http://2009.igem.org/wiki/index.php?title=Special:Contributions/Fanny.c&feed=atom&limit=50&target=Fanny.c&year=&month=2009.igem.org - User contributions [en]2024-03-29T05:25:41ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/Team:Paris/ProjectTeam:Paris/Project2009-10-22T04:00:53Z<p>Fanny.c: /* A.2. The reception system */</p>
<hr />
<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris#top | Home]] > [[Team:Paris/Project#bottom | OMV Project]]<br />
{{Template:Paris2009}}<br />
{{Template:Paris2009_menu}}<br />
== '''Overall project:''' '''''Message in a bubble'''''==<br />
<br />
<center>'''Message in a Bubble: cell-cell communication using vesicles. '''</center><br />
<br />
<br />
<center>''Communication is a "two way" process. When you communicate you perceive the other persons responses and react with your own thoughts and feelings. It is only by paying attention to the other person that you have any idea about what to say or do next.''</center><br />
<br />
<br />
*''Bacterial communication:''<br />
<br />
Bacteria communicate with another one using chemical signal molecules. As in higher organisms, the information supplied by these molecules is critical for synchronizing the activities of large groups of cells. In bacteria, chemical communication involves producing, releasing, detecting, and responding to small hormone-like molecules<br />
(called acylhomoserine lactones, AHL). This process, also known as quorum sensing, allows bacteria to monitor the environment for other bacteria and to alter behavior on a population-wide scale in response to changes in the number and/or species present in a community. Nevertheless, AHL molecules are broken down by other bacteria, and some AHL signals are poorly soluble in water, so '''they cannot travel far in an aqueous environment (this factor limits their potential as a long communication signals)'''. <br />
<br />
<br />
*''Outer membrane vesicules in bacteria''<br />
Growing '''gram-negative bacteria (like ''E.Coli'' ) release vesicles from their outer membranes as a means of delivering toxins to host cells and other bacteria'''. This mecanism is conserved among Gram-negative bacteria. The vesicles consist of a lipid bilayer surrounding an aqueous core and they can therefore transport lipid-soluble toxins (lipopolysaccharide endotoxin) on their surface and protein toxins in their core. They release their content by fusing with the lipid bilayer of target cells. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<center>'''''The project :'''''</center><br />
<br />
We decided to''' improve bacterial communication''' thanks to the vesicles formation process. In this direction our engineered communication platform consists in '''controlling OMV production''' by destabilizing membrane integrity through over-expression of specific periplasmic proteins of the Tol/Pal system. The over-production of TolR (a major protein of the Tol/Pal system which ensure the membrane integrity) has to be controled to avoid the bacteria death. <br />
Another important key point of our project is to obtain a delay between the production of protein of interest and the vesicle formation, to be sure that the produced vesicles carried the different protein required for the recognition of the target bacteria and thus the one essential for the signal transduction.<br />
<br />
<br />
<font color=red>Producing the messenger :</font> <br />
<br />
In order to control and modulate message content, we used fusions with our protein of interest and OmpA signal sequence or the ClyA hemolysin as delivery tags. OmpA is a major protein of the external membrane of ''E.Coli'' and is also localize on OMVs. In this direction OmpA seems to be appropriate to deliver a specific protein to the outer membrane and, by consequence into vesicles. As OmpA, ClyA is an interesting way to explore to send protein to the external membrane.<br />
<br />
<br />
<font color=red>Addressing the message :</font> <br />
<br />
To own the communication between the donnor and the receiver a targeting system was developed. This system is based on the outer-membrane expression of Jun/Fos leucine zippers to control the vesicle flux between donor and recipient cells. Jun was mutated into its leucine zipper-motif to abolished the homodimer formation but to allow the development of heterodimer with Fos. To express these protein to the outer membrane of bacteria, they were merged with AIDA autotransporter. In this direction, the direction and the specificity of communication is controled.<br />
<br />
<br />
<font color=red>Receiving the message :</font><br />
<br />
Once received, the signal from incoming vesicles is transduced through a modified Fec pathway, whereby the receptor is provided by the OMV. Few ABC transporter such as FecABCD (iron transporter) are able to induce a response regardless of the tranlocation, due to the activity of FecA. Moreover some mutant can also have a constitutive expression of FecABCD .So, we would like to use FecA- mutant receiver and FecA+ mutant donor to transfert the constitutive FecA protein to the receiver to transmit to the target cell the message (which comes from vesicles (bubbles)). <br />
<br />
<br />
Computational models provided insight to all of the above steps and suggested directions for system improvement. '''Such reliable communications systems have wide biotechnological implications, ranging from targeted drugs delivery and detoxification to advanced division of labor or even cell-based computing.'''<br />
<br />
<br />
<br />
<br />
All of our constructions are just [[Team:Paris/constructions | here]]<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
<div id="paris_content_boxtop"><br />
</div><br />
<div id="paris_content"><br />
<br />
<br />
==='''A. Plasmid construction'''===<br />
<br />
The plasmid construction is divided into 2 functional modules :<br />
*'''The emission system''', which aims at producing vesicules.<br />
*'''The reception system''' of the signal sent via the vesicules.<br />
<br />
====A.1. The emission system ====<br />
<br />
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.<br />
<br />
<br />
<font color=red>Writing the message: production of signaling proteins </font><br><br />
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.<br />
<br />
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.<br />
<br />
<br />
<font color=red>Preparing the messenger: creation of the vesicles </font><br><br />
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). <br />
<br />
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.<br />
<br />
<br />
* In '''presence of Arabinose''', proteins of interest are created as well as vesicles :<br />
[[Image:Global_On.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
<br />
<br />
*In the '''absence of Arabinose''', the pBad promoter is repressed and there is no production of proteins nor vesicles :<br />
[[Image:Global_Off.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
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.<br />
<br />
====A.2. The reception system====<br />
<br />
