Team:Valencia UPV/Notebook
Starts our work in the lab! Marta, a lab mate gives us a construction, the red toggle swich (E:PIF6:PhyB:VP16:Etr8:luc), we just have to add the
renilla; α2 (GB160) to test it. Make the ligation (step 2 in the protocol): Electroporation (step 3 in the protocol) of E. coli to insert our first construction. Make a petri dish culture with a LB-Agar plate with streptomycin. There is no white colonies, we electroporate again and make petri dish culture. There was just one white colony, make the ligation again. Electroporation of the new ligation. There are white colonies. Make 2 liquid cultures of them (Step 4 in the protocol). Add 3.5ml of LB and 3.5µL of
spectomycin. Make liquid culture of just some glycerinates: Al cultures have grown except for Ω2. Make minipreps (Step 5 in the protocol). Digestion of the minipreps (Step 6 of the protocol). Make the gel: 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. 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? Meeting with Daniel Ramón (Biopolis). Ligations: Digestions: 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. We’ve got white colonies from PIF+Phy and BxbI! Pick two colonies from each construction. Minipreps of the 4 liquid cultures and digestion to see the band patterns. Digestion: Agarose gel was made: 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: 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. Digestion of the ligation of yesterday containing: EPIF6-PhyB-VP16 (C1 y C2) Agarose gel 1%: We see three bands: 7000, 4000, 1900pb Transform optimized ligation PIF-Phy-Luc-Renilla-P19 and make petri dish cultures. Alfredo’s part is not working. Gel: 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. The petri dish with PIF:PhyB:luc was taken out the 37ºC room and put into the fridge to pick colonies tomorrow. Pick colonies to make liquid culture: KDronpa EcoRI 2800 We discovered that the construction with BxbI did not go well because our lab college gives us the wrong piece. Thanks
Alfredo :) Templates PCR: 1+2, 5+6, 7+8PIF, 7+8VP16 These quantities multiplied by 3. 1+2, 5+6, 7+8PIF, 7+8VP16 all in pUPD2 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. Make an agarose gel with all the digestions: FOTO Pick colonies of the plates done yesterday and pass them into a liquid medium: 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. Quantification of DNA: Minipreps of the liquid culture: Digestion of the minipreps and do the gel: Gel: 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. Do ligations: Digestion: Gel: 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. 1?EXPERIMENT. Red toggle. E:PIF6:PhyB and renilla. For more info, click here. 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: Do the minipreps of the liquid cultures that have grown. Do the digestions of the minipreps: Make the gel. Take glycerinated: Do the minipreps of the 4 colonies of LexABD and both glycerinates, 35S and T35S. Do the digestion of the minipreps: Make the gel: Make ligations: 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: Transform Gal4+KDronpa and LacI+KDronpa and make petri dish culture. Miniprep of: Gel: 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.Notebook
27 May 2015
E:PIF6:PhyB:VP16:Etr8:luc+ren; Ω1 1µL E:PIF6:PhyB:VP16:Etr8:luc; α1 1µL renilla; apha2 1µL Ω1 6,8µL H2O 28 May 2015
29 May 2015
30 May 2015
E:PIF6:PhyB:VP16:Etr8:luc+ren; Ω1 0.5µL E:PIF6:PhyB:VP16:Etr8:luc; α1 1µL renilla; apha2 1µL Ω1 7.2µL H2O 1 June 2015
2 June 2015
3 June 2015
