Valencia UPV iGEM 2015
Valencia UPV Team
Be patient, we are under construction
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%
FOTO
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; Ω1 Etr8 CMV+Bxb1_T35S; α1 1µL (GB892) PIF; α1 1µL 1097 (Etr8 CMV) pUPD2 1µL (GB88E) PhyB; α2 1µL Bxb1; pUPD2 1µL Ω1 1µL Tnos PuPD 1.2µL Buffer ligase 1µL α1 1µL Bsmb1 5.8µL H2 O 6.8µL H2 O
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 ? ? FOTO 7 June 2015
We?ve got white colonies from PIF+Phy and Bxb1! 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:
Bxb1 (C1) Bxb1 (C2) EPIF6 + PhyB-PV16 (C1) EPIF6 + PhyB-PV16 (C2) ok ok
FOTO
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-Phy-Luc-Renilla-P19 1 µL vector 0.8 µL dilution ½ GB160 1.7 µL PIF:PhyB 4.15 µL H2 O 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
FOTO
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
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 Agrobacterium EPIF6-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:
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
FOTO
Transformation into Agrobacterium of 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.
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