• 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

Gel:

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