligation:

PIF-PhyB-Luc-Renilla-P19

1 µL vector

0.8 µL dilution ?GB160

1.7 µL PIF:PhyB

4.15 µL H₂O

Ratio 1:2 vector insert

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

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

Problem: domesticator is introduced in an old pUPD2. The new one has different bases.
Change manually the pUPD2 bases in the program (Benchling).

9 June 2015

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

EPIF6-PhyB-VP16

PvuII (green buffer)

3663, 9472pb

Agarose gel 1%:

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

We see three bands: 7000, 4000, 1900pb

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

10 June 2015

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

Alfredo’s part is not working.

Make liquid culture of E:PIF6:PhyB:VP16:luc:ren (C1-C3).

Minipreps of liquid culture (PIF + Phy), colonies C3, C4, C5, C6
Digestion:

PIF+Phy:VP16	PvuII (buffer green 10x)	3663, 9472
PIF+Phy:VP16	BamHI	1939, 2685, 2337, 6674

Agarose gel 1%:

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 into AgrobacteriumEPIF6-PhyB-VP16 + luciferase (GB896) and make petri dish culture. We are not going to have the positive control (renilla+P19) and we won’t be able to quantify and make ratios.

11 June 2015

Minipreps of the culture:

Digestion:

E:PIF6:PhyB:VP16:luc: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)
no	no	no	no

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

12 June 2015

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

13 June 2015

Pick colonies to make liquid culture:

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

BxbI; α1+PhyB; α2

1µl BxbI

1 µl PhyB

1 µl Ω2

4.6 µl H₂O

Transform renilla (160) with pSub plasmid into agrobacterium and make petri dish culture.

15 June 2015

Repeat the ligation BxbI+35S:E-PIF6:tnos because PIF was Ω2

BxbI + 35S:E-PIF6:tnos; Ω1
1µl BxbI
1 µl PhyB
1 µl Ω1
4.6	µl H₂O

KDronpa has arrived:

Centrifuge it 2-5sec at maximum velocity.
Add 50 µl to have a concentration of 20ng/µl
Mix it with the vortex and spin.

Ligation:

KDronpa; pUPD2

1 µl KDronpa

1 µl pUPD2

5.6 µl H₂O

It was not possible to pick colonies of the Agrobacterium transformed with renilla because they did not grow. Maybe the problem is that with tetraciclyn bacterias grow slowly. Wait 1 day more.
Transformation of the ligation, BxbI+35S:E-PIF6:tnos; Ω1, into E. coli.Make petri dish culture.

16 June 2015

Transformation of the ligation, KDronpa, into E. coli.
Pick colonies of BxbI:E-PIF6 and make liquid culture (C1-C3).
Primers had arrived, it has been done the resuspension (dilution 1:10) of all of them.

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 following the temperature gradient.

The templates were in dilution 1:50, exception of KDronpa that was dilution 1:5 and the primers 1:10.

PCR Fusion Taq (50µl)

DNA template (10 µg/µl)

0.5 µl fusion taq

2.5 µl primer F

2.5 µl primer R

2 µl NTPs

31.5 µl H₂O

17 June 2015

Pick colonies and make liquid culture of:

KDronpa (C1-C4)

Ligations with the PCR’s products:

Templates PCR: 1+2, 5+6, 7+8PIF, 7+8VP16, 9+10, 11+12.

Template PCR; pUPD2

0.5µl template

1µl pUPD2

6.1µl H₂O

Minipreps of liquid cultures:

BxbI:E-PIF6 (C1-C3)

Agarose gel with the PCRs:

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 and make petri dishes cultures:

1+2, 5+6, 7+8PIF, 7+8VP16, 11+12

18 June 2015

Minipreps of the liquid cultures:

KDronpa (C1-C4)

Digestions:

KDronpa EcoRI 2800

Gel:

Kdronpa C1	Kdronpa C2	Kdronpa C3	Kdronpa C4
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. Thanks Alfredo :)

Take glicerynates out:

Gal4; pUPD2 (GB731)
Ω2
NoATGPromoter (GB00552)
Renilla (GB160)(GB159)(GB109)

PCR:

NDronpa
2.5 µl (9+10) primer F
2.5 µl (11+12) primer R
2 µl NTPs
0.2 µl Taq
10 µl Buffer
31.5	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
1 µl α1
5.8 µl H₂O

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

19 June 2015

We do a PCR with the normal Taq polymerase.

1µl of DNA’s template (9+10, 9+12 and 11+12)

2µl of specific buffer

2µl of NTPs

1µl primer forward

1µl primer reverse

0.5 µl of Taq

12.5 µl H₂O

These quantities multiplied by 3.

Minipreps of the yesterday’s glycerinated cultures.

Gal4; pUPD2 (GB731)
Ω2
NoATGPromoter (GB00552)
Renilla (GB160)(GB159)(GB109)

Do the glycerinates digestions:

Minipreps:	Enzime	Band pattern
(GB159) pDGB1_Ω2 renilla	EcoRV	2909, 2475,882, 812, 381
Entry vector, Ω2	EcoRV	6652, 621
(GB552) pP35s NoATG; pUPD2	EcoRI	2997, 1090
(GB160) renilla pDGB1, α2	EcoRV	4601, 2475, 381
(GB731) Gal4BD (CDS); pUPD2	EcoRI	2997, 2493
(GB109)	355:renilla:Tnos; α1	EcoRI	2580, 2493

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

159	160	Ω2	552	731	109	9+10	9+12	11+12
ok	ok	ok	ok	ok	ok	no	ok	ok

Transformation into E. coli of ligations:

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

We made an stack of Cloranfenicol petri dishes

250ml LB agar
X-Gal (1:500): 500 µl
IPTG (1:1000): 250 µl
Cloranfenicol (1:2000): 125 µl

20 june 2015

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

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

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

21 June 2015

Pick colonies and make liquid culure of (all colonies are in pUPD2):

Plates : PIF (17/06/15) (C1 and C2)
VP16 (C1 and C3)
LacI (C1-C3)
Plates (19/06/15): BxbI (C1, C2, C3),
VP16 (C4, C5)
LacI (C1, C2)
PIF (C1-C5)
LexA (C1, C2)

We take out two glicerynates of GFP and BFP (of the Alfredo’s box)

22 June 2015

We made minipreps of the liquid culture of the day before:

LacIBD; pUPD2 (C1-C5)
LexABD; pUPD2 (C1, C2)
Etr8(CMV):Bxb1 (C1-C3)
PIF6; pUPD2 (C1-C5)
VP16; pUPD2 (C1, C4, C5)

Make the digestions of all the minipreps:

LacIBD, pUPD2	NotI	2046, 1053
LexABD, pUPD2	NotI	2046, 321
Etr8(CMV):Bxb1	NotI	1532, 1290, 5896
PIF6,pUPD2	NotI	2046, 407
VP16, pUPD2	NotI	2046, 500

Refresh the viral system that a lab mate borrow to us. This is going to be use to agroinfiltrate some plants to make some cool draws to sent to a TV programm so they can watch what are we doing. This cultures consist of three parts divided in three Agrobacteriumcolonies. They are the citoplasm, the fluerescent protein (GFP, DsRed or YFP) and the integrase, in our case PhiC31.

We received the reporter BxbI (RepBxbI)!

500ng of sample
Centrifuge at 3000rpm for 5 seconds (spin).
Add 50 µl H₂O
Shake it and let at 50ºC for 20min

Make a PCR of Gal4 and NDronpa (9-10), the primers of NDronpa are aliquoted.

Make an agarose gel with all the digestions:

LacI C1	LacI C2	LacI C3	LacI C4	LacI C5	LexA C1	LexA C2	BxbI C1	BxbI C2	BxbI C3
Ok	ok	ok	ok	ok	no	no	ok	ok	no
PIF C1	PIF C2	PIF C3	PIF C4	PIF C5	VP16 C1	VP16 C4	VP16 C5
No	no	-	ok	ok	ok	ok	ok
Gal4	NDronpa 1	NDronpa 2

FOTO

We make ligations of:

Etr8(CMV):BxbI; α1 + PhyB:VP16;α2; Ω1
LacIBD;pUPD2 + PIF6BDPless; pUPD2; α1
KDronpa;pUPD2 + LacIBD; pUPD2; α1
Gal4BD; pUPD2
Reporter of BxbI; pUPD2

Tomorrow we have to take out pUPD2 of constitutive promoters, terminators and GFP (CDS).

23 June 2015

Transform into E.Coli the 5 ligations done yesterday and two more transformations of 5+6(1) and 5+6(2) which are the ligations in pUPD2 of the 18/06.

