July 14^th to 21^st

July 7^th to 13^th

Team Marmottes

June 29^th to July 6^th

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)

-> 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)

• pTet/TetR Miniprep

Petri dishes full of colonies.

• MerR receipt

MerR was delivered into a small flask containing bacteria already transformed with MerR.

• MerR culture and PCR checking

The insert amplified by PCR had the expected length.

• Cloning of every first steps of our construction : (Standard Assembly Method)

-> 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.

• Analysis of Sequencing Results :

-> 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 22^nd to 28^th

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

Great Colony Rate on Petri dishes

• Cloning training with RBS-CinI: (3A Assembly Method)

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.

• Cloning of every first steps of our construction:(3A Assembly Method)

-> 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.

• Sequencing order of the two valid constructions :

-> pMerT-GFP
-> RBS-CinR
We ordered on GATC company.

June 15^th to 21^st

Team Marmottes

Still working on Biobrick Transformations. MerR transformations is still giving nothing. pTet/TetR has been considered as a substitut.

• Plasmids and MerR Transformation

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.

• Miniprep

The kit Macherey-Nagel NucleoSpin Extract II was used.

• MerR Electroporation Transformation

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.

• MerR PCR

To verify if there is something in the well.
Nothing on the gel.

• pTet/TetR Transformation

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.

• MerR order

After many trial, merR seems to be absent from the well 7C of the plate 4.

June 8^th to 14^th

Team Marmottes

Computer sequencing.
First manipulations: transformation of the Biobrick just arrived.

• Computer Sequencing

Every intermediate, final and test constructions were listed on DNA Workbench in order to compare PCR and Sequencing Results with this data bank.

• Biobricks Transformation

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!!

• Culture on liquid media

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 1^st to 7^th

Team Marmottes

No manipulation, we just planned the best way to work.

• Plasmid Mapping

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.

• Biobrick Listing

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.

• Manipulation schedule

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.