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.

< /tr>

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.