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 H2O |
| 6.8µL 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 | ? | ? |
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 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 |
FOTO
We see three bands: 7000, 4000, 1900pb
Transform optimized ligation PIF-Phy-Luc-Renilla-P19 and make petri dish cultures.