Team:Grenoble/Notebook
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Revision as of 14:19, 2 August 2011
TEAM:Grenoble
Our Sponsors
Event Calendar
- 2nd September:
3 parvis Louis Néel
38 000 GRENOBLE
FRANCE
Contact Us
Clic here !
July 14th to 21st
July 7th to 13th
Team Marmottes
June 29th to July 6th
Team Godlike
Working on parameters
Team Marmottes
Thanks to the delivery of new MerR material, we will finally be able to include MerR to our System.
Because former clonings gave no results, we carried out the Standard Assembly but results are not significant.
We are having to many problem with cloning, we have to check every steps (Minipreps concentration, digestion results by PCR , purification of the insert, proportion of vector (X1)/insert(X3) during the ligation...)
- New cloning trial CinI-RBS (Standard Assembly)
- pTet/TetR Miniprep
- MerR receipt
- MerR culture and PCR checking
- Cloning of every first steps of our construction : (Standard Assembly Method)
- Analysis of Sequencing Results :
-> 2 different assemblies were achieved
Digestion of the 2 biobricks :
-> RBS (S-P), the plasmid of RBS remains.
-> CinI (X-P) is inserted into the plasmid of RBS.
Digestion of the 2 biobricks :
-> CinI (S-P), the plasmid of CinI remains.
-> RBS (X-P) is inserted into the plasmid of CinI.
Ligation :
First digestion results were individually heat-inactivated at 80°C to eliminate enzymes remaining and then mixed all together.
Spreading over Petri dish
Colonies grown only on the Petri dish containing the construction of CinI inserted into RBS plasmid.
PCRs were performed on 4 colonies from this dish. None of them gave a conclusive result. All inserts are much shorter than expected (400 bps instead of 1040 bps)
Petri dishes full of colonies.
MerR was delivered into a small flask containing bacteria already transformed with MerR.
The insert amplified by PCR had the expected length.
-> 8 clonings
We used the same process as above inserting the biggest Biobrick into the plasmid of the shortest. Thus, the risk to loose the shortest piece is avoided.
-> pMerT-GFP
-> RBS-CinR
Sequences from GATC and Computer Sequencing were compared.
No match between both sequences from GATC and Computer Sequencing for pMerT-GFP construction.
Whereas sequences comparison of RBS-CinR demonstrates a strong similitude in the CinR region. But, RBS region seems to be absent or to have received mutations.
June 22nd to 28th
Team Godlike
Working on Quorum Sensing
- Parameters
- Models and results
We now have a complete set of parameters for our whole network. However physical parameters such as viscosity of AHL are still missing. Accurate predictions are therefore still not possible for the whole system.
We have worked on the whole set of equations for the Quorum Sensing part of our network. We also demonstrated that our system can switch from one way to another with sufficient amount of IPTG/pollutant.
Team Marmottes
3A-Assembly were carried out for all first steps of our constructions but results were inconclusive. Alternative methods should be thought off like inactivate enzymes before mixing them together.
- Stock of electro competent cells
- Cloning training with RBS-CinI: (3A Assembly Method)
- Cloning of every first steps of our construction:(3A Assembly Method)
- Sequencing order of the two valid constructions :
Great Colony Rate on Petri dishes
Digestion of the 3 biobricks
-> RBS (E-S)
-> CinI (X-P)
-> pSB1AC3 (E-P)
Ligation:
digestion results were mixed all together and then enzymes were heat-inactived at 80°C
Growth of red and white colonies.PCR checkings were performed on 3 white colonies. The results demonstrate that the insert is shorter than expected.
-> 10 clonings
Same process as above
Growth of red and white colonies for 6 out of 10 ligation results. PCR checks were performed as above for the 6 valid ligations. Only 2 appeared to have the right size. The sequencing of the 2 valid constructions should corroborate our results.
-> pMerT-GFP
-> RBS-CinR
We ordered on GATC company.
June 15th to 21st
Team Godlike
Working on parameters
- Parameters
- Simulations
- Plasmids and MerR Transformation
- Miniprep
- MerR Electroporation Transformation
- MerR PCR
- pTet/TetR Transformation
- MerR order
- Model equations
- Parameters
- Programs
- Computer Sequencing
- Biobricks Transformation
- Culture on liquid media
- Plasmid Mapping
- Biobrick Listing
- Manipulation schedule
- Models
- Programs
We now have enough parameters for simulating our toggle switch system. But still many parameters missing for a complete model.