To implement our vesicles reception project, we had to take several constrains into account. To put into place all the functionalities we needed, we designed 2 different types of plasmids.<br />
<br />
<br />
<font color=red>Giving the message: fusion of the vesicles with the receiver.</font><br><br />
To merge the OMVs with the targeted bacteria. We have explored two different methods : Jun/Fos dimere and G3P. <br>With Jun/Fos, after mutations into the leucine zipper motif of Jun, we fused it to AIDA to send them to the extern membrane of bacteria. <br>With G3P, we fuse it to the OmpA- Linker protein to target it at the surface of the vesicles.<br />
<br />
<br><br />
<br> On the first plasmid '''donnor''', the part wich interest us is : the fusion system part 1<br />
<br> it include the sequence : RBS / FecA / OmpA signal / Jun / AidA translocator / ter<br />
<br />
<br />
[[Image:PSB1A3paris.png|500px|center]]<br />
<br />
<br />
<br>On the plasmid '''receiver''', the part wich interest us is : the fusion system part 2 <br />
<br> it include the sequence : plac / OmpA-signal / Fos / AidA translocator / ter<br />
<br />
[[Image:PSB2K3paris.png|500px|center]]<br />
<br />
<br />
<font color=red>Transduction: decryption of the message.</font><br />
<br><br />
We plan to use FecA- mutant receiver and FecA+ mutant donor to transfert the constitutive FecA protein to the receiver. In this case the receiver will express the FecABCD operon without being induce by ferric citrate in the medium , and so we could place under the control of the Fec ABCD promoter, which is called pfec, the gene sequence encoding for the response. For the moment a response that would be easy to detect is the fluorescence of the RFP and the biobrick BBa-J61002 is the perfect candidate to test the system. <br />
<br><br />
We also discovered that some fecR and fecI mutants can be use to amplify the signal because they have a constitutive activity. So we put under the control of pfec a FecR and FecI mutated. When they will be expressed, they will be activators of pfec and consequently of RFP. Normaly we would be able to obtain a increasing fluorescence. <br />
<br />
<br><br />
<br>On the plasmid '''receiver''', the the sequences wich interest us are : the fec proteins (transduction) and the promotor pFec (amplification response)<br />
<br />
<br />
[[Image:PSB2K3paris.png|500px|center]]<br />
<br />
<br />
<html><br />
</div><br />
<div id="paris_content_boxtop"><br />
</div><br />
<div id="paris_content"><br />
</html><br />
<br />
<br />
{{Template:Paris2009_guided2|#top|/DryLab}}</div>Fanny.chttp://2009.igem.org/Team:Paris/ProjectTeam:Paris/Project2009-10-22T04:00:06Z<p>Fanny.c: /* A.2. The reception system */</p>
<hr />
<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris#top | Home]] > [[Team:Paris/Project#bottom | OMV Project]]<br />
{{Template:Paris2009}}<br />
{{Template:Paris2009_menu}}<br />
== '''Overall project:''' '''''Message in a bubble'''''==<br />
<br />
<center>'''Message in a Bubble: cell-cell communication using vesicles. '''</center><br />
<br />
<br />
<center>''Communication is a "two way" process. When you communicate you perceive the other persons responses and react with your own thoughts and feelings. It is only by paying attention to the other person that you have any idea about what to say or do next.''</center><br />
<br />
<br />
*''Bacterial communication:''<br />
<br />
Bacteria communicate with another one using chemical signal molecules. As in higher organisms, the information supplied by these molecules is critical for synchronizing the activities of large groups of cells. In bacteria, chemical communication involves producing, releasing, detecting, and responding to small hormone-like molecules<br />
(called acylhomoserine lactones, AHL). This process, also known as quorum sensing, allows bacteria to monitor the environment for other bacteria and to alter behavior on a population-wide scale in response to changes in the number and/or species present in a community. Nevertheless, AHL molecules are broken down by other bacteria, and some AHL signals are poorly soluble in water, so '''they cannot travel far in an aqueous environment (this factor limits their potential as a long communication signals)'''. <br />
<br />
<br />
*''Outer membrane vesicules in bacteria''<br />
Growing '''gram-negative bacteria (like ''E.Coli'' ) release vesicles from their outer membranes as a means of delivering toxins to host cells and other bacteria'''. This mecanism is conserved among Gram-negative bacteria. The vesicles consist of a lipid bilayer surrounding an aqueous core and they can therefore transport lipid-soluble toxins (lipopolysaccharide endotoxin) on their surface and protein toxins in their core. They release their content by fusing with the lipid bilayer of target cells. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<center>'''''The project :'''''</center><br />
<br />
We decided to''' improve bacterial communication''' thanks to the vesicles formation process. In this direction our engineered communication platform consists in '''controlling OMV production''' by destabilizing membrane integrity through over-expression of specific periplasmic proteins of the Tol/Pal system. The over-production of TolR (a major protein of the Tol/Pal system which ensure the membrane integrity) has to be controled to avoid the bacteria death. <br />
Another important key point of our project is to obtain a delay between the production of protein of interest and the vesicle formation, to be sure that the produced vesicles carried the different protein required for the recognition of the target bacteria and thus the one essential for the signal transduction.<br />
<br />
<br />
<font color=red>Producing the messenger :</font> <br />
<br />
In order to control and modulate message content, we used fusions with our protein of interest and OmpA signal sequence or the ClyA hemolysin as delivery tags. OmpA is a major protein of the external membrane of ''E.Coli'' and is also localize on OMVs. In this direction OmpA seems to be appropriate to deliver a specific protein to the outer membrane and, by consequence into vesicles. As OmpA, ClyA is an interesting way to explore to send protein to the external membrane.<br />
<br />
<br />
<font color=red>Addressing the message :</font> <br />
<br />
To own the communication between the donnor and the receiver a targeting system was developed. This system is based on the outer-membrane expression of Jun/Fos leucine zippers to control the vesicle flux between donor and recipient cells. Jun was mutated into its leucine zipper-motif to abolished the homodimer formation but to allow the development of heterodimer with Fos. To express these protein to the outer membrane of bacteria, they were merged with AIDA autotransporter. In this direction, the direction and the specificity of communication is controled.<br />
<br />
<br />
<font color=red>Receiving the message :</font><br />
<br />
