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To implement our vesicles emission project, we had to take several constrains into account. To put into place all the functionalities we needed, we designed 2 different plasmids as shown on the image below. | To implement our vesicles emission project, we had to take several constrains into account. To put into place all the functionalities we needed, we designed 2 different plasmids as shown on the image below. | ||
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<font color=red>Writing the message: production of signaling proteins </font><br> | <font color=red>Writing the message: production of signaling proteins </font><br> | ||
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To create this delay between the creation of proteins and the production of vesicles, we designed a regulatory cascade consisting of the LacI and TetR repressors. The LacI biobrick is placed in the first plasmid, downstream the pBad promoter and once synthesized acts as a repressor on the pLac promoter. The pLac promoter in the second plasmid then stops expressing TetR. The ptet promoter is then no longer repressed and the creation of non functional TolR can start leading to the emission of vesicles. | To create this delay between the creation of proteins and the production of vesicles, we designed a regulatory cascade consisting of the LacI and TetR repressors. The LacI biobrick is placed in the first plasmid, downstream the pBad promoter and once synthesized acts as a repressor on the pLac promoter. The pLac promoter in the second plasmid then stops expressing TetR. The ptet promoter is then no longer repressed and the creation of non functional TolR can start leading to the emission of vesicles. | ||
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<font color=red>Preparing the messenger: creation of the vesicles </font><br> | <font color=red>Preparing the messenger: creation of the vesicles </font><br> | ||
Revision as of 17:14, 21 October 2009
iGEM > Paris > Home > OMV Project
Contents |
Overall project: Message in a bubble
Message in a Bubble: cell-cell communication using vesicles.
The release of membrane vesicles (MVs) is conserved among Gram-negative bacteria (1). Extracellular secretion of products is the major mechanism by which Gram-negative pathogens communicate with cells. These vesicles released from the envelope of growing bacteria serve as secretory vehicles for proteins and lipids of Gram-negative bacteria.
Bibliography
(1) Kuehn and Kesty, 2005 Bacterial outer membrane vesicles and the host-pathogen interaction, Genes Dev. 2005;19(22):2645-55 (lien pubmed 16291643)
A. Plasmid construction
Avant de parler des plasmides que vous avez voulu construire, il faut parler du système entier lui même. Et c'est bien ce que vous faites dans la suite en partie. L'implémentation sur des plasmides particuliers est un point technique qui vient après.
GC: tout a fait d'accord ! Il faut aussi mettre ici (ou juste au dessus) quelques generalités sur la communication en général et chez les bactos en particulier. Mettre l'accent sur les obstacles a la communication (taille des cellules, peu de systeme dedié...) et j'en profiterai pour faire une presentation simple des membranes qui est une forme de contrainte. Les membranes sont centrale dans ce projet et doivent etre presentée clairement et rapidement. Pour le moment, cela ce trouve dans la partie Tol/Pal et je pense que ça serait mieux ici).
En premier lieu faites un schéma, un diagramme du projet complet: production des proteines signal, système d'adressage de ces protéines vers la membraexterne, production des proteins pour l'émission de vésicules,production des vésicules, réception des vésicules, déclenchement d'un accusé de réception etc...
Un tel schéma peut être dynamique hyperliens vers les parties correspondantes. Ayez en vue le discours modulaire à différentes échelles des biobricks: niveau système, niveau device, niveau ADN... on ne parle des plasmides qu'au niveau inférieur.
The plasmid construction is divided into 2 functional modules :
- The emission system, which aims at producing vesicules.
- The reception system of the signal sent via the vesicules.
A.1. The emission system
To implement our vesicles emission project, we had to take several constrains into account. To put into place all the functionalities we needed, we designed 2 different plasmids as shown on the image below.
Writing the message: production of signaling proteins
First of all, before sending vesicles into the surrounding medium, we have to make sure that every molecule and protein that has to be inside the vesicles is already into place before the bacteria starts the creation of vesicles. In other words, the "emitting" bacteria must produce the proteins of interest, the export systems, the FecA proteins as well as the fusion mechanism before creating vesicles.
To create this delay between the creation of proteins and the production of vesicles, we designed a regulatory cascade consisting of the LacI and TetR repressors. The LacI biobrick is placed in the first plasmid, downstream the pBad promoter and once synthesized acts as a repressor on the pLac promoter. The pLac promoter in the second plasmid then stops expressing TetR. The ptet promoter is then no longer repressed and the creation of non functional TolR can start leading to the emission of vesicles.
Preparing the messenger: creation of the vesicles
As the creation of vesicles via the over-expression of TolR disturbs the membrane integrity and can create an important cell lysis, it appeared very important to find a way to avoid a long lasting expression of our TolR biobrick once the input signal is on (presence of arabinose in the medium).
To solve this problem, we decided to place a tag on the LacI protein to speed up its degradation. As a consequence, once the arabinose in the medium is depleted, LacI production stops and the remaining LacI is rapidly degraded. The production of TetR can resume and inhibit vesicle production.
- In presence of Arabinose, proteins of interest are created as well as vesicles :
- In the absence of arabinose, the pBad promoter is repressed and there is no production of proteins nor vesicles :
A more accurate description of the parts used at each step of the creation process (including links to the parts registry and references) can be found in the different subdivision of the project.
A.2. The reception system
A FAIRE RAPIDEMENT
B. Modeling overview
Cette image est pas mal. Et c'est quelque chose dans cet esprit qui devrait être tout au début pour présenter le système global de votre projet. Ce n'est pas seulement une image de modélisation.
Mais ce diagramme est à clarifier. Protein exported -> Exported "message" protein production Export system -> (il s'agit bien de l'addessage des protéines ci-dessus vers la membrane externe?) Addressing system of the message protein to the "bottle", sending the message to the bottle TolRIII -> Production of the message bootle (or of the envelope)
On ne comprend pas Chaperone Action. Ce diagramme doit être lisible pour comprendre les mécanismes, sans détails des molécules sous-jacentes. Il peut-être complété par un niveau plus détaillé, faisant apparaître la couche sous-jacente (les protéines particulières impliquées, les construction de plasmides, etc...)
Le même diagramme aussi pour la réception des signaux.
