Team:Valencia UPV/Notebook

Valencia UPV iGEM 2015

The project
Notebook

CONSTRUCTIONS

5 June 2015

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

Agrobacterium culture of promoter less: Luciferase + Renilla

Minipreps

Digestion with BamHI and EcoRV

Agarose gel 1%

How to ask and make primers?

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

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

Meeting with Daniel Ramón (Biopolis).

Ligation with part 2 and 24 of task sheet.

PIF6 + PhyB; ?1	Etr8 CMV_Bxb1_T35S
1µL 892 (PIF α1)	1µL 1097 (Etr8 CMV) Pupd2
1µL 88E (Phy α2)	1µL Bxb1 (PuPD)
1µL ?1	1µL Tnos PuPD
1.2µL Buffer ligase	1µL α1
1µL Bsmb1	5.8µL H2O
6.8µL H2O

If we make a digestion of 160 (35S:Renilla:tNOS-35S:P19:tNOS) with EcoRV, we obtain: 2475, 381, 4601 pb.

If we make a digestion of 896 (Luc:PIF6:PhyB)with EcoRV, we obtain: 11608, 3942 pb.

June 2015

Transform to E.coli from PIF+Phy and Bxb1

1.5µL of ligation

Cuvette on ice
Competent cells + 1.5µL of ligation
Pulse (electroporator) at 1500V
Add 300µL shock medium and put Eppendorf 1h at 37ºC

Culture on petri dishes the ligations.

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

Agarose gel.

7 June 2015

We’ve got white colonies! (from PIF+Phy and Bxb1)

Pick two colonies from each construction.

4 tubes

3.5µL LB each tube

2) 2 tubes + 3.5µL Kanamycin (K)

8 June 2015

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

Digestion:

Etr8(CMV):Bxb1:Tnos; ?1	EcoRI	6345, 238
EPIF6 + PhyB-PV16; ?1	BamHI	6686, 1439, 2685, 2237

Agarose gel was made:

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

Repeat digestion (errors).

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

Optimized ligation of PIF-Phy-Lac-Renilla-P19

1 µL vector
0.8 µL dilution ½ 160
1.7 µL big part
1.2 µL BSA
1.2 µL buffer
1 µL BsbmI
1 µL ligase
4.15 µL H2O
Ratio 1:2 vector insert

As Bxb1 was good at the digestion we put 1 µL of LB and 1 µL of Kanamicyne on the tube where it had grown and store at 37ºC to glycerinate later.

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

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

9 June 2015

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

EPIF6-PhyB-VP16

PvuII (green buffer)

3663, 9472pb

Agarose gel 1%:

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

We see three bands: 7000, 4000, 1900pb

Transform optimized ligation (yesterday 8/6)

3.5 µL ligation product (EPIF6-PhyB-VP16 + Luciferase + Renille + P19)
40 µL electrocompetent cells.
Pulse of 1500V
Store 1h at 37ºC
Plate culture at agar with Spectinomycin: 50 µL of the transformation.

10 June 2015

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

Alfredo’s part is not working.

Pick colonies of transformation and make liquid culture of E:PIF6:PhyB:VP16:luc:ren (C1-C3).

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

PIF + Phy:VP16 PvuII (buffer green 10x) 3663, 9472

PIF + Phy:VP16 BamHI 1939, 2685, 2337, 6674

Agarose gel 1% (10 samples, 8 + 2 molecular markers)

PIF + Phy (PvuII) C3 PIF + Phy (PvuII) C4 PIF + Phy (PvuII) C5 PIF + Phy (PvuII) C6

no ok no No

PIF + Phy (BamHI) C3 PIF + Phy (BamHI) C4 PIF + Phy (BamHI) C5 PIF + Phy (BamHI) C6

no ok no No

Transformation of Agrobacterium (C58) of the 896 construction (EPIF6-PhyB-VP16 + luciferase). We are not going to have the positive control and we won’t be able to quantify (we don’t have Renilla + P19).

11 June 2015

Minipreps of the culture:

Digestion:

E:PIF6:PhyB:VP16:luc:ren BamHI 4209, 3756, 6100, 6674

EcoRV 3942, 2989, 2475, 381, 10952

Gel:

PIF6:PhyB:VP16:luc:
ren C1 (BamHI)	PIF6:PhyB:VP16:luc:
ren C3 (BamHI)	PIF6:PhyB:VP16:luc:ren
C1 (EcoRV)	PIF6:PhyB:VP16:luc:ren
C3 (EcoRV)
??

Transformation in Agrobacterium of Renilla (160) due to that we could not join this with PIF:phyB and so we will do a cotransfection of both plasmids. Make petri dish culture with kanamicyn and rifampicin.