Etr8(CMV):BxbI; α1 + PhyB:VP16;α2; Ω1
LacIBD;pUPD2 + PIF6BDPless; pUPD2; α1
KDronpa;pUPD2 + LacIBD; pUPD2; α1
Gal4BD; pUPD2
Reporter of BxbI; pUPD2
LexABD (5+6), pUPD2 (1 and 2)

We have taken out of the -80ºC fridge the glycerinate of GFP; pUPD2 (GB0059)/ampicilin.
The liquid culture of Renilla (ryfampicin/kanamycin/tetracyclin) does not grow after the two days required. So we decide to refresh two new colonies, one of them in a tube with the three antibiotics and another with rifampicina and kanamicine. Asun says that the tetracycline slow down the growth of Agro.
The 4 liquid cultures of LexA+IPTG/+gal are all blue: throw them.
We ordered again the primer n?0 (NDronpa R1). Changing one codon in 3?and delete another in 5?

24 June 2015

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

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

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

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

Ligation:

ETR8(CMV):BxbI; α1+PhyB:VP16; α2; Ω1	Gal4BD(pcr) + pUPD2
1.5 µl Etr8:BxbI	1 µl Gal4 PCR
1.5 µl 88E (PhyB:VP16)	1 µl pUPD2
1 µl Ω1	5,6 µl H₂O
3.6µl H₂O

Quantification of DNA:

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

25 June 2015

Minipreps of the liquid culture:

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

Digestion of the minipreps and do the gel:

Gal4BD; pUPD2	NotI	2046, 282
RepBxbI; pUPD2	NotI	2046, 460
Etr8(CMV):BxbI:PhyB; α1	BamHI	6674, 2237, 2806, 1174
LexABD; pUPD2	NotI	2046, 321
9+10; pUPD2	NotI	464

Gel:

Etr8:BxbI	LexA C1	LexA C2	LexA C3	LexA C4	RepBxbI C1	RepBxbI C2	RepBxbI C3	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 NDronpa (R1).
Refresh the cultures of Agrobacteriumwith the viral system. Add only ryfampicin and kanamycin.
Ligations:

N-dronpa; pUPD2	RepBxbI; α1	Gal4BD, pUPD2	LexABD; pUPD2
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 µl α1
4,6 µl H₂O	3,6 µl H₂O	5,6 µl H₂O	5,6 µl H₂O

Etr8:BxbI+PhyB; Ω1
1 µl Etr8:BxbI
1 µl 88E
1µl Ω1
3,6 µl H₂O

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

26 June 2015

Do ligations:

RepBxbI+GFP; α2	LacIBD+PIF6; α1
1 µl RepBxbI	1 µl LacIBD, pUPD2
1 µl promoter without ATG	1 µl PIF6, pUPD2
1 µl Tnos	1 µl promoter
1µl GFP (0059)	1 µl T35
1 µl α2	1 µl α1
2.6 µl H₂O	2.6 µl H₂O

Digestion:

LacIBD+PIF6; α1

EcoRI

6345, 1997, 641

Gel:

LacIBD+PIF C1	LacIBD+PIF C2
no	no

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

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

PhyB:PIF6:luc: 0.35 (1:2)	0.35	1.429 µl
Ren+P19: 0.34 (1:2)	0.34	1.412 µl

Ligation of:

LacIBD; pUPD2+KDronpa; pUPD2; α1

1 µl 35S

1 µl LacIBD;pUPD2

1 µl KDronpa; pUPD

1 µl T35S

1 µl α1

2.6 µl H₂O

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

27 June 2015

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

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

We make liquid culture of:

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

28 June 2015

Do the minipreps of the liquid cultures that have grown.

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

Do the digestions of the minipreps:

LacIBD+PIF; α1	EcoRI	6345, 1997, 641
RepBxbI:GFP; Ω2	HindIII	6345, 2683
Gal4BD; pUPD2	NotI	2681, 644
NDronpa; pUPD2	NotI	2046, 744

Make the gel.

RepBxbI:GFP C1	RepBxbI: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:KDronpa and RepBxb1:GFP, adding to the agar plates 100 µl of each transformation.

29 June 2015

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

Do the digestion of the minipreps:

LexABD; 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+KDronpa+promoter+termi; α1	Gal4BD+KDonpa+prom+ter; α1	LexABD+KDronpa+prom+term; α1
1 µl LacI; pUPD2	1 µl Gal4; pUPD2	1 µl Gal4; pUPD2
1 µl KDronpa; pUPD2	1 µl KDronpa; pUPD2	1 µl KDronpa; pUPD2
1 µl 35S (GB0030)	1 µl 35S (GB0030)	1 µl 35S (GB0030)
1 µl T35S (GB0036)	1 µl T35S (GB0036)	1 µl T35S (GB0036)
2.6 µl H₂O	2.6 µl H₂O	2.6 µl H₂O
1 µl α1	1 µl α1	1 µl α1

NDronpa+VP16; α2	Gal4BD+PIF6; α1	LacIBD+PIF6; α1
1 µl NDronpa; 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)
2.6 µl H₂O	2.6 µl H₂O	2.6 µl H₂O
1 µl α2	1 µl α1	1 µl α1

LexABD+PIF6; α1

1 µl LexABD; pUPD2

1 µl PIF6; pUPD2

1 µl 35S (GB0030)

1 µl T35S (GB0036)

2.6 µl H₂O

1 µl α2

Transform all the ligations into E.Coli. Gal4BD+K-Dronpa and LacIBD+K-Dronpa went wrong and we have to do it again.

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

Quantification of DNA:

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

30 June 2015

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

Miniprep of:

RepBxbI+GFP (C1-C3)

RepBxb1+GFP; Ω2

HindIII

6345, 2683

Gel:

RepBxbI+GFP C1	RepBxbI+GFP C2	RepBxbI+GFP C3
No	no	no

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

Make liquid culture of:

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

NDronpa+VP16; α2 (C1 and C2)

Gal4BD+PIF6; α1 (C1 and C2)

LacIBD+PIF6; α1 (C1 and C2)

LexABD+PIF6; α1 (C1 and C2)

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

July

1 July 2015

Do the minipreps of the 10 liquid cultures.

Do the digestions:

LexABD+KDronpa;α1	EcoRI	6345, 2296
NDonpa+VP16; α2	HindIII	6345, 2427
Gal4BD+PIF6; α1	EcoRI	6345, 1867
LacI+PIF; α1	EcoRI	6345, 2638
LexABD+PIF6; α1	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; Ω1 (C1)
LacI+PIF; Ω1 (C1)
LexA+K-Dronpa (C1)
Gal4+PIF; Ω1 (C1)
VP16, pUPD2 (C1)
LexABD, pUPD2 (C2)
PIF6, pUPD2 (C5)
LacIBD, pUPD2 (C1)

EXPERIMENT 1.

Luciferase essay.

Pick colonies and make liquid culture of:

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

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

2 July 2015

Minipreps of:

Gal4+KDronpa (C1 and C2)
LacI+KDronpa (C1 and C2)
RepBxbi+GFP (C4-C6)

Gal4+KDronpa	EcoRI	6345, 3028
LacI+KDronpa	EcoRI	6345, 2257
RepBxbI+GFP	HindIII	6300, 2400

Do the gel:

LacI+KDronpa C1	LacI+KDronpa C2	Gal4+KDronpa C1	Gal4+KDronpa C2
ok	ok	ok	ok
RepBxb1+GFP C4	RepBxb1+GFP C5	RepBxb1+GFP C6
ok	ok	ok

Make ligations:

LexA:Kdonpa+NDronpa; Ω1	LacI:Kdronpa+NDronpa; Ω1	Gal4:Kdronpa+NDronpa; Ω1
1 µl LexA:Kdronpa	1 µl LacI:Kdronpa	1 µl Gal4:Kdronpa
1 µl NDronpa	1 µl NDronpa	1 µl NDronpa
1 µl Ω1	1 µl Ω1	1 µl Ω1
4.6 µl H₂O	4.6 µl H₂O	4.6 µl H₂O

LexA:PIF+PhyB:VP16; Ω1	LacI:PIF+PhyB:VP16; Ω1	Gal4:PIF+PhyB:VP16; Ω1
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 Ω1	1 µl Ω1	1 µl Ω1
4.6 µl H₂O	4.6 µl H₂O	4.6 µl H₂O

35S:BxbI+RepBxbI:GFP; Ω1	E-PIF+phyB+luc+ren; Ω1
1 µl 35s:Bxb1:T35S (alfredo’s)	0.5 µl PIF:PhyB:luc (GB0896)
1 µl NoATGProm:RepBxbI:GFP	1 µl Renilla GB0160
1 µl Ω1	1 µl Ω1
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 NDronpa have the desired mutation.

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

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

4 July 2015

Make the 2nd refresh of the culture of Agrobacterium.

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

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

5 July 2015

Ligations:

LacI:PIF+PhyB:VP16; Ω1	Gal4:PIF+PhyB:VP16; Ω1
1 µl LacI:PIF	1 µl Gal4:PIF
1 µl PhyB:VP16	1 µl PhyB:VP16
1 µl Ω1	1 µl Ω1
4.6	µl H₂O	4.6 µl H₂O

Minipreps of the liquid cultures.