Once received, the signal from incoming vesicles is transduced through a modified Fec pathway, whereby the receptor is provided by the OMV. Few ABC transporter such as FecABCD (iron transporter) are able to induce a response regardless of the tranlocation, due to the activity of FecA. Moreover some mutant can also have a constitutive expression of FecABCD .So, we would like to use FecA- mutant receiver and FecA+ mutant donor to transfert the constitutive FecA protein to the receiver to transmit to the target cell the message (which comes from vesicles (bubbles)). <br />
<br />
<br />
Computational models provided insight to all of the above steps and suggested directions for system improvement. '''Such reliable communications systems have wide biotechnological implications, ranging from targeted drugs delivery and detoxification to advanced division of labor or even cell-based computing.'''<br />
<br />
<br />
<br />
<br />
All of our constructions are just [[Team:Paris/constructions | here]]<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
<div id="paris_content_boxtop"><br />
</div><br />
<div id="paris_content"><br />
<br />
<br />
==='''A. Plasmid construction'''===<br />
<br />
The plasmid construction is divided into 2 functional modules :<br />
*'''The emission system''', which aims at producing vesicules.<br />
*'''The reception system''' of the signal sent via the vesicules.<br />
<br />
====A.1. The emission system ====<br />
<br />
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.<br />
<br />
<br />
<font color=red>Writing the message: production of signaling proteins </font><br><br />
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.<br />
<br />
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.<br />
<br />
<br />
<font color=red>Preparing the messenger: creation of the vesicles </font><br><br />
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). <br />
<br />
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.<br />
<br />
<br />
* In '''presence of Arabinose''', proteins of interest are created as well as vesicles :<br />
[[Image:Global_On.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
<br />
<br />
*In the '''absence of Arabinose''', the pBad promoter is repressed and there is no production of proteins nor vesicles :<br />
[[Image:Global_Off.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
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.<br />
<br />
====A.2. The reception system====<br />
<br />
To implement our vesicles reception project, we had to take several constrains into account. To put into place all the functionalities we needed, we designed 2 different types of experiments.<br />
<br />
<br />
<font color=red>Giving the message: fusion of the vesicles with the receiver.</font><br><br />
To merge the OMVs with the targeted bacteria. We have explored two different methods : Jun/Fos dimere and G3P. <br>With Jun/Fos, after mutations into the leucine zipper motif of Jun, we fused it to AIDA to send them to the extern membrane of bacteria. <br>With G3P, we fuse it to the OmpA- Linker protein to target it at the surface of the vesicles.<br />
<br />
<br><br />
<br> On the first plasmid '''donnor''', the part wich interest us is : the fusion system part 1<br />
<br> it include the sequence : RBS / FecA / OmpA signal / Jun / AidA translocator / ter<br />
<br />
<br />
[[Image:PSB1A3paris.png|500px|center]]<br />
<br />
<br />
<br>On the plasmid '''receiver''', the part wich interest us is : the fusion system part 2 <br />
<br> it include the sequence : plac / OmpA-signal / Fos / AidA translocator / ter<br />
<br />
[[Image:PSB2K3paris.png|500px|center]]<br />
<br />
<br />
<font color=red>Transduction: decryption of the message.</font><br />
<br><br />
We plan to use FecA- mutant receiver and FecA+ mutant donor to transfert the constitutive FecA protein to the receiver. In this case the receiver will express the FecABCD operon without being induce by ferric citrate in the medium , and so we could place under the control of the Fec ABCD promoter, which is called pfec, the gene sequence encoding for the response. For the moment a response that would be easy to detect is the fluorescence of the RFP and the biobrick BBa-J61002 is the perfect candidate to test the system. <br />
<br><br />
We also discovered that some fecR and fecI mutants can be use to amplify the signal because they have a constitutive activity. So we put under the control of pfec a FecR and FecI mutated. When they will be expressed, they will be activators of pfec and consequently of RFP. Normaly we would be able to obtain a increasing fluorescence. <br />
<br />
<br><br />
<br>On the plasmid '''receiver''', the the sequences wich interest us are : the fec proteins (transduction) and the promotor pFec (amplification response)<br />
<br />
<br />
[[Image:PSB2K3paris.png|500px|center]]<br />
<br />
<br />
<html><br />
</div><br />
<div id="paris_content_boxtop"><br />
</div><br />
<div id="paris_content"><br />
</html><br />
<br />
<br />
{{Template:Paris2009_guided2|#top|/DryLab}}</div>Fanny.chttp://2009.igem.org/Team:Paris/ProjectTeam:Paris/Project2009-10-22T03:57:44Z<p>Fanny.c: /* A.2. The reception system */</p>
<hr />
<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris#top | Home]] > [[Team:Paris/Project#bottom | OMV Project]]<br />
{{Template:Paris2009}}<br />
{{Template:Paris2009_menu}}<br />
== '''Overall project:''' '''''Message in a bubble'''''==<br />
<br />
<center>'''Message in a Bubble: cell-cell communication using vesicles. '''</center><br />
<br />
<br />
<center>''Communication is a "two way" process. When you communicate you perceive the other persons responses and react with your own thoughts and feelings. It is only by paying attention to the other person that you have any idea about what to say or do next.''</center><br />
<br />
<br />
*''Bacterial communication:''<br />
<br />
Bacteria communicate with another one using chemical signal molecules. As in higher organisms, the information supplied by these molecules is critical for synchronizing the activities of large groups of cells. In bacteria, chemical communication involves producing, releasing, detecting, and responding to small hormone-like molecules<br />
(called acylhomoserine lactones, AHL). This process, also known as quorum sensing, allows bacteria to monitor the environment for other bacteria and to alter behavior on a population-wide scale in response to changes in the number and/or species present in a community. Nevertheless, AHL molecules are broken down by other bacteria, and some AHL signals are poorly soluble in water, so '''they cannot travel far in an aqueous environment (this factor limits their potential as a long communication signals)'''. <br />
<br />
<br />
*''Outer membrane vesicules in bacteria''<br />
Growing '''gram-negative bacteria (like ''E.Coli'' ) release vesicles from their outer membranes as a means of delivering toxins to host cells and other bacteria'''. This mecanism is conserved among Gram-negative bacteria. The vesicles consist of a lipid bilayer surrounding an aqueous core and they can therefore transport lipid-soluble toxins (lipopolysaccharide endotoxin) on their surface and protein toxins in their core. They release their content by fusing with the lipid bilayer of target cells. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<center>'''''The project :'''''</center><br />
