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 (New England Biolabs).

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

Our deterministic model is now finished. The whole system is properly simulated and we simplificated it. We will now work on the stochastic approach. Stochastic is very important for our system as it will define the precision of the whole measure.

This is now the main part of our work. We have to look for the best set of parameters for our system to properly modelize it. We dissect litterature as well as other and former teams results to get a whole set. For the time being we do not have proper set.
The parameters we obtained are often contradictory from one set to another. We will try to characterize some of the parameters ourselves. We are working with biologists on experiments to characterize those parameters.

-> 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.

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.

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.

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 :

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 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.

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.

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 " to use biobricks" is waiting at 4°C and an other one is remaining at -80°C.

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.

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, 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