Team:KULeuven/NotebookDaily

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KULeuven iGEM 2011

Weekly notebook Daily notebook

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)

monday 18th 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. 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.

thursday 21th of July
Minipreparation of A, B, G, H, I, U, V, W, X and 3 (FIG.5).

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).

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.

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).

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.

monday 1th of August
A new restriction was performed with 2000ng instead of 500ng of DNA. After DNA gel electrophoresis, only U (= constitutive promoter, Bba_j23119) and W (= pBAD, Bba_I13453) showed high enough concentration to cut them out of the gel. Since these were the only fragments, we didn’t start ligation.

tuesday 2nd of August
A new minipreparation of all the promoters and GFP was done, this time according to the CTAB method. However, this didn’t result in high enough concentrations to go on with restriction and we decided to make new cultures for a minipreparation tomorrow.

E. Coli MG 1655 cells were made competent and can be used for GFP-testing later.

wednesday 3th of August
Another minipreparation was done and again we didn’t get any results.

thursday 4th of August
Contamination on the agar plates was discovered and we decided to start all over again with the experiments, since we didn’t get any good results lately.

A transformation of promoters W (=pBAD), U(= constitutive promoter), R(=pLac-Lux), S(=plambda CI and LuxR regulated), B (= pLac/Mnt) and insert G (= GFP) was done in DH5alpha cells and TOP10 cells.

friday 5th of August
The transformation of DH5alpha cells seemed to have worked and precultures were made for miniprepping.

saturday 6th of August
The new miniprep didn’t result in DNA.

monday 8th of August
Minipreparations of the promoters and GFP were carried out by Lies Vandesteene of the Molecular Physiology of Plants and Micro-organisms Section. She applied the CTAB method and managed to get good results. She also did a restriction and ligation of the promoters with GFP in TOP10 cells.

We did a transformation of biobricks H (Crt-EBI), I (RBS-CI repressor), X (ribokey) and K (terminator) in TOP10 cells.

Tuesday 9th of August
Instructor Erwin Swinnen carried out a DNA purification of the same biobricks as Lies did the day before. A high DNA yield was obtained. We have enough DNA of the promoters and GFP in stock to go on with restriction and ligation.

10 colonies per ligated fragment were inoculated in LB medium with ampicillin so another minipreparation can be done tomorrow. 3 other biobricks were also inoculated in LB medium (O= hybB promoter, BBa_K410000; T= LuxR, Bba_C0062 and Y= MelA, Bba_K193602) but instead of ampicillin they were inoculated with a different antibiotic.

Wednesday 10th of August
Minipreparations of the ligated fragments were done, followed by restriction digest to check if there are any fragments that are ligated correctly. We used EcoRI and SpeI as restriction enzymes. No correct ligated fragments were discovered and we decided to test these ligations again tomorrow with other restriction enzymes. The 3 other biobricks (pHybB, LuxR and MelA) were also purified and restricted.

Thursday 11th of August
A restriction digest with XbaI and PstI was carried out on the purified DNA of 10/08. There were still no positive clones identified and we decided to do some more minipreparations and test another 40 colonies tomorrow.

A PCR of the HybB promoter (BBa_K410000) was carried out and seemed successful (FIG.9).

Friday 12th of August
The 40 inoculated colonies were miniprepped and restricted and still no positive clone was found. We will switch again to the 3A assembly method next week to get less false positive colonies and we hope this time we will have some more success.

Monday 15th of August
We started the day with restrictions to make sure that the minipreparation of CrtEBI, CI repressor, terminator and the ribokey3d from the past week had been successful. The bands on the gel were inconclusive. We decided to do the restriction again but opted to put the samples on gel on Tuesday. We also PCRed MelA and 4 vectors (psB1A3, pSB1K3, psB1C3 and psB1T3). We also ligated all the promoters excluding hybB with GFP.

Tuesday 16th of August
The restriction started the day before was completed by putting the samples on gel. (We decided to make 2 different gels; a 2% and a 4% gel because of low amount of base pairs of the restricted fragments.) The PCR we did on Monday showed, after loading 3µL onto a gel, no results. We therefore decided to do 2 PCRs: one of psB1A3 and another of psB1A3 containing pBAD. After putting them on gel we saw a band in the lane which contained the plasmid which was PCRed from the plasmid with pBAD.

Wednesday 17th of August
We started the day off with a CTAB minipreparation of the ligated constitutive promoter with GFP and INP and afterwards we did a restriction and purified the samples out of the gel. We also did a restriction of the plasmid which was PCRed the day before.

Thursday 18th of August
The purification protocol usually used has a very low success rate thus today a restriction was purified from gel with a different protocol to see if this one has a higher success rate. The vectors pSB1C3, pSB1K3 and pSB1A3 were miniprepped and restricted. Restrictions were also done on LuxR, pBAD, Ribolock CeaB and ribokey ( no purifications were done). pBAd was ligated to GFP and a transformation of LuxI was also done.

Friday 19th of August
GOOD NEWS: a ligation was successful!!! We started on making the data page.

Monday 22th of August
Restriction was done on the constitutive and HybB promoters. This was followed with ligation of LuxR and pBAD, Ribolock-CeaB and Terminator and HybB and Ribokey-terminator. During the day we also did a 10 minipreparation; 8 of the ligated fragments we ligated last week and 2 of LuxI. The vectors psB1A2, psB1C3 and psB1K3 were PCRed.

Wednesday 24th of August
Two ligations were attempted; 1.Ribokey-terminator and HybB promoter and 2. Terminator and INP. Restriction of these biobricks were done prior to the ligations.