Digestion of the minipreps:

LacI:Kdronpa+NDronpa	BamHI	6674, 5437
35S:BxbI+RepBxbI:GFP	BamHI	6674, 3859, 1782
Gal4:Kdronpa+NDronpa	BamHI	6674, 4666
LexA:Kdonpa+NDronpa	BamHI	6674, 4705
LexA:PIF+PhyB:VP16	BamHI	6674, 3513, 2337
E:PIF6:PhyB:VP16	BamHI	4209, 3756, 6100, 6674

Agarose gel (1%):

LacI:KNDronpa C1	LacI:KNDronpa C2	BxbI:Rep:GFP C1	BxbI:Rep:GFP C2	Gal4:KNDronpa C1	Gal4:KNDronpa C2
ok	ok	ok	ok	ok	ok
LexA:KNDronpa C1	LexA:KNDronpa C2	LexA:PIF:Phy C1	LexA:PIF:Phy C2	Red toggle
ok	ok	no	no	no

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

Take out the glycerinate 88C (GB1098), Etr8:luc:Tnos. We will use it like a negative control in the second luciferase essay.
Calculation of the ODs:

Renilla	0.22	182 µl of sample + 1.818 ml MES
E:PIF6:PhyB:Luc	0.26	154 µl of sample + 1.646 ml MES

EXPERIMENT 2.

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

6 July 2015

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

Etr8:luc:Tnos
BxbI:ReporterBxbI:GF

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

7 July 2015

Prepare this cultures for agroinfiltration:

Renilla
E:PIF6:PhyB:Luc

Renilla	0.29	0.138ml of sample + 1.862ml of MES
PhyB+PIF+luc	0.69	0.145ml of sample + 1.855ml of MES

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

Digestions:

Red toggle	BamHI	4209, 3756, 6100, 6674
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.

Resuspended with 50µl of H₂O.

Ligations:

AsLOVpep; pUPD2	Red Toggle	LacI:Kdronpa:Ndronpa:VP16+renilla; α1	Gal4:Kdronpa:Ndronpa:VP16+renilla; α1
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
5.6 µl H₂O	1 µl Ω1	1 µl α1	1 µl α1
	4.6 µl H₂O	4.6 µl H₂O	4.6 µl H₂O

LexA:Kdronpa:Ndronpa:VP16+renilla; α1	LexA:PIF:Phy:VP16+renilla; α1
1 µl LexA:Kdronpa:Ndronpa:VP16	1 µl LexA:PIF:Phy:VP16
1 µl GB159	1 µl GB159
1 µl α1	1 µl α1
4.6 µl H₂O	4.6 µl H₂O

8 July 2015

EXPERIMENT 2. Change the ligth conditions of the plants.

Transform into E. coli last ligations.

9 July 2015

Pick colonies and make liquid culture of:

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

Repeat the ligations.

AsLOVpep, as before.

Red toggle (PIF+PhyB+luc+ren)

0.5 µl PIF+phy+luc (896)

1 µl renilla (160)

1 µl Ω1

5.1 µl H₂O

EXPERIMENT 2.

Luciferase essay.

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

10 July 2015

Minipreps of:

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

Digestions of:

LexABD:PIF:PhyB:VP16+Renilla; α1	EcoRI	6345, 5487, 4891
LexA:Kdronpa:Ndronpa+renilla; α1	EcoRI	9333, 6345
Gal4:Kdronpa:Ndronpa+renilla; α1	EcoRI	6345, 9194
LacI:Kdronpa:Ndronpa+renilla; α1	EcoRI	6345, 9965
LacIBD+PIF+PhyB; Ω1	BamHI	6674, 4245, 2337
Gal4BD+PIF+PhyB; Ω1	BamHI	6674, 3474, 2337

Make the gel:

LacIBD+PIF+PhyB	Gal4BD+PIF+PhyB	LexA:PIF:PhyB:VP16+Ren C1	LexA:PIF:PhyB:VP16+Ren C2
Ok	Ok	ok	ok
LexA:KNdronpa+ren C1	LexA:KNdronpa+ren C2	Gal4:KNdronpa+ren C1	Gal4:KNdronpa+ren C2
Ok	Ok	ok	Ok
Gal4:KNdronpa+ren C3	LacI:Kdronpa:Ndronpa+ren C1	LacI:Kdronpa:Ndronpa+ren 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).

Ligations:

PhiC31; pUPD2	Reporter PhiC31; pUPD2	LacI:PIF:PhyB:VP16+ren; α1
1 µl PhiC31	1 µl RepPhiC31	1 µl LacI:PIF:PhyB:VP16
1µl pUPD2	1µl pUPD2	1 µl renilla (GB159)
5.6 µl H₂O	5.6 µl H₂O	1 µl α1
		4.6 µl H₂O

Gal4:PIF:Phy:VP16+ren; α1	LexA:PIF:PhyB:ren+OpLex:luc; Ω1	LexA:KNdronpa:ren+OpLex:Luc; Ω1
1 µl Gal4:PIF:phy:VP16	1 µl LexA:PIF:phy:ren	1 µl LexA:KNdronpa:ren
1 µl renilla (GB159)	1 µl opLex:luc (GB151)	1 µl OpLex:Luc (GB151)
1 µl α1	1 µl Ω1	1 µl Ω1
4.6 µl H₂O	4.6 µl H₂O	4.6 µl H₂O

Gal4:KNdronpa:ren+UAS:luc; Ω1	LacI:KNdronpa:ren+OpLacI:luc; Ω1
1 µl Gal4:KNdronpa:ren	1 µl LacI:KNdronpa:ren
1 µl OpUAS:luc (GB227)	1 µl OpLacI:luc (GB152)
1 µl Ω1	1 µl Ω1
4.6 µl H₂O	4.6 µl H₂O

11 July 2015

Prepare glycerinates of:

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

Do miniprep of:

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

Digestions:

E:PIF6:PhyB:luc:ren	BamHI	6674, 6100, 4209, 3756
AsLOVpep	NotI	2558, 512

AsLOVpep C1	AsLOVpep C2	Red toggle C2
no	no	no

Do transformation of DHSa and yesterday ligations:

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

12 July 2015

Pick colonies and make liquid culture:

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

Ligations that were repeated:

Gal4:PIF:phy:VP16+ren; α1	LacI:KNdronpa:ren+OpLacI:luc; Ω1
1 µl Gal4:PIF:phy:VP16	1 µl LacI:KNdronpa:ren
1 µl renilla (GB159)	1 µl OpLacI:luc (GB152)
4.6 µl H₂O	4.6 µl H₂O
1 µl α1	1 µl Ω1

Refresh the liquid cultures of Agrobacterium:

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

13 July 2015

All the liquid cultures have grown, do minipreps.

Do digestions:

phyC31;pUPD2	NotI	2046, 1899
Reporter phiC31; pUPD2	NotI	2046, 475
LacI:PIF:Phy:VP16+ren; α1	EcoRI	6345, 5623, 5487
LexA:PIF:phy:ren+opLex:luc; Ω1	BamHI	9431, 6674, 3531
LexA:KNdronpa:ren+OpLex:Luc; Ω1	BamHI	1199, 6674
Gal4:KNdronpa:ren+UAS:luc; Ω1	BamHI	11582, 6674

Agarose gel:

PhiC31 C1	PhiC31 C2	PhiC31 C3	RepPhiC31 C1
no	no	no	ok
RepPhiC31 C2	LacI:PIF:PhyB:VP16+ren C1	LacI:PIF:PhyB:VP16+ren C2	LacI:PIF:PhyB: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+OpUAS:luc C1
no

The Agrobacterium cultures refreshed yesterday were store in the fridge.

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

EXPERIMENT 4. Recombinase BxbI.

Mesurement of the OD’s:

35S:BxbI+reporterBxbI:GFP: 0.28

143 µl of culture+1857 µl of MES

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

The liquid cultures of this constructions were repeated:

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

14 July 2015

Do minipreps of:

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

Do digestions:

phyC31;pUPD2	NotI	2046, 1899
Reporter phiC31; pUPD2	NotI	2046, 475
LacI:PIF:Phy:VP16+ren; α1	EcoRI	6345, 5623, 5487
LexA:PIF:phy:ren+opLex:luc, Ω1	BamHI	9431, 6674, 3531
LexA:KNdronpa:ren+OpLex:Luc; Ω1	BamHI	1199, 6674
Gal4:KNdronpa:ren+UAS:luc; Ω1	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+OpUAS: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
PhiC31: 4.6 ng/µl
AsLOVpep: 30.3 ng/µl
OpLexA:luc (GB151): 40 ng/µl
Gal4:PIF:PhyB:ren (C1): 124.4 ng/µl
LacI:PIF:PhyB:VP16 (C1): 126 ng/µl
Renilla (GB159): 42 ng/µl
Gal4:Kdronpa:Ndronpa:renilla (C3): 172.8 ng/µl
OpUAS:luc (GB227): 6.3 ng/µl

Pick colonies and make liquid culture of LacI:KDronpa:NDronpa:ren:luc (C4 and C5). The colonies of Gal4:PIF:phy:VP16+ren were all blue.