<br />
We decided to''' improve bacterial communication''' thanks to the vesicles formation process. In this direction our engineered communication platform consists in '''controlling OMV production''' by destabilizing membrane integrity through over-expression of specific periplasmic proteins of the Tol/Pal system. The over-production of TolR (a major protein of the Tol/Pal system which ensure the membrane integrity) has to be controled to avoid the bacteria death. <br />
Another important key point of our project is to obtain a delay between the production of protein of interest and the vesicle formation, to be sure that the produced vesicles carried the different protein required for the recognition of the target bacteria and thus the one essential for the signal transduction.<br />
<br />
<br />
<font color=red>Producing the messenger :</font> <br />
<br />
In order to control and modulate message content, we used fusions with our protein of interest and OmpA signal sequence or the ClyA hemolysin as delivery tags. OmpA is a major protein of the external membrane of ''E.Coli'' and is also localize on OMVs. In this direction OmpA seems to be appropriate to deliver a specific protein to the outer membrane and, by consequence into vesicles. As OmpA, ClyA is an interesting way to explore to send protein to the external membrane.<br />
<br />
<br />
<font color=red>Addressing the message :</font> <br />
<br />
To own the communication between the donnor and the receiver a targeting system was developed. This system is based on the outer-membrane expression of Jun/Fos leucine zippers to control the vesicle flux between donor and recipient cells. Jun was mutated into its leucine zipper-motif to abolished the homodimer formation but to allow the development of heterodimer with Fos. To express these protein to the outer membrane of bacteria, they were merged with AIDA autotransporter. In this direction, the direction and the specificity of communication is controled.<br />
<br />
<br />
<font color=red>Receiving the message :</font><br />
<br />
Once received, the signal from incoming vesicles is transduced through a modified Fec pathway, whereby the receptor is provided by the OMV. Few ABC transporter such as FecABCD (iron transporter) are able to induce a response regardless of the tranlocation, due to the activity of FecA. Moreover some mutant can also have a constitutive expression of FecABCD .So, we would like to use FecA- mutant receiver and FecA+ mutant donor to transfert the constitutive FecA protein to the receiver to transmit to the target cell the message (which comes from vesicles (bubbles)). <br />
<br />
<br />
Computational models provided insight to all of the above steps and suggested directions for system improvement. '''Such reliable communications systems have wide biotechnological implications, ranging from targeted drugs delivery and detoxification to advanced division of labor or even cell-based computing.'''<br />
<br />
<br />
<br />
<br />
All of our constructions are just [[Team:Paris/constructions | here]]<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
<div id="paris_content_boxtop"><br />
</div><br />
<div id="paris_content"><br />
<br />
<br />
==='''A. Plasmid construction'''===<br />
<br />
The plasmid construction is divided into 2 functional modules :<br />
*'''The emission system''', which aims at producing vesicules.<br />
*'''The reception system''' of the signal sent via the vesicules.<br />
<br />
====A.1. The emission system ====<br />
<br />
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.<br />
<br />
<br />
<font color=red>Writing the message: production of signaling proteins </font><br><br />
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.<br />
<br />
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.<br />
<br />
<br />
<font color=red>Preparing the messenger: creation of the vesicles </font><br><br />
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). <br />
<br />
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.<br />
<br />
<br />
* In '''presence of Arabinose''', proteins of interest are created as well as vesicles :<br />
[[Image:Global_On.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
<br />
<br />
*In the '''absence of Arabinose''', the pBad promoter is repressed and there is no production of proteins nor vesicles :<br />
[[Image:Global_Off.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
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.<br />
<br />
====A.2. The reception system====<br />
<br />
To implement our vesicles reception project, we had to take several constrains into account. To put into place all the functionalities we needed, we designed 2 different types of experiments.<br />
<br />
<br />
<font color=red>Giving the message: fusion of the vesicles with the receiver.</font><br><br />
To merge the OMVs with the targeted bacteria. We have explored two different methods : Jun/Fos dimere and G3P. <br>With Jun/Fos, after mutations into the leucine zipper motif of Jun, we fused it to AIDA to send them to the extern membrane of bacteria. <br>With G3P, we fuse it to the OmpA- Linker protein to target it at the surface of the vesicles.<br />
<br />
<br><br />
<br> On the first plasmid '''donnor''', the part wich interest us is : the fusion system part 1<br />
<br> it include the sequence : AidA translocator-Jun fusion, OmpA signal-FecA<br />
<br />
<br />
[[Image:PSB1A3paris.png|500px|center]]<br />
<br />
<br />
<br>On the plasmid '''receiver''', the part wich interest us is : the fusion system part 2 <br />
<br> it include the sequence : plac / OmpA-signal / Fos / AidA translocator<br />
<br />
[[Image:PSB2K3paris.png|500px|center]]<br />
<br />
<br />
<font color=red>Transduction: decryption of the message.</font><br />
<br><br />
We plan to use FecA- mutant receiver and FecA+ mutant donor to transfert the constitutive FecA protein to the receiver. In this case the receiver will express the FecABCD operon without being induce by ferric citrate in the medium , and so we could place under the control of the Fec ABCD promoter, which is called pfec, the gene sequence encoding for the response. For the moment a response that would be easy to detect is the fluorescence of the RFP and the biobrick BBa-J61002 is the perfect candidate to test the system. <br />
<br><br />
We also discovered that some fecR and fecI mutants can be use to amplify the signal because they have a constitutive activity. So we put under the control of pfec a FecR and FecI mutated. When they will be expressed, they will be activators of pfec and consequently of RFP. Normaly we would be able to obtain a increasing fluorescence. <br />
<br />
<br><br />
<br>On the plasmid '''receiver''', the the sequences wich interest us are : the fec proteins (transduction) and the promotor pFec (amplification response)<br />
<br />
<br />
[[Image:PSB2K3paris.png|500px|center]]<br />
<br />
<br />
<html><br />
</div><br />
<div id="paris_content_boxtop"><br />
</div><br />
<div id="paris_content"><br />
</html><br />
<br />
<br />