12 June 2015

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

13 June 2015

Pick colonies to make liquid culture:

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

Ligation:

BxbI; alfa1+phyB; alfa2

1µl BxbI

1 µl phyB

1 µl ?2

4.6 µl H2O

Transform renilla (160) into agrobacterium and make

15 June 2015

Repeat the ligation BxbI+35S:E-PIF6:tnos because PIF was ?2

BxbI + 35S:E-PIF6:tnos; ?1

1µl BxbI

1 µl phyB

1 µl ?1

4.6 µl H2O

KDronpa has arrived:

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

Ligation: en la libreta pone que se liga a pUPD2

KDronpa; pUPD2

1 µl KDronpa

1 µl pUPD2

5.6 µl H2O

It was not possible to pick colonies of the Agrobacterium transformed because they did not grow. Maybe the problem is that with tetraciclyn bacterias grow slowly. Wait 1 day more.
Transformation of the ligation, BxbI + 35S:E-PIF6:tnos; ?1, into E.coli.
Make an agar culture in petri dish and let grow 16h at 37ºC.

16 June 2015

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

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

A PCR with all the primers and the fragments was done, the samples were put in order with the temperature.

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

PCR Fusion Taq (50µl)

DNA template (10 µg/µl)

0.5 µl fusion taq

2.5 µl primer F

2.5 µl primer R

2 µl NTPs

31.5 µl H2O

17 June 2015

Pick colonies and make liquid culture of:

KDronpa (C1-C4)
Ligations with the PCR’s products:
Templates PCR: 1+2, 5+6, 7+8PIF, 7+8VP16, 9+10, 11+12.

Template PCR; pUPD2

0.5µl template

1µl pUPD2

6.1µl H2O

Minipreps of liquid cultures:

BxbI:E-PIF6 (C1-C3)

Agarose gel with the PCRs:

Template 1+2 5+6 7+8PIF 7+8VP16 9+10 11+12

Band pattern 1017 284 391 478 464 290

Gel result ok ok ok ok No DNA ok

Transformation in E.coli of the correct ligations:

1+2, 5+6, 7+8PIF, 7+8VP16, 11+12
Put in cloranfenicol petri dishes.

18 June 2015

Minipreps of the liquid cultrures:

KDronpa (C1-C4)

Digestions:

KDronpa EcoRI 2800

Gel:

Kdronpa C1 Kdronpa C2 Kdronpa C3 KdronpaC4

no no ok no

Etr8:BxbI:phyB C1 Etr8:BxbI:phyB C2 Etr8:BxbI:phyB C3

No no no

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

Take glicerynates out:

Gal4; pUPD (731)
?2
PromotersinATG (GB00552)
Renilla (160)(159)(109)

PCR:

NDronpa

2.5 µl (9+10) primer F

2.5 µl (11+12) primer R

2 µl NTPs

0.2 µl Taq

10 µl Buffer

31.5 µl H2O

Ligations:

Etr8:BxbI:T35S; α1 Template PCR; pUPD2

1 µlEtr8 0.5µl template

1 µl BxbI 1µl pUPD2

1 µl T35S 6.1µl H2O

5.8 µl H2O

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.
To do the digestions we add in each eppendorf.

Minipreps: Enzime Band pattern

159 pDGB1_?2 renilla EcoRV 2909, 2475,882, 812, 381

Entry vector, ?2 EcoRV 6652, 621

552 pP35s NoATG, pUPD EcoRI 2997, 1090

160 renilla pDGB1, a2 EcoRV 4601, 2475, 381

731 pUPD pGal4BD (CDS) EcoRI 2997, 2493

109 GB1_a1 355:renilla:Tnos EcoRI 2580, 2493

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

LexA:PIF:Phy:ren:luc	BamHI	9431, 6674, 3513
PhyC31	NotI	2046, 1899

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

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

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

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

Team:Valencia UPV/Notebook

Notebook

20 june 2015

21 June 2015

22 June 2015

23 June 2015

24 June 2015

25 June 2015

26 June 2015

27 June 2015

28 June 2015

29 June 2015

30 June 2015

1 July 2015

2 July 2015

4 July 2015

5 July 2015

6 July 2015

7 July 2015

8 July 2015

9 July 2015

10 July 2015

11 July 2015

12 July 2015

13 July 2015

14 July 2015

15 July 2015

16 July 2015

17 July 2015

18 July 2015

19 July 2015

20 July 2015

21 July 2015

22 July 2015

23 July 2015

24 July 2015

26 July 2015

27 July 2015

28 July 2015

29 July 2015

30 July 2015

31 July 2015

1 August 2015

3 August 2015

4 August 2015

5 August 2015

6 August 2015

7 August 2015

8 August 2015

9 August 2015

10 August 2015

11 August 2015

12 August 2015

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

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

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

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

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

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

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

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

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