15 July 2015

Miniprep of LacI:KDronpa:NDronpa:ren:luc (C4 and C5).
Digestion:

LacI:KDronpa:NDronpa:ren: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:ren: 209.0 ng/µl

Repeat the ligations:

PhiC31;pUPD2	AsLOVpep; pUPD2	LacI:PIF:PhyB:VP16+ren; α1
1 µl PhiC31	1 µl AsLOVpep	1.5 µl LacI:PIF:PhyB:VP16
1µl pUPD2	1µl pUPD2	2.5 µl renilla (159)
5.6 µl H₂O	5.6 µl H₂O	0.5 µl α1
	4.6 µl H₂O

Gal4:PIF:phy:VP16+ren; α1	LexA:PIF:phy:ren+opLex:luc; Ω1	LacI:KNdronpa:ren+OpLex:Luc; Ω1
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)
2.6 µl H₂O	2.6 µl H₂O	3 µl H₂O
1 µl α1	0.5 µl Ω1	0.5 µl Ω1

Gal4:KNdronpa:ren+OpLex:Luc; Ω1	PhyB:VP16+PIF6; Ω1
1 µl Gal4:KNdronpa:ren	1 µl PhyB:VP16 (88E)
4 µl OpUAS:Luc (227)	2 µl PIF6 (170)
3 µl H₂O	3.6 µl H₂O
0.5 µl Ω1	1 µl Ω1

These Agrobacterium cultures have been refreshed:

E:PIF:PhyB:luc
Renilla
Pnos
Etr8

16 July 2015

Yesterday ligations have been transformed into E. coli:

phyC31;pUPD2
AsLOVpep; pUPD2
LacI:PIF:Phy:VP16+ren; α1
Gal4:PIF:phy:VP16+ren; α1
LexA:PIF:phy:ren+opLex:luc; Ω1
LacI:KNdronpa:ren+OpLex:Luc; Ω1
Gal4:KNdronpa:ren+OpLex:Luc; Ω1
PhyB:VP16+PIF6; Ω1

EXPERIMENT 4.

The agroinfiltrated leaf with BxbI:rep:GFP has been observed in the magnifying glass.

Second refresh of the Agrobacterium cultures:

E:PIF:PhyB:luc
Renilla
Pnos
Etr8

Miniprep of these cultures.
Digestion of the minipreps:

E:PIF:PhyB:luc; Ω1	EcoRI	??
Renilla; Ω2	HindIII	7453
Pnos; α1	EcoRI	2997, 353
Etr8; Ω1	EcoRI	2742

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:

Pnos	Etr8:luc	Etr8:luc C+	PIF6:PhyB:luc
no	ko	ok	ok
PIF6:PhyB:luc C+	renilla	renilla C+
ok	no	ok

Make liquid cultures of yesterday ligations, three colonies per cultures.
OD’s mesurement for the agroinfiltration.

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

EXPERIMENT 5. Red toggle experiement:

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

EXPERIMENT 4.

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

18 July 2015

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

Digestions of the minipreps:

PhiC31;pUPD2	NotI	2046, 1899
AsLOVpep; pUPD2	NotI	2046, 521
LacI:PIF:PhyB:VP16+ren; α1	EcoRI	6345, 5623, 5487
Gal4:PIF:phy:VP16+ren; α1	EcoRI	6345, 5487, 4852
LexA:PIF:phy:ren+OpLex:luc; Ω1	BamHI	9431, 6674, 3513
LacI:KNdronpa:ren+OpLex:Luc; Ω1	BamHI	12632, 6574
Gal4:KNdronpa:ren+OpLex:Luc; Ω1	BamHI	11582, 6674
PhyB:VP16+PIF6; Ω1	BamHI	6674, 2685, 2337, 1439

Gel has been done:

phiC31 C1	phiC31 C2	phiC31 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; α1
LexA:PIF:phy:ren+opLex:luc; Ω1

New digestions with new enzymes:

phiC31;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; α1 is accepted the construction.

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

FOTO

19 July 2015

The incredible mistery of the lost notebook papers...

20 July 2015

Another day...

21 July 2015

EXPERIMENT 5. Red toggle.

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

Time lapses:

-19:00=t0

-1:00=t1

-7:00= t2

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

Minipreps of the colonies that were in 37ºC.

LexA:PIF:Phy:ren:luc (C1 and C2)
PhyC31 (C1, C2, C4 and C5)

LexA:PIF:Phy:ren:luc	BamHI	9431, 6674, 3513
PhiC31	NotI	2046, 1899

Gel with the digestions:

PhiC31 C1	PhiC31 C2	PhiC31 C4	PhiC31 C5
Ok?	ok	ok	ok
LexA:PIF:PhyB:ren:luc C1	LexA:PIF:PhyB: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 (227)
OpLacI:luc (152)

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

It was made ligations:

35S:Gal4:AsLOVpep:T35S; α1	35S:LacI:AsLOVpep:T35S; α1	35S:LexA:AsLOVpep:T35S; Ω1
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 α1	1 µl α1	1 µl α1
2.6 µl H₂O	2.6 µl H₂O	2.6 µl H₂O

PsinATG:RepPhiC31:GFP:T35S; α2	LacI:PIF:PhyB:ren+luc; Ω1	PIF:PhyB+renilla; α2
1 µl PsinATG (552)	1.5 µl LacI:PIF:PhyB:ren; α1	1.5 µl PIF:PhyB
1 µl ReporterPhiC31	3 µl OpLacI:luc (152); α2	2.5 µl renilla (159)
1 µl GFP (0059)	0.5 µl Ω1	0.5 µl α2
1 µl T35S (0036)	2.6 H₂O	3 µl H₂O
1 µl α2
2.6 µl H₂O

EXPERIMENT 5.

Luciferase essay.

22 July 2015

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

Digestion of the miniprep:

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

Gel was made:

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

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

Made liquid culture of the ReporterBxbI:GFP in Agrobacterium.

Transform the ligations into E. coli:

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

EXPERIMENT 6.

Luciferase essay:

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

23 July 2015

EXPERIMENT 7.

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

FOTO

Make liquid cultures of yesterday transformations.

24 July 2015

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

Digestion of the minipreps:

Gal4: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:RepPhiC31:GFP C1	PsinATG:RepPhiC31:GFP C2
ok	ok	ok	ok	no
PsinATG:RepPhiC31:GFP C3	LacI:AsLOV C1	LacI:AsLOV C2	LexA:AsLOV C1	LexA:AsLOV C2
no	ok	No	no	ok
LexA:AsLOV C3	LexA:PIF:PhyB:ren+luc C1	LexA:PIF:PhyB:ren+luc C2
no	no	Ok

PhiC31 (C1)	PhiC31 (C2)	PhiC31 (C3)	PhiC31 (C4)	PhiC31 (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 vectors (citoplasm, integrase)
GFP
Dsred

27 July 2015

Sent to sequence:

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

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

Ligations:

Gal4:PIF:phiB + ren; α1	LacI:PIF:phiB:ren + luc; Ω2	PIF:PhyB+renilla; α2
1.5 µl Gal4:PIF:PhyB	1.5 µl LacI:PIF:PhyB:ren	1.5 µl PIF:PhyB
2 µl Renilla (GB159)	2 µl OpLacI:luc	2.5 µl Renilla (GB159)
0.5 µl α1	0.5 µl Ω2	0.5 µl α2
3.6 µl H₂O	2.6 µl H₂O	3 µl H₂O

OD’s measurements to agroinfiltrate:

Cytoplasm: 0.39 (viral)	0.25ml
Integrase: 0.35 (viral)	0.28ml
GFP: 0.32 (viral)	0.31ml
Dsred: 0.31 (viral)	0.32ml
BxbI:reporter: 0.41	0.49ml
Reporter: 0.26	0.77ml

EXPERIMENT 8.

BxbI:RepBxbI:GFP
RepBxbI:GFP

EXPERIMENT 9.

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

Ligations to join the negative controls with renilla:

Etr8:luc+staffer(SF)	OpLexA:luc+SF	OpLacI:luc+SF	OpUAS: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; α2	1 µl SF; α2	1 µl SF; α2	1 µl SF;α2
1 µl Ω1	1 µl Ω1	1 µl Ω1	1 µl Ω1
6.1 µl H₂O	6.1 µl H₂O	6.1 µl H₂O	6.1 µl H₂O

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

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

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

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

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

28 July 2015

Miniprep of the liquid culture: ePDZ.

Transformation into E. coli of the ligations:

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

EXPERIMENT 9.

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

FOTO

Transformation in Agrobacterium the ReporterPhiC31:GFP.

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

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

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

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

Ligation:

LTB; pUPD2

1 µl LTB

1 µl pUPD2

5.6 µl H₂O

29 July 2015

Miniprep of yesterday liquid culture:

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

Digestion:

LacI:PIF:phiB:ren: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)
OpUAS:luc:SF (C1-C3)
Gal4:PIF:phyB:ren (C1-C3)

LacI:PIF:phy:ren:luc (C1-C3)
PIF:PhyB:ren (C1-C3)

Etr8:luc:staffer(SF)	BamHI	6674, 2766
OpLexA:luc:SF	BamHI	6674, 2746
OpLacI:luc:SF	BamHI	6674, 2847
OpUAS: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:

Primers have arrived:

ePDZ reverse and forward and phiC31.