{{Template:Paris2009_guided2|#top|/DryLab}}</div>Fanny.chttp://2009.igem.org/Team:Paris/ProjectTeam:Paris/Project2009-10-22T03:50:00Z<p>Fanny.c: /* A.2. The reception system */</p>
<hr />
<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris#top | Home]] > [[Team:Paris/Project#bottom | OMV Project]]<br />
{{Template:Paris2009}}<br />
{{Template:Paris2009_menu}}<br />
== '''Overall project:''' '''''Message in a bubble'''''==<br />
<br />
<center>'''Message in a Bubble: cell-cell communication using vesicles. '''</center><br />
<br />
<br />
<center>''Communication is a "two way" process. When you communicate you perceive the other persons responses and react with your own thoughts and feelings. It is only by paying attention to the other person that you have any idea about what to say or do next.''</center><br />
<br />
<br />
*''Bacterial communication:''<br />
<br />
Bacteria communicate with another one using chemical signal molecules. As in higher organisms, the information supplied by these molecules is critical for synchronizing the activities of large groups of cells. In bacteria, chemical communication involves producing, releasing, detecting, and responding to small hormone-like molecules<br />
(called acylhomoserine lactones, AHL). This process, also known as quorum sensing, allows bacteria to monitor the environment for other bacteria and to alter behavior on a population-wide scale in response to changes in the number and/or species present in a community. Nevertheless, AHL molecules are broken down by other bacteria, and some AHL signals are poorly soluble in water, so '''they cannot travel far in an aqueous environment (this factor limits their potential as a long communication signals)'''. <br />
<br />
<br />
*''Outer membrane vesicules in bacteria''<br />
Growing '''gram-negative bacteria (like ''E.Coli'' ) release vesicles from their outer membranes as a means of delivering toxins to host cells and other bacteria'''. This mecanism is conserved among Gram-negative bacteria. The vesicles consist of a lipid bilayer surrounding an aqueous core and they can therefore transport lipid-soluble toxins (lipopolysaccharide endotoxin) on their surface and protein toxins in their core. They release their content by fusing with the lipid bilayer of target cells. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<center>'''''The project :'''''</center><br />
<br />
We decided to''' improve bacterial communication''' thanks to the vesicles formation process. In this direction our engineered communication platform consists in '''controlling OMV production''' by destabilizing membrane integrity through over-expression of specific periplasmic proteins of the Tol/Pal system. The over-production of TolR (a major protein of the Tol/Pal system which ensure the membrane integrity) has to be controled to avoid the bacteria death. <br />
Another important key point of our project is to obtain a delay between the production of protein of interest and the vesicle formation, to be sure that the produced vesicles carried the different protein required for the recognition of the target bacteria and thus the one essential for the signal transduction.<br />
<br />
<br />
<font color=red>Producing the messenger :</font> <br />
<br />
In order to control and modulate message content, we used fusions with our protein of interest and OmpA signal sequence or the ClyA hemolysin as delivery tags. OmpA is a major protein of the external membrane of ''E.Coli'' and is also localize on OMVs. In this direction OmpA seems to be appropriate to deliver a specific protein to the outer membrane and, by consequence into vesicles. As OmpA, ClyA is an interesting way to explore to send protein to the external membrane.<br />
<br />
<br />
<font color=red>Addressing the message :</font> <br />
<br />
To own the communication between the donnor and the receiver a targeting system was developed. This system is based on the outer-membrane expression of Jun/Fos leucine zippers to control the vesicle flux between donor and recipient cells. Jun was mutated into its leucine zipper-motif to abolished the homodimer formation but to allow the development of heterodimer with Fos. To express these protein to the outer membrane of bacteria, they were merged with AIDA autotransporter. In this direction, the direction and the specificity of communication is controled.<br />
<br />
<br />
<font color=red>Receiving the message :</font><br />
<br />
Once received, the signal from incoming vesicles is transduced through a modified Fec pathway, whereby the receptor is provided by the OMV. Few ABC transporter such as FecABCD (iron transporter) are able to induce a response regardless of the tranlocation, due to the activity of FecA. Moreover some mutant can also have a constitutive expression of FecABCD .So, we would like to use FecA- mutant receiver and FecA+ mutant donor to transfert the constitutive FecA protein to the receiver to transmit to the target cell the message (which comes from vesicles (bubbles)). <br />
<br />
<br />
Computational models provided insight to all of the above steps and suggested directions for system improvement. '''Such reliable communications systems have wide biotechnological implications, ranging from targeted drugs delivery and detoxification to advanced division of labor or even cell-based computing.'''<br />
<br />
<br />
<br />
<br />
All of our constructions are just [[Team:Paris/constructions | here]]<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
<div id="paris_content_boxtop"><br />
</div><br />
<div id="paris_content"><br />
<br />
<br />
==='''A. Plasmid construction'''===<br />
<br />
The plasmid construction is divided into 2 functional modules :<br />
*'''The emission system''', which aims at producing vesicules.<br />
*'''The reception system''' of the signal sent via the vesicules.<br />
<br />
====A.1. The emission system ====<br />
<br />
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.<br />
<br />
<br />
<font color=red>Writing the message: production of signaling proteins </font><br><br />
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.<br />
<br />
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.<br />
<br />
<br />
<font color=red>Preparing the messenger: creation of the vesicles </font><br><br />
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). <br />
<br />
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.<br />
<br />
<br />
* In '''presence of Arabinose''', proteins of interest are created as well as vesicles :<br />
[[Image:Global_On.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
<br />
<br />
*In the '''absence of Arabinose''', the pBad promoter is repressed and there is no production of proteins nor vesicles :<br />
[[Image:Global_Off.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
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.<br />
<br />
====A.2. The reception system====<br />
<br />
To implement our vesicles reception project, we had to take several constrains into account. To put into place all the functionalities we needed, we designed 2 different types of experiments.<br />
<br />
<br />