α1 - - α2 - - Ω1 - - pUPD2 - - E:PIF6:NLS; pUPD2 (GB0288) EcoRI 3000, 1000 E:PIF6:NLS; α1 (GB892) EcoRI 6300, 2500 E:PIF6:NLS; Ω2 (GB893) EcoRV 1800, 6600, 900 E:PIF6:NLS:luc:PhyB; α1 (GB896) EcoRI 3600, 6300, 5600 Luc:PhyB; Ω1 (GB890) BamHI 2300, 6300, 4200 PhyB:VP16; pUPD2 (GB289) EcoRI 3000, 2000, 500 PhyB:VP16; α2 (GB88E) HindIII 2100, 6300, 1800 Etr8:CMVmini; pUPD2 (GB1097) EcoRI 3000, 480 OpLexA:mini35S; pUPD2 (GB733) EcoRI 3000, 460 OpLexA:mini35S:luc:Tnos; α2 (GB151) HindIII 2500 LexABD; pUPD2 (GB0732) EcoRI 3000, 300 LacI for N-Tfusion; pUPD2 (GB858) EcoRI 3000, 1000 Linker:LacIBD; pUPD2 (GB704) EcoRI 3000, 1000 OpLacI:mini35S:luc:Tnos; α2 (GB152) HindIII 2500, 2600 OpLacI:mini35S; pPUD2 (GB534) EcoRI 3000, 560 E:PIF6:PhyB:VP16:Etr8:luc+ren; Ω1 BamHI 3700, 6100, 6600, 4200 E:PIF6:PhyB:VP16:Etr8:luc+ren; Ω1 EcoRV 11000, 400, 2500, 3000, 4000
pUPD2 Alf Alpha1 288 289 534 ok ok ok ok ok ? 704 732 733 858 892 896 ok ok ok ok ok ok 1097 Alpha2 88E 151 152 Omega1 ok ok ok ok ok ok 890 893 Red toggle (C1) (EcoRV) Red toggle (C1) (BamHI) Red toggle (C2)
(EcoRV) Red toggle (C2) (BamHI) ok ok ok no no no 4 June 2015
5 June 2015
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
(GB160) 35S:Renilla:tNOS-35S:P19:tNOS EcoRV 2475, 381, 4601 (GB896) Luc:PIF6:PhyB EcoRV 11608, 3942 6 June 2015
GB160 289 PIF+PhyB BxbI ok no ? ?
7 June 2015
8 June 2015
Etr8(CMV):Bxb1:Tnos; α1 EcoRI 6345, 238 EPIF6 + PhyB-PV16; Ω1 BamHI 6686, 1439, 2685, 2237
BxbI (C1) BxbI (C2) E:PIF6+PhyB-VP16 (C1) E:PIF6+PhyB-PV16 (C2) ok ok no no
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 9 June 2015
EPIF6-PhyB-VP16 PvuII (green buffer) 3663, 9472pb
EPIF6-PhyB-VP16 (C1) EPIF6-PhyB-VP16 (C2) no no
10 June 2015
PIF+Phy:VP16 PvuII (buffer green 10x) 3663, 9472 PIF+Phy:VP16 BamHI 1939, 2685, 2337, 6674
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 11 June 2015
E:PIF6:PhyB:VP16:luc:ren BamHI 4209, 3756, 6100, 6674 EcoRV 3942, 2989, 2475, 381, 10952
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
12 June 2015
13 June 2015
BxbI; α1+PhyB; α2 1µl BxbI 1 µl PhyB 1 µl Ω2 4.6 µl H2O 15 June 2015
BxbI + 35S:E-PIF6:tnos; Ω1 1µl BxbI 1 µl PhyB 1 µl Ω1 4.6 µl H2O
KDronpa; pUPD2 1 µl KDronpa 1 µl pUPD2 5.6 µl H2O 16 June 2015
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
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
Template PCR; pUPD2 0.5µl template 1µl pUPD2 6.1µl H2O
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 18 June 2015
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
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
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 19 June 2015
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
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
159 160 Ω2 552 731 109 9+10 9+12 11+12 <
/tr>
ok ok ok ok ok ok no ok ok
20 june 2015
21 June 2015
22 June 2015
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
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 23 June 2015
24 June 2015
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 25 June 2015
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
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
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 26 June 2015
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
LacIBD+PIF6; α1 EcoRI 6345, 1997, 641
LacIBD+PIF C1 LacIBD+PIF C2 no no
PhyB:PIF6:luc: 0.35 (1:2) 0.35 1.429 µl Ren+P19: 0.34 (1:2) 0.34 1.412 µl
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 27 June 2015
28 June 2015
LacIBD+PIF; α1 EcoRI 6345, 1997, 641 RepBxbI:GFP; Ω2 HindIII 6345, 2683 Gal4BD; pUPD2 NotI 2681, 644 NDronpa; pUPD2 NotI 2046, 744
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
29 June 2015
LexABD; pPPD2 NotI 2358, 312 35S; pUPD2 NotI 2981, 1074 T35S; pPUD2 NotI 2981, 304
LexA C1 LexA C2 LexA C3 LexA C4 P35S T35S ok ok ok ok Ok? Ok?
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 30 June 2015
RepBxb1+GFP; Ω2 HindIII 6345, 2683
RepBxbI+GFP C1 RepBxbI+GFP C2 RepBxbI+GFP C3 No no no