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Team:Paris/Conclusion
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=== Controling vesicle production and addessing proteins into them === | === Controling vesicle production and addessing proteins into them === | ||
| - | The strategy was here to destabilize the outer membrane by playing with the tolAR system. | + | The strategy was here to destabilize the outer membrane by playing with the tolAR system. We either worked with tolA- strains or we tried to overexpress protein domains of tolR in order to disrupt the Tol protein complex essential for membrane stability. |
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| - | As OMVs are very small (around 100nm) they are impossible to see with optical microscopy. As | + | As OMVs are very small (around 100nm) they are impossible to see with optical microscopy. As we did not dispose of an electron microscope we tried to see if our clyA-mRFP1 when expressed in a tolA mutant could lead to the presence of red fluorescence into vesicles. Here are images of what could be fluorescent vesicles. Nevertheless we are just above background fluorescence and more controls would be required for a real proof. |
| + | DEMANDER A ARIEL SI IL A REUSSI A FAIRE DES IMAGES DE VESICULES ET LES METTRE ICI | ||
=== Transducing a signal from a protein in the outer membrane to a reporter gene in the receiver === | === Transducing a signal from a protein in the outer membrane to a reporter gene in the receiver === | ||
Revision as of 23:01, 21 October 2009
iGEM > Paris > Home > Conclusion & Results
Contents |
Results
In order to prove the feasibility of our project 5 main points needed to be adressed:
1. Addressing proteins to the sender bacteria outer membrane
2. Controlling the production of outer membrane vesicules
3. Addressing proteins to outer membrane vesicles
4. Fusing outer membrane vesicles of a sender population with the outer membrane of receiver cells
5. Transducing a signal from a protein in the outer membrane to a reporter gene in the receiver
We provided experimental proof for 3 out of 5 points:
Addressing proteins into the sender bacteria outer membrane
Our strategy was here to take advantage of the clyA outer membrane protein by fusing a fluorophore to N or C terminal of the ClyA protein.
METTRE L'IMAGE DE LA CONSTRUCTION
FAIRE LE LIEN AVEC LE DETAIL AILLEURS DANS LE WIKI
Our clyA-mRFP1 fusion was cloned under the control of a pBAD promoter (FAIRE LE LIEN AVEC LA CONSTRUCTION DANS LA REGISTRY). We can in this microscopy pictures clearly observe the membrane localization of fluorescence.
METTRE LES IMAGES
Controling vesicle production and addessing proteins into them
The strategy was here to destabilize the outer membrane by playing with the tolAR system. We either worked with tolA- strains or we tried to overexpress protein domains of tolR in order to disrupt the Tol protein complex essential for membrane stability.
METTRE LIEN VERS DETAILS ET METTRE LES IMAGES DES CONSTRUCTIONS ET LIEN VERS LA REGISTRY
As OMVs are very small (around 100nm) they are impossible to see with optical microscopy. As we did not dispose of an electron microscope we tried to see if our clyA-mRFP1 when expressed in a tolA mutant could lead to the presence of red fluorescence into vesicles. Here are images of what could be fluorescent vesicles. Nevertheless we are just above background fluorescence and more controls would be required for a real proof.
DEMANDER A ARIEL SI IL A REUSSI A FAIRE DES IMAGES DE VESICULES ET LES METTRE ICI
Transducing a signal from a protein in the outer membrane to a reporter gene in the receiver
Conclusion
After a great summer of hard work, it's time to conclued on our project.
Concerning the laboratory experiment we learnt few things... The first one is that the bench work takes a lot of time, so even if our project is designed on paper, it's not so easy in the "practical" life.
Ethics is really important when a new discipline is created. In this direction, it was an evidence for our team to inclued the ethics as a part in our project. In this direction we were able to think about the synthetic biology and its applications. [Report Synthethics PDF]
About the collaboration, it was a great pleasure and it was reallu appreciable to work with other iGEM team for different part of our project (modelisation, ethics or i-phone tool). This collaboration allow us to contribute to the iGEM spirit.
