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Team:KULeuven/NotebookDaily
From 2011.igem.org
tuesday 5th of July
Cell culture medium and agar plates containing antibiotics (ampicillin, kanamycin, tetracyclin and chloramphenicol) were prepared. A precul- ture of competent cells was made (protocol competent cells).
wednesday 6th of July
Competent cells were prepared according to the CaCl2 method.
thursday 7th of July
Biobricks were transformed in competent cells. List of biobricks:
1. pLux-CI promotor BBa K091107
2. pLac/Mnt Hybrid Promoter BBa K091104
3. cspA Promoter BBa K328001
4. luxI Bba C0061
5. luxR Bba C0062
6. pconst-RBS-MelA Bb K193602
7. CrtEBI Bba K274100
8. CI repressor BBa R0051
9. Mnt repressor BBa C0072
10. Colicin E2 operon without SOS promotor BBa K131009
11. ribolock3c BBa J23031
12. ribokey3c Bba J23008
13. ribosome binding site BBa B0034
14. terminator BBa B0015
15. OmpA Bba K103006
16. TEV cleavage site Bba K128002
17. GFP Generator BBa E0240
These transformed cells were grown on agar plates containing the cor- rect antibiotic for selection.
friday 8th of July
We observed some colonies on the plates, so the transformation of bio- bricks worked.
tuesday 12th of July
Colonies were picked and grown in liquid medium overnight at 37°C. Another transformation of competent cells with new biobricks was done:
1. BAD promotor BBa I13453 2. ribokey3d BBa J23066
The Cu promotor was replaced by the BAD promotor.
wednesday 13th of July
Minipreparations of the 17 biobricks were performed according to the protocol of the kit (promega miniprep kit) (Fig.1).
thursday 14th of July
New cultures for minipreparations were prepared since the DNA con- centrations of the previous miniprep were too low to go on with restric- tion. A new transformation of the competent cells was performed: luxI BBa C0261 (this will be our new luxI instead of Bba C0061 which we transformed more early).
friday 15th of July
6ml cultures were miniprepped and after DNA gel electrophoresis the concentration of the different biobricks was estimated. The DNA con- centrations were higher than the first miniprep and suffcient to proceed with restriction. (Fig.2)
Fig.1: Miniprep 13/07: L, A= pLux-CI promotor, B= pLac/Mnt Hybrid Promoter, D= Ribolock-Coilicin E2 Dnase, E= ribolock3c, F= ribokey3d, G= GFP Generator, H= CrtEBI, I= CI repressor, J= ribosome binding site, K1= terminator, K2= pconst-RBS-MelA, Z= CspA Promoter, 1= luxI, 2= luxR , 3= Mnt repressor, 4= OmpA , 5= TEV cleavage site, L
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Fig.2: Miniprep 15/07: labelling is the same as in Fig.1 except for K= pconst-RBS-MelA and Y= terminator.
monday18th of July
A restriction digest of the miniprepped biobricks was performed. 500ng of DNA was used for most biobricks, except for those which still had low concentration (a maximum volume of 15µl DNA in a total volume of 20µl was used here). Biobricks A, B, G, H1 (XbaI and PstI), H2 (EcoRI and SpeI), 31 (XbaI and PstI) and 32 (EcoRI and SpeI) were succesfully cut by the restriction enzymes (Fig.3) and gel purified.
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Fig.3: Restriction 18/07: A= pLux-CI promotor, B= pLac/Mnt Hybrid Promoter, I= CI repressor, G= GFP generator, H= CrtEBI, 3= Mnt repressor
Transformation of a constitutive promotor (Bba J23119) was performed.
tuesday 19th of July
DNA gel electrophoresis of the purified DNA fragments was performed and showed there was no DNA left at all. This means we have to do the restriction digest again.