However, with such parameters we can at least start working on the final device main features.
Obtained our first (meaningful) curves ! The simulated genetical network does switch indeed :
Team Marmottes
Still working on Biobrick Transformations. MerR transformations is still giving nothing. pTet/TetR has been considered as a substitut.
In case our colony issues come from a manipulation mistake, we tried again to Transform our Biobricks with the Standard protocole.
MerR transformation gave nothing. From plasmid backbones resulted Red colonies. But after looking on iGEM website plasmid backbones appears to have a RFP-gene as default Biobrick.
The kit Macherey-Nagel NucleoSpin Extract II was used.
Because the efficiency of the Standard Transformation is much lower than the Electroporation Transformation, we performed the latter.
As previously, we get nothing on our Petri dish.
To verify if there is something in the well.
Nothing on the gel.
Because we still have nothing with merR, we are looking for alternative to the couple pMerT/MerR. As usual, the Standard protocole was used.
Petri dishes full of colonies.
After many trial, merR seems to be absent from the well 7C of the plate 4.
June 8th to 14th
Team Godlike
Still worked on model equations
We deducted our equations from chemical and physical mechanisms and worked on simplifications.
One of the most importants part of our work is finding parameters for our model in order to match real behaviour of our cells as precisely as possible. Half of the needed parameters obtained up to now.
Minor changes on the algorithm, minor bugs fixed and some plotting features added.
Team Marmottes
Computer sequencing.
First manipulations: transformation of the Biobrick just arrived.
Every intermediate, final and test constructions were listed on DNA Workbench in order to compare PCR and Sequencing Results with this data bank.
To achieve it, the Standard Transformation protocole was performed.
3 out 21 transformation gave colonies on Petri dishes:
- MerR transformation
- 2 plasmid backbone
Red colonies resulted from all plasmid backbones, looks odd!!
All transformation that gave colonnies were resuspended except plasmid backbones.
A stock of "ready to use biobricks" is waiting at 4°C and an other one is remaining at -80°C.
June 1st to 7th
Team Marmottes
No manipulation, we just planned the best way to work.
Toggle Switch: (4 final plasmids)
1 plasmid was considered for each toggle switch way (MerR or LacI), so 2 final plasmids.
But those 2 plasmids will include the 2 different couples cinI/cinR and luxI/luxR. The most efficient construction will be used.
Coloration Generator: (4 plasmids)
Coloration is induced by the activation of the promoter pLux or pCin depending on which couple cinI/cinR or luxI/luxR picked. As previously, the best coloring agent hasn't been decided yet. So, 4 plasmids will be produced: 1 for pCin, 1 for pLux and both followed either by GFP or Lycopène.
Tests: (6 plasmids)
The test of the two ways (MerR and LacI) of our toggle will be achieved by replacing pLac or pMerT by a constitutive promoter. So, 4 plasmids: 2 ways of the toggle switch, 2 different couples (luxI/luxR, cinI/cinR).
Efficiency of both promoters will be tested separately by associating them to GFP.
21 Biobricks will be necessary to achieve only the toggle switch and the coloration generator:
- 14 Biobricks
- 7 plasmid backbones
This includes biobricks which will be used for the tests (for example for the promoters) and as intermediate constructions.
Steps were defined for each construction depending on the level of assembly required. For example the assembly of two existing biobricks corresponds to a first level. Whereas the assembly of two newly obtained corresponds to a 2nd our 3rd level. Each level should be achieve at the time.
Team Godlike
Essentially worked on the models to use and how to simulate them
Toggle Switch : Worked on the equations that would modelize our system the best. We now have the equations for the toggle switch and a first Matlab script that we can base our work on. reference : Gardner, T.R., Cantor, C.R. & Collins, J.J., Construction of a genetic toggle switch in Escherichia coli, Nature 403, 339 - 342 (2000)
Simulations : We are mainly working on a deterministic model for our network, we use basic Matlab ODE solvers for now. Our plan for the final device is a plate with our bacteria. We will have to adapt our code for this particular device