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

ePDZ PCR

10µl Buffer HF

31.5 µl H₂O

2 µl dNTPs

2.5 µl Primer forward

2.5 µl primer reverse

1 µl ePDZ (dilution 1:50)

0.5 µl Taq phunion

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

Ligations:

ePDZ; pUPD2	PIF:phyB+ren; α2	OpUAS:luc:SF+ren; α1	OpLexA:luc:SF+ren; α1
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 α2	1 µl α1	1 µl α1
	4.6 µl H₂O	4.6 µl H₂O	4.6 µl H₂O

OpEtr8:luc:SF+ren; α1	Op:LacI:luc:SF+ren; α1
1 µl OpEtr8:luc:SF	1 µl OpLacI:luc:SF
1 µl ren	1 µl ren
1 µl α1	1 µl aplha1
4.6 µl H₂O	4.6 µl H₂O

Make liquid culture (E. coli) of:

LTB; pUPD2 (C1-C3)

31 July 2015

Ligation:

LacI:PIF:PhyB:ren+OpLacI:luc; Ω2

1.5 µl LacI:PIF:phyB:ren

3 µl OpLacI:luc

0.5 µl Ω2

2.6 µl H₂O

EXPERIMENT 8.

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

Foto!

Minipreps of liquid culture LTB (C1 and C2)

Digestion:

LTB; pUPD2

NotI

2046, 474

Gel:

LTB (C1)	LTB (C2)	PCR ePDZ
ok	ok	ok

Take out glicerynates of Paloma, lab mate:

Sip rotavirus CH₂
Sip rotavirus CH₂-CH₃

For E. coli and Agrobacterium.

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

We had miniprep of IFN in pUPD. Make ligations.

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

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

Refresh agro cultures to agroinfiltrate tomorrow:

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

August and September

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.

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

EXPERIMENT 9.

PhiC31
RepPhiC31:GFP
BxbI:RepBxbI:GFP
RepBxbI:GFP
Gal4:NDronpa:KDronpa:luc:ren.
Pnos

Look the petri dishes culture:

PIF:phyB+ren; α2
OpLexA:luc:SF+ren; α1
Op:LacI:luc:SF+ren; α1

ePDZ; pUPD2
OpUAS:luc:SF+ren; α1
OpEtr8:luc:SF+ren; α1
LacI:PIF:PhyB:ren+OpLac:luc; α2

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 and make liquid culture of:

ePDZ; pUPD2 (C1-C3)
OpUAS:luc:SF+ren; α1(C1-C3)
OpEtr8:luc:SF+ren; α1(C1-C3)
LacI:PIF:phyB:ren+OpLacI:luc; Ω2 (C1-C3) Added X-gal and IPTG.

Repeat ligations:

OpLexA:luc:SF+ren; α1	Op:LacI:luc:SF+ren; α1	E-PIF:phyB+ren; α2
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 α1	1 µl α1	1 µl α2
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; Ω2 (GB159) and make liquid culture.

EXPERIMENT 9.

We have made all the leaf discs and put in order in the plates.

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; α1	EcoRI	6345, 7096
OpEtr8:luc:SF+ren; α1	EcoRI	6345, 7294
LacI:PIF:phyB:ren+OpLacI:luc; Ω2	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 and make petri dish culture:

OpLexA:luc:SF+ren; α1
Op:LacI:luc:SF+ren; α1
E-PIF:phyB+ren; α2

Ligations:

35S+ePDZ+VP16+T35S;α2	35S+LTB+T35S; α1
1µl ePDZ	1µl 35S (30)
1 µl VP16	1µl T35S (36)
1 µl 35S (30)	1µl LTB
1 µl T35S (36)	1µl α1
1 µl α2	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; α2
35S+LTB+T35S; α1

Pick colonies and make liquid cultures cultures of:

OpLexA:luc:SF+ren; α1 (C1-C3)
Op:LacI:luc:SF+ren; α1 (C1-C3)
E-PIF:phyB+ren; α2 (C1)
35S+ePDZ+VP16+T35S; α2 (C1 and C2)
35S+LTB+T35S; α1 (C1 and C2)

EXPERIMENT 9.

Make luciferase essay of red and blue toggle:

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; Ω1	LacI:AsLOVpep+ePDZ;Ω1	Gal4:AsLOVpep+ePDZ; Ω1
1 µl LexA:AsLOVpep; α1	1 µl LacI:AsLOVpep; α1	1 µl Gal4:AsLOVpep;α1
1 µl ePDZ; α2	1 µl ePDZ; α2	1 µl ePDZ; α2
1 µl BsmBI	1 µl BsmBI	1 µl BsmBI
1 µl Ω1	1 µl Ω1	1 µl Ω1
4.6 µl H₂O	4.6 µl H₂O	4.6 µl H₂O

Refresh Agrobacteriumcultures to agroinfiltrate:

PhiC31:RepPhiC31:GFP
RepPhiC31:GFP
BxbI:RepBxbI:GFP
RepBxbI:GFP
IFN (interferon)
Sip-CH₂
Sip-CH₂-CH₃
Pnos
P19 (this culture has 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 of PhiC31:

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 (LacI:KDronpa:NDronpa:ren:OpLacI:luc)

EXPERIMENT 10.

PhiC31:RepPhiC31:GFP
RepPhiC31:GFP
BxbI:RepBxbI:GFP
RepBxbI:GFP
IFN (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)

EXPERIMENT 11.

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

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; α1	LacI:AsLOVpep+ePDZ+ren; α1	Gal4:AsLOVpep+ePDZ+ren; α1
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 α1	1 µl α1	1 µl α1
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; α1
LacI:AsLOVpep+ePDZ+ren; α1
Gal4:AsLOVpep+ePDZ+ren; α1

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

EXPERIMENT 10.

Take the samples of the agroinfiltrated plants that were in darkness 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)

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+ren colonies 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)

*The three last lines are the only specify bellow.

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 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)	LacI:AsLOVpep:ePDZ:renC2	LacI:AsLOVpep:ePDZ:ren(C3)	LacI:AsLOVpep:ePDZ:ren(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

EXPERIMENT 11.

Take the samples of the agroinfiltrated plants that were in darkness to change the conditions.

EXPERIMENT 12.

Citoplasm
RepPhiC31
35S:LTB:T35S
PhiC31
GFP
RepBxbI
PsinATG:RepBxbI:GFP
PhiC31
DsRed
P19

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 Agrobacteriumand 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 Ω1.

Ligations:

LexA:AsLOVpep:ePDZ+ren; α1	Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc; Ω1	LacI:AsLOVpep:ePDZ:ren+OpLacI:luc; Ω1
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 α1	1 µl Ω1	1 µl Ω1
4.6 µl H₂O	4.6 µl H₂O	4.6 µl H₂O

PROTOPLAST 1: Do for the first time. PROTOCOL, click here.

13 August 2015

Analysis of the AsLOVpep PCR’s products that were set to sequencing:

The primer AsLOVpep F (forward) works well.
The primer AsLOVpep Ri (reverse intern): it is possible that occurs a deletion in the position 147.
The primer AsLOVpep Fi (forward intern): change in one base A to C in the position 446, last base in the codon, the amino acid (alanine) still the same.
The primer AsLOVpep R (reverse): it is ok but the las 50bp are no sequenced.

The primer PhiC31 Fi (forward intern): did not work well. 4 consecutive deletions and changes in the nucleotides from 470 to 990.
The primer PhiC1 Ri2 (reverse intern 2): corrects the errors in the sequencing of the las primer. There is not deletion in the zone between 470 till 990 but is diffuse.
The primer PhiC31 Fi2 (forward intern 2): possible deletion in 1063.
The primer PhiC31 Fi3 (forward inter 3): ok
The primer PhiC31 R (reverse): ok

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

LexA:AsLOVpep:ePDZ+ren; ?1
Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc; ?1
LacI:AsLOVpep:ePDZ:ren+OpLacI:luc; ?1

PROTOPLAST 1:We try to obtain the protoplast but we make a mistake, repeat.

PROTOPLAST 2. Start the procedure again.

EXPERIMENT 11. Luciferase essay of blue and red toggle.

Ligations of the PCR products:

AsLOVpep (BB); pUPD2

1µl AsLOVpep1

1µl AsLOVpep2

1µl pUPD2

1µl BsmbI

4.6µl H₂O

14 August 2015

Repeat the digestions of the negative controls:

OpLexA:luc:SF:ren	SacII	13619
	PouI	2495, 2055, 5937, 3152
OpUAS:luc:SF:ren	PouI	2475, 2055, 5937, 2474
OpEtr8.luc:SF:ren	PouI	2637, 2055, 5937, 3010
OpLacI:luc:SF:ren	SacII	12930, 790
OpLacI:luc:SF:ren	PouI	2475, 2055, 5937, 3253

Make the gel:

OpUAS:luc:SF:ren C1	OpUAS:luc:SF:ren C2	OpUAS:luc:SF:ren C3	OpEtr8.luc:SF:ren
ok	no	ok	ok
OpLacI:luc:SF:ren (PouI)	OpLacI:luc:SF:ren (SacII)	OpLexA:luc:SF:ren (PouI)	OpLexA:luc:SF:ren (SacII)
ok	ok	ok	ok

Transform into E. coli AsLOVpep (BB) and make petri dish culture.