<font color=red>Giving the message: fusion of the vesicles with the receiver.</font><br><br />
To merge the OMVs with the targeted bacteria. We have explored two different methods : Jun/Fos dimere and G3P. <br>With Jun/Fos, after mutations into the leucine zipper motif of Jun, we fused it to AIDA to send them to the extern membrane of bacteria. <br>With G3P, we fuse it to the OmpA- Linker protein to target it at the surface of the vesicles.<br />
<br />
On the first plasmid '''donnor''', the part wich interest us is : the fusion system part 1<br />
<br> it include the sequence :<br />
<br />
<br />
[[Image:PSB1A3paris.png|500px|center]]<br />
<br />
<br />
<font color=red>Transduction: decryption of the message.</font><br />
<br><br />
We plan to use FecA- mutant receiver and FecA+ mutant donor to transfert the constitutive FecA protein to the receiver. In this case the receiver will express the FecABCD operon without being induce by ferric citrate in the medium , and so we could place under the control of the Fec ABCD promoter, which is called pfec, the gene sequence encoding for the response. For the moment a response that would be easy to detect is the fluorescence of the RFP and the biobrick BBa-J61002 is the perfect candidate to test the system. <br />
<br><br />
We also discovered that some fecR and fecI mutants can be use to amplify the signal because they have a constitutive activity. So we put under the control of pfec a FecR and FecI mutated. When they will be expressed, they will be activators of pfec and consequently of RFP. Normaly we would be able to obtain a increasing fluorescence. <br />
<br />
On the plasmid '''receiver''', the the sequences wich interest us are : the fec proteins (transduction) and the promotor pFec (amplification response)<br />
<br />
<br />
[[Image:PSB2K3paris.png|500px|center]]<br />
<br />
<br />
<html><br />
</div><br />
<div id="paris_content_boxtop"><br />
</div><br />
<div id="paris_content"><br />
</html><br />
<br />
<br />
{{Template:Paris2009_guided2|#top|/DryLab}}</div>Fanny.chttp://2009.igem.org/Team:Paris/ProjectTeam:Paris/Project2009-10-22T03:39:44Z<p>Fanny.c: /* A.2. The reception system */</p>
<hr />
<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris#top | Home]] > [[Team:Paris/Project#bottom | OMV Project]]<br />
{{Template:Paris2009}}<br />
{{Template:Paris2009_menu}}<br />
== '''Overall project:''' '''''Message in a bubble'''''==<br />
<br />
<center>'''Message in a Bubble: cell-cell communication using vesicles. '''</center><br />
<br />
<br />
<center>''Communication is a "two way" process. When you communicate you perceive the other persons responses and react with your own thoughts and feelings. It is only by paying attention to the other person that you have any idea about what to say or do next.''</center><br />
<br />
<br />
*''Bacterial communication:''<br />
<br />
Bacteria communicate with another one using chemical signal molecules. As in higher organisms, the information supplied by these molecules is critical for synchronizing the activities of large groups of cells. In bacteria, chemical communication involves producing, releasing, detecting, and responding to small hormone-like molecules<br />
(called acylhomoserine lactones, AHL). This process, also known as quorum sensing, allows bacteria to monitor the environment for other bacteria and to alter behavior on a population-wide scale in response to changes in the number and/or species present in a community. Nevertheless, AHL molecules are broken down by other bacteria, and some AHL signals are poorly soluble in water, so '''they cannot travel far in an aqueous environment (this factor limits their potential as a long communication signals)'''. <br />
<br />
<br />
*''Outer membrane vesicules in bacteria''<br />
Growing '''gram-negative bacteria (like ''E.Coli'' ) release vesicles from their outer membranes as a means of delivering toxins to host cells and other bacteria'''. This mecanism is conserved among Gram-negative bacteria. The vesicles consist of a lipid bilayer surrounding an aqueous core and they can therefore transport lipid-soluble toxins (lipopolysaccharide endotoxin) on their surface and protein toxins in their core. They release their content by fusing with the lipid bilayer of target cells. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<center>'''''The project :'''''</center><br />
<br />
We decided to''' improve bacterial communication''' thanks to the vesicles formation process. In this direction our engineered communication platform consists in '''controlling OMV production''' by destabilizing membrane integrity through over-expression of specific periplasmic proteins of the Tol/Pal system. The over-production of TolR (a major protein of the Tol/Pal system which ensure the membrane integrity) has to be controled to avoid the bacteria death. <br />
Another important key point of our project is to obtain a delay between the production of protein of interest and the vesicle formation, to be sure that the produced vesicles carried the different protein required for the recognition of the target bacteria and thus the one essential for the signal transduction.<br />
<br />
<br />
<font color=red>Producing the messenger :</font> <br />
<br />
In order to control and modulate message content, we used fusions with our protein of interest and OmpA signal sequence or the ClyA hemolysin as delivery tags. OmpA is a major protein of the external membrane of ''E.Coli'' and is also localize on OMVs. In this direction OmpA seems to be appropriate to deliver a specific protein to the outer membrane and, by consequence into vesicles. As OmpA, ClyA is an interesting way to explore to send protein to the external membrane.<br />
<br />
<br />
<font color=red>Addressing the message :</font> <br />
<br />
To own the communication between the donnor and the receiver a targeting system was developed. This system is based on the outer-membrane expression of Jun/Fos leucine zippers to control the vesicle flux between donor and recipient cells. Jun was mutated into its leucine zipper-motif to abolished the homodimer formation but to allow the development of heterodimer with Fos. To express these protein to the outer membrane of bacteria, they were merged with AIDA autotransporter. In this direction, the direction and the specificity of communication is controled.<br />
<br />
<br />
<font color=red>Receiving the message :</font><br />
<br />
Once received, the signal from incoming vesicles is transduced through a modified Fec pathway, whereby the receptor is provided by the OMV. Few ABC transporter such as FecABCD (iron transporter) are able to induce a response regardless of the tranlocation, due to the activity of FecA. Moreover some mutant can also have a constitutive expression of FecABCD .So, we would like to use FecA- mutant receiver and FecA+ mutant donor to transfert the constitutive FecA protein to the receiver to transmit to the target cell the message (which comes from vesicles (bubbles)). <br />
<br />
<br />