Thursday 25th of August
The ligation of Ribokey-terminator and HybB promoter was successful in contrast to the ligation of the terminator and INP. Restriction of the terminator was because of this repeated.

Monday 29th of August
The GFP testing was continued. The PCR of LuxI, done last week with the general primers, keep failing thus we suspect that the functionality of the primers is the cause of the continual failure. To test this we PCRed LuxI and HybB with the general primers and when we put the PCR on gel, we saw no bands and have concluded that the general primers are faulty.

Tuesday 30th of August
We did minipreparations of Ribolock CeaB – Terminator and INP – Terminator. Afterwards the fragments were restricted and then ligated to a promoter. Before the restriction we put the fragments on gel to make sure that the ligation was successful. A transformation of AFP was also done.

Wednesday 31th of August
The multiple restrictions and ligations were done; INP-Terminator with pLac-Lux and constitutive promoter and AFP with constitutive and Lambda cI promoter. A control ligation was made for all the promoters. In preparation for the following day the restriction of GFP and LuxR were done too.

Thursday 1th of September
On the plates with the ligated INP were successful in contrast to the AFP ligations. We opted to repeat the ligation of AFP with Lambda cI. A CTAB minipreparation was also done followed by a control restriction.

Monday 5th of September
A control restriction was done on the CTAB products from last Friday. Due to the failure of Fridays ligations, we did the ligations again together with the restriction and ligation of AFP with the constitutive promoter.

Wednesday 7th of September
We miniprepped the ligated products and did a control restriction with EcroRI and PstI. Unfortunately the results were not what we expected. The results of GFP test from the past week were inconclusive so we decided to repeat it. GFP testing was done on the promoters pBAD-GFP and constitutive promoter – GFP (Both induced with arabinose in two types of ''E.coli'' TOP10’ and MG1655).

Thursday 8th of September
The ligations of AFP and INP-Terminator to their promoters was repeated. We did GFP testing on pLAc-Lux – GFP and Constitutive promoter – GFP, both induced with IPTG again in the two types of ''E.coli'').

Friday 9th of September
The ligation of INP – Terminator were unsuccessful again so we repeated the ligation again together with the ligation of GFP to the HybB promoter.

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.

monday 1st of August
We have designed some constructs of biobricks for the detailed project description.

tuesday 2nd of August
Working on project animations.

thursday 4th of August
Working on detailed project description. We started with antifreeze construct.

friday 5th of August
Still working on detailed project description. (antifreeze)

monday 8th of August
Still working on detailed project description. (cell death)

tuesday 9th of August
Still working on detailed project description(regulation system). Tomorrow we start working on the real job! exit: project description

wednes- & thursday 10th & 11th of August
making simulations of cell death mechanism and freezecomponent in matlab. The lay-out of our model is messed up and simbiology has no undo button! Whaaaa...

tuesday 16th of August
Making simulations of antifreeze component in matlab. Meeting with instructor for sensitivity analysis.

tuesday 30th of August
Meeting with instructor Lyn, discussing finished description of the cell death model.

wednesday 31st of August
Working on description of freeze subsystem. Doing some sensitivity analysis.

thursday 1st of September
Searching more accurate kinetic parameters and references.

tuesday 6th of September
Small presentation to give the other teammembers an update in modeling. Good remark of instructor on our model, we made a mistake in the working of LuxR-AHL induction/inhibition. Brainstorm on how to put modeling on the wiki. Brainstorm on how to make an introduction movie for iGEM jamboree.

wednesday 8th of September
Made corrections to description of modeling. Wiki division for modeling is finished by designers. We will put a first version of the model on the wiki soon, although this should still be updated with the LuxR/AHL complex. Thank you, designteam, for making the modeling page, ready for us!

thursday 9th of September
Worked on the full model today, included the LuxR/HSL complex. The iGEM-team of 2008 KULeuven also used it in their system, so it was just copy/pasting. The antifreeze and cell death descriptions are corrected for a second time. Two slides for jamboree in Amsterdam are prepared.

@@ Line 437: / Line 437: @@
 <span class="notebook-dayno">Thursday 1th of September</span><br>
 <span class="notebook-txt">On the plates with the ligated INP were successful in contrast to the AFP ligations. We opted to repeat the ligation of AFP with Lambda cI. A CTAB minipreparation was also done followed by a control restriction.</span><hr class="notebook-divider">
+<span class="notebook-dayno">Monday 5th of September</span><br>
+<span class="notebook-txt">A control restriction was done on the CTAB products from last Friday. Due to the failure of Fridays ligations, we did the ligations again together with the restriction and ligation of AFP with the constitutive promoter. </span><hr class="notebook-divider">
+<span class="notebook-dayno">Wednesday 7th of September</span><br>
+<span class="notebook-txt">We miniprepped the ligated products and did a control restriction with EcroRI and PstI. Unfortunately the results were not what we expected. The results of GFP test from the past week were inconclusive so we decided to repeat it. GFP testing was done on the promoters pBAD-GFP and constitutive promoter – GFP (Both induced with arabinose in two types of ''E.coli'' TOP10’ and MG1655).</span><hr class="notebook-divider">
+<span class="notebook-dayno">Thursday 8th of September</span><br>
+<span class="notebook-txt">The ligations of AFP and INP-Terminator to their promoters was repeated. We did GFP testing on pLAc-Lux – GFP and Constitutive promoter – GFP, both induced with IPTG again in the two types of ''E.coli'').</span><hr class="notebook-divider">
+<span class="notebook-dayno">Friday 9th of September</span><br>
+<span class="notebook-txt">The ligation of INP – Terminator were unsuccessful again so we repeated the ligation again together with the ligation of GFP to the HybB promoter.</span><hr class="notebook-divider">
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