Make liquid cultures of:

LexA:AsLOVpep:ePDZ+ren; ?1 (C1-C4)
Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc; ?1 (C1-C4)
LacI:AsLOVpep:ePDZ:ren+OpLacI:luc; ?1 (C1-C4)

Make liquid cultures of agrobacterium cultures in petri dishes:

LexA:AsLOVpep:ePDZ
Gal4:AsLOVpep:ePDZ
LacI:AsLOVpep:ePDZ
Gal4:PIF6:phyB:VP16
LacI:PIF6:phyB:VP16

We make triple strake method to obtain isolated colonies of LexA:PIF6:PhyB:VP16.

PROTOPLAST 2:

Continue with the procedure to obtain protoplasts. We finally see in the microscope the protoplasts.

Transform protoplasts. How? Click here.

E:PIF6:PhyB:luc+ren
E:PIF6:PhyB:luc+ren

EXPERIMENT 12.

Prepare samples of both recombinases and its negative control to see them in the confocal microscope. We see samples of plants agroinfiltrated 7 days ago and other agroinfiltrated 5 days ago with the viral silencing repressor, P19.

FOTO!!

PROTOPLAST 2:

Take samples at time 8h of protoplasts.

Samples of both toggles and in all the conditions.

15 August 2015

Minipreps of:

LexA:AsLOVpep:ePDZ+ren; α1
Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc; Ω1
LacI:AsLOVpep:ePDZ:ren+OpLacI:luc; Ω1

Minipreps digestions:

LexA:AsLOVpep:ePDZ+ren; α1	EcoRI	8837, 6345
Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc; Ω1	BamHI	6674, 11186
LacI:AsLOVpep:ePDZ:ren+OpLacI:luc; Ω1	BamHI	6674, 12236

Make the gel:

LexA:AsLOVpep:ePDZ+ren C1	LexA:AsLOVpep:ePDZ+ren C2	LexA:AsLOVpep:ePDZ+ren C3	LexA:AsLOVpep:ePDZ+ren C4
ok	ok	ok	Ok
Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc C1	Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc C2	Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc C3	Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc C4
no	no	no	No
LacI:AsLOVpep:ePDZ:ren+OpLacI:luc C1	LacI:AsLOVpep:ePDZ:ren+OpLacI:luc C2	LacI:AsLOVpep:ePDZ:ren+OpLacI:luc C3	LacI:AsLOVpep:ePDZ:ren+OpLacI:luc C4
ok	ok	ok	ok

We keep LexA:AsLOVpep:ePDZ+ren C1 and LacI:AsLOVpep:ePDZ:ren+OpLacI:luc C4.

Make liquid cultures of (added X-gal + IPTG):

AsLOvpep (BB); pUPD2 (C1-C4)

PROTOPLASTS 3.

We cut 3 leafs into strips and let this overnight shaking at minimum.

PROTOPLAST 2.

Take protoplasts samples at time 16h. Samples of both toggles and in all the conditions.

Take also time 24h.

16 August 2015

All liquid cultures of AsLOvpep (BB); pUPD2 (C1-C4) were blue.

Pick 4 more colonies also with added X-gal + IPTG

Ligations:

Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc; Ω1	AsLOVpep; pUPD2	LexA:AsLOVpep:ePDZ:ren+OpLexA:luc; Ω1
1µl Gal4:AsLOVpep:ePDZ:ren	1µl AsLOVpep1	1µl Lex!:AsLOVpep:ePDZ:ren
1µl OpUAS:luc (227)	1µl AsLOVpep2	1µl OpLexA:luc (151)
1µl Ω1	1µl pUPD2	1µl Ω1
4.6µl H₂O	4.6µl H₂O	4.6µl H₂O

PROTOPLASTS 3.

Finish the protoplasts started yesterday, we obtain good concentrations.

Transform them:

LacI:AsLOVpep:ePDZ:ren:OpLacI:luc
LacI:KDronpa:NDronpa:ren:OpLacI:luc
OpLacI:luc:ren

PROTOPLAST 2. Luciferase essay.

Do a luciferase essay with protoplasts, click here.

Transform into agro and make petri dish culture of:

LacI:AsLOVpep:ePDZ:ren:OpLacI:luc; Ω1

Refresh agrobacterium liquid culture of:

Viral system (cytoplasm, integrase, Dsred).

Pnos, to make a miniprep tomorrow and obtain the DNA construction.

17 August 2015

PROTOPLASTS 3.

Take protoplasts samples at time 16h. Big disapoint here, we dscovered hat they were cloroplasts... ha, ha.

Liquid cultures of AsLOVpep (BB); pUPD2 were all blue. The ligation was made again.

Miniprep of Pnos in Agrobacterium.

Transform the ligations in E. coli:

Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc; Ω1
AsLOVpep; pUPD2
LexA:AsLOVpep:ePDZ:ren+OpLexA:luc; Ω1

EXPERIMENT 13.

Prepare the viral system in agrobacterium cultures to infiltrate into sunflowers sprouts by vacuum.

Measurement of the ODs:

DsRed	0.14	71.4ml
Integrase	0.14	71.4ml
Citoplasm	0.12	83.3ml

Make 60ml of medium for each infiltration.

New pieces have arrived:

1st part Lactoferrina
Primer PhiC31 F

EXPERIMENT 11 and 12.

Take samples of leads agroinfiltrated with LTB, IFN and sip rotavirus CH₂ and CH₂-CH₃.

18 August 2015

Make PCRs of PhiC31 and AsLOVpep to eliminate the enzyme recognition sites of Biobricks (BB).

PhiC31 (BB)	AsLOVpep (BB)
1 µl PhiC31	1 µl AsLOVpep
2.5 µl PhiC31 F (1:10)	2.5 µl AsLOVpep nested
2.5 µl PhiC31 Rint (1:10)	2.5 µl AsLOVpep Fint
Tm=44ºC	Tm=59ºC

Program: 96ºC-10’; 96ºC-30’’; Tm-30’’ and 72ºC-30’’

Ligation of the PCRs products:

PhiC31 (BB); pUPD2	AsLOVpep (BB); pUPD2
1µl Phi1	1µl AsLOVpep1
1µl Phi2	1µl AsLOVpep nested
1µl Phi3	1µl pUPD2
1µl Phi4	4.6µl H₂O
1µl pUPD2
2.6µl H₂O

Transform in Agrobacterium:

OpLexA:luc:SF:ren; α1
OpUAS:luc:SF:ren; α1
OpEtr8:luc:SF:ren; α1
OpLacI:luc:SF:ren; α1

Make liquid culture of:

Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc; Ω1 (C1 and C2)
LexA:AsLOVpep:ePDZ:ren+OpLexA:luc; Ω1 (C1-C3)

We start to do the western to see if any drugs had been produced in our plants.

The gel order is:

IFN 1	IFN2	LTB1 (10µL)	LTB1 (10 µL)	LTB2 (30 µL)

Sip 1 CH₂	Sip2 CH₂	Sip 1 CH₂-CH₃	Sip 2 CH₂-CH₃

FOTO

Make a gel with the PCRs products:

PhiC31 and AsLOVpep modified for BioBricks. NO SE LAS BANDAS ESPERADAS.

We change a little bit the protocol to make protoplasts, now we follow a protocol found in a Nature paper. The important changes are that during the digestion of the cell wall there is no agitation.

Transform into E. coli the PCRs products:

PhiC31 (BB)
AsLOVpep (BB)

Prepare liquid culture of:

LTB; α1
Gal4:AsLOVpep:ePDZ; Ω1
LacI:AsLOVpep:ePDZ; Ω1
LexA:AsLOVpep:ePDZ; Ω1
ePDZ; pPUD2
AsLOVpep; pUPD2
NDronpa; pUPD2
Gal4:AsLOVpep; α1
RepPhiC31; pPUPD2
LexA:AsLOVpep; α1

19 August 2015

Miniprep of the liquid cultures:

Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc; Ω1 (C1 and C2), did not grow.