Since one of the biobricks (CI repressor BBa R0051) miniprepped be- fore has never shown any measurable concentrations, we decided to do this transformation again.
wednesday 20th of July
A new restriction digest was performed (Fig.4). Restriction purification of the DNA fragments didn't result in measurable DNA concentrations.
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Fig.4: Restriction 20/07: A= pLux-CI promotor, B= pLac/Mnt Hybrid Promoter, H2= RBS-CrtEBI, K= terminator, Z= cspA Promoter, 31= Mnt repressor were all cut with EcoRI and SpeI. V= luxI, K= terminator, 31= Mnt repressor, H1= RBS-CrtEBI and G= GFP generator were all cut with XbaI and PstI.
thursday 21th of July
Minipreparation of A, B, G, H, I, U, V, W, X and 3 (Fig.5).
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Fig.5: Miniprep 21/07: A= pLux-CI promotor, B= pLac/Mnt Hybrid Promoter, G= GFP generator, H= CrtEBI, I= CI repressor, U= constitutive promotor, V= LuxI, W= BAD promotor, X= Ribokey3d, 3= Mnt repressor
friday 22th of July
Discussing the results of last week and preparing experiments of next week.
monday 25th of July
Searching 2 new promotors since 2 promotors (bricks BBa K091107 and BBa K091104) we planned to use seemed to be something else than shown on the iGEM website. We decided to use pLac-lux hybrid BBa K091100 and lambda cI and luxR regulated-hybrid BBa R0065. We transformed these bricks into competent cells and grew these cells on agar plates. A new restriction digest was performed, this time ac- cording to the standard assembly instead of the 3A assembly method. Restriction of U, W, X, K and Z (Fig.6).
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Fig.6: Restriction 25/07: U= Constitutieve promotor (PstI and SpeI), W= BAD promoter (PstI and SpeI), Z= CspA promotor (PstI and SpeI) K= Terminator (EcoRI and XbaI), X= ribokey3d (EcoRI and XbaI)
All these fragments were gel purified.
tuesday 26th of July
DNA gel electrophoresis of the gel purified fragments of 25/07 (Fig.7).
wednesday 27th of July
Another restriction digest was performed, this time with biobricks GFP generator, LuxR, LuxI, pLac-lux hybrid, lambda cI and luxR regulated- hybrid, the constitutive promotor, BAD promotor and CrtEBI.
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Fig.7: Results of gel purification: U= Constitutive promotor (PstI and SpeI), W= BAD promoter (PstI and SpeI), Z= CspA promotor (PstI and SpeI), K= Terminator (EcoRI and XbaI), X= ribokey3d (EcoRI and XbaI).
thurday 28th of July
Since the restriction of 27th July again didn't gave measurable DNA concentrations after gel purification, a new restriction digest of the same biobricks was performed (Fig.8).
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Fig.8: Restriction 28/07: G= GFP generator, T= LuxR, V= LuxI
The primers of Eurogentec arrived and were used to start PCR of INP, MelA(Bba K193602), Ribolock (BBa J23031) and the 4 IGEM vectors.
friday 29th of July
A new PCR was performed with some adjustments to the protocol to give better results. This PCR resulted in amplification of the 4 vectors and Ribolock.
friday 22th of July
After reformatting the laptop, it took windows 2 hours to update! Installation of maya, an amazing 3D-visualising program, provided by iGEM sponsor the mathworks. We checked the price and normally it costs $3,251! Time to watch tutorials... But first an evening dinner with our team.
monday 25th of July
Importation of 3D- structures of icenucleating protein and anti-freezeprotein in maya: check! Found some cool video made in maya. So, it should be possible to make one, but how long will it takes us?
tuesday 26th of July
Working in simbiology.
wednesday 27th of July
We made a simulation for a hybrid promotor lac_lux (BBa_R091100) which is almost synthesized in the wet lab coupled to GFP. Values of parameters are estimated, a recurring theme in kinetic modelling?
thursday 28th of July
Today we brainstormed on how we could make a molecular movie about are project.