Computational models provided insight to all of the above steps and suggested directions for system improvement. '''Such reliable communications systems have wide biotechnological implications, ranging from targeted drugs delivery and detoxification to advanced division of labor or even cell-based computing.'''<br />
<br />
<br />
<br />
<br />
All of our constructions are just [[Team:Paris/constructions | here]]<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
<div id="paris_content_boxtop"><br />
</div><br />
<div id="paris_content"><br />
<br />
<br />
==='''A. Plasmid construction'''===<br />
<br />
The plasmid construction is divided into 2 functional modules :<br />
*'''The emission system''', which aims at producing vesicules.<br />
*'''The reception system''' of the signal sent via the vesicules.<br />
<br />
====A.1. The emission system ====<br />
<br />
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.<br />
<br />
<br />
<font color=red>Writing the message: production of signaling proteins </font><br><br />
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.<br />
<br />
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.<br />
<br />
<br />
<font color=red>Preparing the messenger: creation of the vesicles </font><br><br />
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). <br />
<br />
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.<br />
<br />
<br />
* In '''presence of Arabinose''', proteins of interest are created as well as vesicles :<br />
[[Image:Global_On.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
<br />
<br />
*In the '''absence of Arabinose''', the pBad promoter is repressed and there is no production of proteins nor vesicles :<br />
[[Image:Global_Off.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
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.<br />
<br />
====A.2. The reception system====<br />
<br />
To implement our vesicles reception project, we had to take several constrains into account. To put into place all the functionalities we needed, we designed 2 different types of experiments.<br />
<br />
<br />
<font color=red>Giving the message: fusion of the vesicles with the receiver.</font><br><br />
To fusion the OMVs with the targeted bacteria. We have explored two differents methods : Jun/Fos dimere and G3P. <br>With Jun/Fos, after mutations into the leucine zipper motif of Jun, we fused it to AIDA to send them to the extern membrane of bacteria. <br>With G3P, we fuse it to the OmpA- Linker protein to target it at the surface of the vesicles.<br />
<br />
<br />
<br />
<font color=red>Transduction: decryption of the message.</font><br />
<br><br />
We plan to use FecA- mutant receiver and FecA+ mutant donor to transfert the constitutive FecA protein to the receiver. In this case the receiver will express the FecABCD operon without being induce by ferric citrate in the medium , and so we could place under the control of the Fec ABCD promoter, which is called pfec, the gene sequence encoding for the response. For the moment a response that would be easy to detect is the fluorescence of the RFP and the biobrick BBa-J61002 is the perfect candidate to test the system. <br />
<br><br />
We also discovered that some fecR and fecI mutants can be use to amplify the signal because they have a constitutive activity. So we put under the control of pfec a FecR and FecI mutated. When they will be expressed, they will be activators of pfec and consequently of RFP. Normaly we would be able to obtain a increasing fluorescence. <br />
<br />
<br />
<br />
<br />
<html><br />
</div><br />
<div id="paris_content_boxtop"><br />
</div><br />
<div id="paris_content"><br />
</html><br />
<br />
<br />
{{Template:Paris2009_guided2|#top|/DryLab}}</div>Fanny.chttp://2009.igem.org/Team:Paris/ProjectTeam:Paris/Project2009-10-22T03:38:56Z<p>Fanny.c: /* A.2. The reception system */</p>
<hr />
<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris#top | Home]] > [[Team:Paris/Project#bottom | OMV Project]]<br />
{{Template:Paris2009}}<br />
{{Template:Paris2009_menu}}<br />
== '''Overall project:''' '''''Message in a bubble'''''==<br />
<br />
<center>'''Message in a Bubble: cell-cell communication using vesicles. '''</center><br />
<br />
<br />
<center>''Communication is a "two way" process. When you communicate you perceive the other persons responses and react with your own thoughts and feelings. It is only by paying attention to the other person that you have any idea about what to say or do next.''</center><br />
<br />
<br />
*''Bacterial communication:''<br />
<br />
Bacteria communicate with another one using chemical signal molecules. As in higher organisms, the information supplied by these molecules is critical for synchronizing the activities of large groups of cells. In bacteria, chemical communication involves producing, releasing, detecting, and responding to small hormone-like molecules<br />
(called acylhomoserine lactones, AHL). This process, also known as quorum sensing, allows bacteria to monitor the environment for other bacteria and to alter behavior on a population-wide scale in response to changes in the number and/or species present in a community. Nevertheless, AHL molecules are broken down by other bacteria, and some AHL signals are poorly soluble in water, so '''they cannot travel far in an aqueous environment (this factor limits their potential as a long communication signals)'''. <br />
<br />
<br />
*''Outer membrane vesicules in bacteria''<br />
Growing '''gram-negative bacteria (like ''E.Coli'' ) release vesicles from their outer membranes as a means of delivering toxins to host cells and other bacteria'''. This mecanism is conserved among Gram-negative bacteria. The vesicles consist of a lipid bilayer surrounding an aqueous core and they can therefore transport lipid-soluble toxins (lipopolysaccharide endotoxin) on their surface and protein toxins in their core. They release their content by fusing with the lipid bilayer of target cells. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<center>'''''The project :'''''</center><br />
<br />
We decided to''' improve bacterial communication''' thanks to the vesicles formation process. In this direction our engineered communication platform consists in '''controlling OMV production''' by destabilizing membrane integrity through over-expression of specific periplasmic proteins of the Tol/Pal system. The over-production of TolR (a major protein of the Tol/Pal system which ensure the membrane integrity) has to be controled to avoid the bacteria death. <br />
Another important key point of our project is to obtain a delay between the production of protein of interest and the vesicle formation, to be sure that the produced vesicles carried the different protein required for the recognition of the target bacteria and thus the one essential for the signal transduction.<br />
<br />
<br />
<font color=red>Producing the messenger :</font> <br />
<br />
In order to control and modulate message content, we used fusions with our protein of interest and OmpA signal sequence or the ClyA hemolysin as delivery tags. OmpA is a major protein of the external membrane of ''E.Coli'' and is also localize on OMVs. In this direction OmpA seems to be appropriate to deliver a specific protein to the outer membrane and, by consequence into vesicles. As OmpA, ClyA is an interesting way to explore to send protein to the external membrane.<br />