Digestions:

LTB; α1	EcoRI	6345, 1684
Gal4:AsLOVpep:ePDZ; Ω1	BamHI	6674, 4270
LacI:AsLOVpep:ePDZ; Ω1	BamHI	6674, 5041
LexA:AsLOVpep:ePDZ; Ω1	BamHI	6674, 4309
ePDZ; pPUD2	NotI	2046, 642
AsLOVpep; pPUD2	NotI	2046, 521
NDronpa; pPUD2	NotI	2046, 735
Gal4:AsLOVpep; α1	EcoRI	6345, 1972
RepPhiC31: pPUD2	NotI	2046, 475
LexA:AsLOVpep; α1	EcoRI	6345, 2016
LexA:AsLOVpep:ePDZ:ren+OpLexA:luc; Ω1	BamHI	6674, 11403

Gel:

LTB; α1	NDronpa; pPUD2	RepPhiC31: pPUD2	AsLOVpep; pPUD2
ok	ok	ok	ok
LexA:AsLOVpep	Gal4:AsLOVpep	LTB; α1	LexA:AsLOVpep; α1
ok	ok	ok	Ok
LacI:AsLOVpep:ePDZ	Gal4:AsLOVpep:ePDZ	LexA:AsLOVpep:ePDZ:ren+OpLexA:luc C2	LexA:AsLOVpep:ePDZ:ren+OpLexA:luc C3
ok	ok	ok	Ok

Make liquid culture of:

PhiC31 (BB) (C1-C9)
AsLOVpep (BB) (C1-C3)

Also had grown the liquid cultures:

LTB; pUPD2
LacI:AsLOVpep:ePDZ:ren; α1 (C1 and C2)
Gal4;pUPD2 and RepBxbI; α2 did not grow.

20 August 2015

All the liquid cultures of AsLOVpep (BB) and PhiC31 (BB) had grown. Do 12 minipreps.

Digestions of the minipreps:

AsLOVpep (BB)	NotI	2046, 518
PhiC31 (BB)	NotI	2046, 1899

Do the gel:

AsLOV C1	AsLOV C2	AsLOV C3	Phi C1	Phi C2	Phi C3
Ok	ok	ok	no	no	no
Phi C4	Phi C5	Phi C6	Phi C7	Phi C8	Phi C9
No	no	Ok?	Ok?	no	No

We keep the three colonies of AsLOVpep but we make another digestion with different enzymes of PhiC31 colonies C6 and C7.

Optimized ligation:

Gal4:AsLOVpep:ePDZ:ren+OpUAS:luc; Ω1

2.5 µl Gal4:AsLOVpep:ePDZ:ren

6 µl OpUAS:luc

0.5 µl Ω1

0 µl H₂O

Make glycerinates of:

ePDZ; pUPD2
NDronpa; pUPD2
RepPhiC31; pUPD2
AsLOVpep; pUPD2
LexA:AsLOVpep; α1
Gal4:AsLOVpep; α1
LexA:AsLOVpep:ePDZ; Ω1
LacI:AsLOVpep:ePDZ; Ω1
Gal4:AsLOVpep:ePDZ; Ω1
LexA:AsLOVpep:ePDZ:ren:OpLexA:luc; Ω1

Make liquid culture of positive controls in Agro:

OpUAS:luc:ren
OpLexA:luc:ren
OpLacI:luc:ren
OpEtr8:luc:ren

Prepare the liquid cultures of Agrobacteriumto agroinfiltrate:

IFN	0.26	77 µl/ng
LTB	0.22	91 µl/ng
Sip CH₂-CH₃	0.12	167 µl/ng
Sip CH₂	0.09	223 µl/ng

The total volume of preparation will be 6ml each drug.

EXPERIMENT 14.

IFN
LTB
Sip CH₂-CH₃
Sip CH₂

Refresh more colonies to make more glycerinates and pick two colonies of the petri dish culture:

Gal4:KDronpa:NDronpa:ren:OpUAS:luc; Ω1
LexA:KDronpa:NDronpa:ren:OpLexA:luc; Ω1
LTB; pUPD2
LTB; α1
Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc; Ω1 (C1 and C2)

Miniprep of the liquid culture:

LacI:AsLOVpep; α1 (C1 and C2)

Digestion:

LacI:AsLOVpep

EcoRI

8345, 2743

New digestion to PhiC31 (BB):

PhiC31 (BB)	AatII	2635, 1310
PhiC31 (BB)	BanI	3497, 448

Make the gel:

PhiC31(BB) C6 (banI)	PhiC31(BB) C7 (BanI)	PhiC31(BB) C6 (AatII)	PhiC31(BB) C7 (AatII)
Ok	no	ok	no

Transformation in Agrobacteriumand make petri dishe culture of :

LexA:AsLOVpep:ePDZ:ren:OpLexA:luc; Ω1

21 August 2015

Miniprep of:

Gal4:KDronpa:NDronpa:ren:OpUAS:luc
LexA:KDronpa:NDronpa:ren:OpLexA:luc
LTB; pUPD2
LTB; α1
Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc; Ω1 (C1 and C2)

Digestions of the minipreps:

Gal4:KDronpa:NDronpa:ren:OpUAS:luc; Ω1	BamHI	6674, 11582
LexA:KDronpa:NDronpa:ren:OpLexA:luc; Ω1	BamHI	6674, 11799
LTB; pUPD2	NotI	2046, 474
LTB; α1	EcoRI	1684, 6345
Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc; Ω1	BamHI	6674, 11186

Do the gel:

LTB; pUPD2	LTB; α1	LTB; α1	Gal4:KDronpa:NDronpa:ren:OpUAS:luc; Ω1
No	no	no	ok
LexA:KDronpa:NDronpa:ren:OpLexA:luc; Ω1	Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc; Ω1 C1	Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc; Ω1 C2
Ok	no	no

We make glicerynates of:

Gal4:KDronpa:NDronpa:ren:OpUAS:luc; Ω1
LexA:KDronpa:NDronpa:ren:OpLexA:luc; Ω1

Transform into E. coli and make Petri dish culture:

Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc; Ω1

Refresh this cultures to agroinfiltrate:

Pnos
LacI:AsLOVpep:ePDZ:ren:OpLacI:luc

Still trying how to make protoplasts… We will win this battle!

22 August 2015

Make liquid culture of the culture of Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc; Ω1. There was only one white colony, nothing hopeful…

We fount that the culture of Pnos in Agrobacterium did not grow, so we decided not to carry on with the experiment because it was our control. Refresh again two different cultures of Pnos that were found in the fridge and do the experiment tomorrow.

Transform into Agrobacteriumthe following constructions:

E:PIF6:PhyB
LexA:PIF6:PhyB
LacI:PIF6:PhyB
Gal4:PIF6:PhyB

Miniprep of:

Pnos1 (agro)
Pnos2 (Agro)
PhiC31 (BB); pUPD2 (C6)

Digestion:

PhiC31 (BB); pUPD2	NotI	2046, 1899
Pnos	EcoRI	7317, 634, 11
LTB; pUPD2	NotI	2046, 474
LTB; α1	EcoRI	1684, 6345

Gel:

LTB; α1	LTB; pUPD2	Pnos 1	Pnos 2	PhiC31 (BB)
Ok	no	?	?	Ok

As the Pnos construction was given to us form a lab partner, we do not surely that the expected bands were correct because we choose the construction at the golden braid register parts.

We can not make any glicerynate because it was not the necessary amount of liquid culture to make it. Refresh again: PhiC31 (BB); pUPD2; LTB; α1.

23 August 2015

Prepare the Agrobacteriumcultures to agroinfiltrate:

Pnos and LacI:AsLOVpep:ePDZ:ren:OpLacI:luc.

Pnos	0.30	1ml/15ml
LacI:AsLOVpep:ePDZ:ren:OpLacI:luc	0.34
OpLacI:luc:ren	0.47

EXPERIMENT 15.

Pnos
LacI:AsLOVpep:ePDZ:ren:OpLacI:luc.
OpLacI:luc

PhiC31 (BB); pUPD2
Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc; Ω1
LTB; α1 did not grow.

Make liquid culture of agro colonies:

LexA:AsLOVpep:ePDZ:ren:OpLexA:luc; Ω1

24 August 2015

Digestion of yesterday minipreps:

Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc	BamHI	11186, 6674
PhiC31 (BB)	NotI	2096, 1899

Do a gel:

Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc	PhiC31 (BB)
Ok	ok

It had arrived the second part of the lactoferrin.

Resuspend them 100 µl.

Ligation into pUPD2 of both parts of the lactoferrin and other ligation:

Lactoferrina; pUPD2	PhiC31; α1
1 µl Part1 Lactoferrin	1 µl PhiC31
1 µl Part2 Lactoferrin	1 µl 35S
1 µl pUPD2	1 µl T35S
4.6 µl H₂O	1 µl α1
	3.6 µl H₂O

Transform into E. coli the ligations.

25 August 2015

Refresh PhiC31 (BB) and Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc to make glycerinates.

Transfrorm into Agrobacteriumthe construction:

Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc; Ω1

Keep trying protoplasts, this time we see them in the microscope without coverslip. We can see them alive, maybe this was the error. Also we decided to leave the digestion enzymes overnight because after only 3 hours the amount of protoplasts was very little.

EXPERIMENT 14.

Take leafs agroinflitrated with the drugs

EXPERIMENT 15.

Do discs of the agroinfiltrated lefs with the blue toggle (LacI:AsLOVpep:ePDZ:ren:OpLacI:luc) and the control Pnos.

