Digestions: Because we are having issues to get 2nd-step constructions. We tried both 3A Assembly and Standard protocols.

Cheking and purification gels: We add a cheking step, before performing ligations on Standard Protocol. To maximise our chance to get 2nd-step constructions, we tried to purified the inserts.

On purification gel, plasmids seem to be digested but inserts is missing. On checking gel, cinI wasn't digested correctly.

RBS-TetR RBS-LuxI RBS-CinR RBS-LacI pLac-GFP pCin-GFP pMerT-GFP pLux-Lyco

6 out of 8 sequence alignments failed.
To perform this constructions, we used standard protocol: we kept the plasmid of the shortest biobrick which we inserted the longest biobrick.
The problem was identified as coming from a wrong identification of antibiotic resistants. In most constructions below, vector and inserts had the same antibiotic resistance. So, even wrong constructions were selected on Petri Dishes.

Because we had no results with the latest cloning session, we tested our enzymes.

We tried to digest each site individually. We obtained linearized plasmid. So, enzymes are well working.

Digestions: a gel checking of the restriction results were performed.

Spreading over Petri dish

On the Restriction Gel, We obtain the right size for all inserts.
Because pSB1AC3 is a double resistance plasmid (ampicillin and chloramphenicol) and RBS plasmid is on ampicillin, as previously the right constructions won't be selected.

Preculture: To perform this new cloning session, we first selected 5 colonies from the Petri dish of MerR-CinR construction.

Digestions: a gel checking of the restriction results were performed.

Purification: In order to check and extract the wright insert.

Ligations : Standard Protocol was performed. MerR-CinR was inserted into pLac and pConst plasmids.

Spreading over Petri dish

Digestions: a gel checking of the restriction results were performed.

Purification: In order to check and extract the wright insert.

Ligations : Standard Protocol was performed. MerR-CinR was inserted into pLac and pConst plasmids.

Spreading over Petri dish

On Puritication gel, 4 biobricks were not correctly digested: CinI, LuxI, LuxR, RBS-LuxR. All the other were apparently well purified.
Ligations were performed as usual.
Results on Petri Dishes were unsatifying: we obtained an agglomerate of colonies. And this in all Petri Dishes. Does the issue come from too old Petri Dishes? Or LB pre culture?
So, we tried a 2nd-spreding on Petri Dishes and the result was the same.
We also checked by PCR if we could work anyway with those colonies. But the results were not conclusive.

We spread again -80°C stock in order to start new miniprep of the troubling biobricks:
Lycopene
RBS
Fha
TetR
LuxR
LuxI
CinI
CinR
pTet
pMerT
pCin
pConst
pLux

Digestions: a gel checking of the restriction results were performed.

Purification: In order to check and extract the wright insert.

Ligations : Standard Protocol was performed. MerR-CinR was inserted into pLac and pConst plasmids. We add a control for the ligations: plasmids without its inserts (pLux, pCin, Fha, RBS).

Spreading over Petri dish

Stochastic program is implemented for the toggle switch and works fine. We adapted the program for the whole system so we can implement the quorum sensing stochastic model very quickly when we have the parameters (about 1/2 day of work)

We have coherent sets of parameters, but we still can't compare our models to the real system and are waiting for the results of characterization experiments.

Stochastic program is implemented for the toggle switch and works fine. We adapted the program for the whole system so we can implement the quorum sensing stochastic model very quickly when we have the parameters (about 1/2 day of work). The current model and parameters prove that we do have a bi-modal distribution when IPTG and pollutant concentrations are equal :

On the X-axis we can see the concentration of lacI (neg) or merR (positive) in cells. On the Y-axis we can see the amount of cells in one way or another.

We have coherent sets of parameters, but we still can't compare our models to the real system and are waiting for the results of characterization experiments.

Ligations:
Because we still have a low cloning rate we tried different proportions between vectors and inserts:
1 vector/1 insert
1 vector/2 inserts
1 vector/3 inserts

Transformations with electrocompetent cells
Spreading over Petri Dish

RESULTS :
Best results on Petri Dishes with the proportion 1/3. All constructions seem to have the right size except MerR-LuxR.

RBS-TetR RBS-LuxI RBS-CinR RBS-LacI pLac-GFP pCin-GFP pMerT-GFP pLux-Lyco

For ligations, digestions, PCR, MiniPreps, MidiPreps

The final RBS (Fha1) which allows to keep the toggle off has just been delivered. So, all first steps constructions including RBS were made once again.
Because the efficiency of Fha1 hasn't been completly demonstrated, we keep on cloning with the standard RBS. So, RBS and Fha1 constructions are made in parallel.

RESULTS :
Every constructions gave colonies on Petri Dishes.
But PCR cheking showed that nothing were amplified on every Fha1 constructions with VF2 and VR primers. Thus, Fha1 was provided in a PCR blent plasmid instead of an iGEM's plasmid.
The insert of the test construction is having the right size.
2 out of 3 2nd step constructions had the right size.
We still have issues to clone the 1st step construction: RBS-CinI.

RESULTS : Great Transformation Rate

DO checking PCR cheking

We changed our strategy: clonings were performed with enzymes recommended by iGEM ().

One Petri Dish was free of colony. We performed PCRs on 5 colonies from each Petri Dish. According to PCR results, 5 constructions out of 11 seem to have the right size.

Only digestions were performed because of a lack of equipments.
We decided to use the Standard Protocol for the first step constructions because the assembly combinates a short and a long gene. The long gene is inserted in the plasmid of the small gene.
But the Second step constructions were performed with the 3A Assembly Protocol.

First results with the stochastic model. Our whole model does work like we expected it. But parameters are needed more than ever now.

We got incomplete sets of parameters from either litterature or iGem teams. Some very useful sets can be found in Aberdeen 09, Brown 10, ETHZ 07, BCCS Bristol 08

We are taking advantage that the school year is over to move in the CIME, a teaching platform of biology.

OD cheking was performed on all our Biobrick stock to have an idea of the DNA rate into our preparation. We also made a PCR cheking to make sure that the DNA rate really corresponds to our Biobrick.

We got an average rate of 100 ng/µl. Which is a much lower than expect, so 10 times more DNA products would had been necessary to get great constructions.

We hope to increase the digestion efficiency.

As previously, inserts were shorter than expected, except the construction RBS-TetR.

In order to send it to the sequencing.

Stochastic model is on its way. We base it on Gillespie's algorithm. It's mainly about getting familiar with the math for now. Also working on Hysteresis and isoclines subparts of the deterministic models.

Still a lot of work going on there. The problem is we can not compare our sets to experimental results as the whole system is not finished yet. We will then try to characterize some of the promoters we use and figure out how to obtain some degradation rates as well