FOTO DE LA PLACA

Make liquid cultures of yesterday transformations:

Lactoferrina; pUPD2 (C1-C3)
PhiC31; α1 (C1-C3)

26 August 2015

Minipreps of:

Lactoferrina; pUPD2 (C1-C3)
PhiC31; α1 (C1-C3)
PhiC31; pUPD2 (BB)
Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc

Digestions of the minnipeps:

Lactoferrina; pUPD2	NotI	2046, 2142
PhiC31; α1	EcoRI	6345, 3127
PhiC31; pUPD2 (BB)	NotI	2046, 1899
Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc	BamHI	6674, 11191

Gel:

PhiC31; α1 C1	PhiC31; α1 C2	PhiC31; α1 C3	PhiC31; α1 C4	Lactoferrina C1
ok	no	ok	ok	ok
Lactoferrina C2	Lactoferrina C3	Lactoferrina C4	PhiC31; pUPD2	Gal4:AsLOVpep:ePDZ:
ren:OpUAS:luc
ok	ok	ok	ok	ok

Do glycerinates of:

Gal4:AsLOVpep:ePDZ:ren:OpUAS:luc; Ω1
PhiC31; pUPD2 (BB)

Ligations:

PhiC31+RepPhiC31:GFP; Ω1	35S+Lactoferrin+T35S; α1
1µl PhiC31	1µl 35S
1µl RepPhiC31:GFP	1µl T35S
1µl Ω1	1µl Lactoferrin; pUPD2
4.6µl H₂O	1µl α1
	3.6µl H₂O

Take samples of the glycerinates:

35S:GFP:T35S (this is necessary to transform into protoplasts and see if the expressed the constructions that we introduce to them)
Alpha1
Omega1

Refresh the Agrobacteriumculture to make agroinfiltration tomorrow of:

Gal4:KDronpa:NDronpa:ren:OpUAS:luc

27 August 2015

Make liquid culture of the Agrobacteriumcolony of Gal4:AsLOVpep:ePDZ:ren:OPUAS:luc; Ω1.

Minipreps of:

Gal4:KDronpa:NDronpa:ren:OpUAS:luc
35S:GFP:T35S
Alpha1
Omega1

Digestions:

Gal4:KDronpa:NDronpa:ren:OpUAS:luc	BamHI	6674, 11191
35S:GFP:T35S	EcoRI	2580, 2274
Alpha1	Nothing	6953
Omega1	Nothing	7295

Gel:

Alpha1	Omega1	35S:GFP:T35S	Gal4:KDronpa:NDronpa:
ren:OpUAS:luc
ok	ok	ok	Ok

Transform into E. coli yesterday ligations:

PhiC31+RepPhiC31:GFP; Ω1
35S+Lactoferrin+T35S; α1

Take samples of the glycerinates and make liquid culture:

Signal peptide (GB0393), in E. coli

Make a glycerinates of Gal4:KDronpa:NDronpa:ren:OpUAS:luc; Ω1

EXPERIMENT 15.

Take samples at 48h and store them at -80ºC in the box “The rebellion of AsLOV”.

PROTOPLASTS 4.

Do protoplasts to transform them tomorrow.

28 August 2015

Miniprep of: 35S:GFP:Tnos.

Digestion of the miniprep:

35S:GFP:Tnos

NotI

2981, 140

Gel:

Correct.

Ligation:

Lactoferrina; α1

1µl Lactoferrina

1µl 35S

1µl T35S

1µl GB393

2.6µl H₂O

Measure the OD of GFP: 157.5. It the right curve.

Make liquid culture of the only white colony of PhiC31+RepPhiC31:GFP; Ω1

PROTOPLASTS 4.

Finish the preparation of protoplasts and transform them with 40 µl of 35S:GFP:Tnos at a concentration of 157.5ng/ml.

After the transformation of the protoplast we see them at the microscope and there were no protoplasts.

Take samples of glycerinates and make liquid cultures of:

OpLexA:mini35S (GB0733)
OpLacI:mini35S (GB534)
OpUAS:mini35S (GB0672)
DsRed (GB0672)

Transform and make petri dish culture of Lactoferrin; α1 ligation.

29 August 2015

Miniprep of the glycerinates’ liquid cultures:

OpLexA:mini35S (GB0733)
OpLacI:mini35S (GB534)
OpUAS:mini35S (GB0672)
DsRed (GB0672)
35S:GFP:Tnos; α1

Refresh the culture of Gal4:KDronpa:NDronpa:ren:OpUAS:luc; Ω1 due to that in the last transformation of protoplasts we run out all the DNA.

Refresh and make liquid culture of

Dsred (GB672) in case the DNA concentration of the miniprep was low.
Staffer fragment in Ω2.

Digestion of the minipreps:

OpLexA:mini35S (GB0733); pUPD2	NotI	2981, 477
OpLacI:mini35S (GB534); pUPD2	NotI	2981, 878
OpUAS:mini35S (GB0672); pUPD2	NotI	2981, 477
DsRed (GB0672); α1	EcoRI	2580, 2274
35S:GFP:Tnos; α1	EcoRI	2580, 2274

Do the gel:

OpUAS:mini35S (GB0672); pUPD2	OpLexA:mini35S (GB0733); pUPD2	OpLacI:mini35S (GB534); pUPD2	OpLacI:mini35S (GB534); pUPD2	35S:GFP:Tnos; α1
ok	ok	ok	ok	ok

Make liquid culture of Lactoferrin; α1 (C1-C4)

30 August 2015

Miniprep of the liquid cultures, lactoferrin C1 and C4 did not grow. We do 5 minipreps.

Digestion of the minipreps:

Gal4:KDronpa:NDronpa:ren:OpUAS:luc; Ω1	BamHI	6674, 11191
Dsred (GB672); α 1	EcoRI
Staffer fragment in Ω2	EcoRV
Lactoferrin; α1	EcoRI	6345, 3436

Gel(we repeat it because yesterday gel was very bad):

OpLacI:mini35S (GB534); pUPD2	OpLexA:mini35S (GB0733); pUPD2	OpUAS:mini35S (GB0672); pUPD2	DsRed (GB0672); pUPD2	Mini35S:Dsred; α1
ok	ok	ok	ok	Ok
Lactoferrin; α1 (C2)	Lactoferrin; α1 (C3)	Gal4:KDronpa:NDronpa:ren:OpUAS:luc; Ω1	Staffer fragment in Ω2
no	no	ok	ok

Ligations: (we make this because the pieze Operator+mini35S can not be ligated with the other constructions in Benchlig so we will join them all in this new ligations).

OpLacI:mini35S+Dsred+T35S; α2	OpLexA:mini35S+DsRed+T35S; α2	OpUAS:mini35S+DsRed+T35S; α2
1µl OpLacI	1µl OpLexA	1µl OpUAS
1µl Dsred	1µl Dsred	1µl Dsred
1µl T35S	1µl T35S	1µl T35S
1µl mini35S	1µl mini35S	1µl mini35S
1µl α2	1µl α2	1µl α2
3.6 µl H₂O	3.6 µl H₂O	3.6 µl H₂O

Gal4:KDronpa:NDronpa+SF(Ω2); α1	LexA:KDronpa:NDronpa+SF(Ω2); α1	LacI:KDronpa:NDronpa+SF(Ω2); α1
1µl Gal4:KDronpa:NDronpa	1µl LexA:KDronpa:NDronpa	1µl LacI:KDronpa:NDronpa
1µl SF(Ω2)	1µl SF(Ω2)	1µl SF(Ω2)
1µl α1	1µl α1	1µl α1
4.6µl H₂O	4.6µl H₂O	4.6µl H₂O

Lactoferrin; α1

1µl Lactoferrin

1µl 35S

1µl T35S

1µl α1

2.6 µl H₂O

31 August 2015

Transform the ligations into E. coli adding IPTG and X-Gal.

Ligation:

PhiC31+RepPhiC31:GFP; Ω1

1µl PhiC31 (BB); α1

1µl RepPhiC31:GFP; α2

1µl Ω1

4.6µl H₂O

The collaborations part of the Norwich iGEM team have arrived:

This are the Mofippers (cloranfenicol resistance):

0408	Promoters	224 ng/µl	7 µl
0409	CDs	77 ng/µl	3 µl
0410	terminator	159 ng/µl	10 µl
0411	TUs	158 ng/µl	10 µl

Añadir una foto de las placas en la wiki y enlace de la wiki de los the norwich

This are the constructions (carbenicillin resistance) that we are going to prove in our plant.

584-MAA with YFP
596-RV3034c with YFP
598-RV3037c with YFP
600-RV3030 with YFP

Transform the Moflippers into E. coli to have later more DNA to make the ligations and be able to send the parts with the necessary characteristics to the BioBricks requirements.

Transform the functional constructions into Agrobacteriumand make petri dishes cultures.

Refresh liquid cultures of Agrobacteriumto make an experiment with red toggle:

LexA:PIF6:PhyB
Gal4:PIF6:PhyB
LacI:PIF6:PhyB
OpLexA:ren:luc
OpLacI:ren:luc
OpUAS:ren:luc
Pnos

Go to Protocols

Scroll

@@ Line 10: / Line 10: @@
 <section id="banner">
-	<h2><b style="color:white">Notebook</b></h2>
+	<h2>Notebook</h2>
 	<p>Daily notebook</p>
 	<ul class="actions">