<br />
<br />
<font color=red>Addressing the message :</font> <br />
<br />
To own the communication between the donnor and the receiver a targeting system was developed. This system is based on the outer-membrane expression of Jun/Fos leucine zippers to control the vesicle flux between donor and recipient cells. Jun was mutated into its leucine zipper-motif to abolished the homodimer formation but to allow the development of heterodimer with Fos. To express these protein to the outer membrane of bacteria, they were merged with AIDA autotransporter. In this direction, the direction and the specificity of communication is controled.<br />
<br />
<br />
<font color=red>Receiving the message :</font><br />
<br />
Once received, the signal from incoming vesicles is transduced through a modified Fec pathway, whereby the receptor is provided by the OMV. Few ABC transporter such as FecABCD (iron transporter) are able to induce a response regardless of the tranlocation, due to the activity of FecA. Moreover some mutant can also have a constitutive expression of FecABCD .So, we would like to use FecA- mutant receiver and FecA+ mutant donor to transfert the constitutive FecA protein to the receiver to transmit to the target cell the message (which comes from vesicles (bubbles)). <br />
<br />
<br />
Computational models provided insight to all of the above steps and suggested directions for system improvement. '''Such reliable communications systems have wide biotechnological implications, ranging from targeted drugs delivery and detoxification to advanced division of labor or even cell-based computing.'''<br />
<br />
<br />
<br />
<br />
All of our constructions are just [[Team:Paris/constructions | here]]<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
<div id="paris_content_boxtop"><br />
</div><br />
<div id="paris_content"><br />
<br />
<br />
==='''A. Plasmid construction'''===<br />
<br />
The plasmid construction is divided into 2 functional modules :<br />
*'''The emission system''', which aims at producing vesicules.<br />
*'''The reception system''' of the signal sent via the vesicules.<br />
<br />
====A.1. The emission system ====<br />
<br />
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.<br />
<br />
<br />
<font color=red>Writing the message: production of signaling proteins </font><br><br />
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.<br />
<br />
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.<br />
<br />
<br />
<font color=red>Preparing the messenger: creation of the vesicles </font><br><br />
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). <br />
<br />
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.<br />
<br />
<br />
* In '''presence of Arabinose''', proteins of interest are created as well as vesicles :<br />
[[Image:Global_On.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
<br />
<br />
*In the '''absence of Arabinose''', the pBad promoter is repressed and there is no production of proteins nor vesicles :<br />
[[Image:Global_Off.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
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.<br />
<br />
====A.2. The reception system====<br />
<br />
To implement our vesicles reception 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.<br />
<br />
<br />
<font color=red>Giving the message: fusion of the vesicles with the receiver.</font><br><br />
To fusion the OMVs with the targeted bacteria. We have explored two differents methods : Jun/Fos dimere and G3P. <br>With Jun/Fos, after mutations into the leucine zipper motif of Jun, we fused it to AIDA to send them to the extern membrane of bacteria. <br>With G3P, we fuse it to the OmpA- Linker protein to target it at the surface of the vesicles.<br />
<br />
<br />
<br />
<font color=red>Transduction: decryption of the message.</font><br />
<br><br />
We plan to use FecA- mutant receiver and FecA+ mutant donor to transfert the constitutive FecA protein to the receiver. In this case the receiver will express the FecABCD operon without being induce by ferric citrate in the medium , and so we could place under the control of the Fec ABCD promoter, which is called pfec, the gene sequence encoding for the response. For the moment a response that would be easy to detect is the fluorescence of the RFP and the biobrick BBa-J61002 is the perfect candidate to test the system. <br />
<br><br />
We also discovered that some fecR and fecI mutants can be use to amplify the signal because they have a constitutive activity. So we put under the control of pfec a FecR and FecI mutated. When they will be expressed, they will be activators of pfec and consequently of RFP. Normaly we would be able to obtain a increasing fluorescence. <br />
<br />
<br />
<br />
<br />
<html><br />
</div><br />
<div id="paris_content_boxtop"><br />
</div><br />
<div id="paris_content"><br />
</html><br />
<br />
<br />
{{Template:Paris2009_guided2|#top|/DryLab}}</div>Fanny.chttp://2009.igem.org/Team:Paris/ProjectTeam:Paris/Project2009-10-22T03:13:13Z<p>Fanny.c: /* A.2. The reception system */</p>
<hr />
<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris#top | Home]] > [[Team:Paris/Project#bottom | OMV Project]]<br />
{{Template:Paris2009}}<br />
{{Template:Paris2009_menu}}<br />
== '''Overall project:''' '''''Message in a bubble'''''==<br />
<br />
<center>'''Message in a Bubble: cell-cell communication using vesicles. '''</center><br />
<br />
<br />
<center>''Communication is a "two way" process. When you communicate you perceive the other persons responses and react with your own thoughts and feelings. It is only by paying attention to the other person that you have any idea about what to say or do next.''</center><br />
<br />
<br />
*''Bacterial communication:''<br />
<br />
Bacteria communicate with another one using chemical signal molecules. As in higher organisms, the information supplied by these molecules is critical for synchronizing the activities of large groups of cells. In bacteria, chemical communication involves producing, releasing, detecting, and responding to small hormone-like molecules<br />
(called acylhomoserine lactones, AHL). This process, also known as quorum sensing, allows bacteria to monitor the environment for other bacteria and to alter behavior on a population-wide scale in response to changes in the number and/or species present in a community. Nevertheless, AHL molecules are broken down by other bacteria, and some AHL signals are poorly soluble in water, so '''they cannot travel far in an aqueous environment (this factor limits their potential as a long communication signals)'''. <br />
<br />
<br />
*''Outer membrane vesicules in bacteria''<br />
Growing '''gram-negative bacteria (like ''E.Coli'' ) release vesicles from their outer membranes as a means of delivering toxins to host cells and other bacteria'''. This mecanism is conserved among Gram-negative bacteria. The vesicles consist of a lipid bilayer surrounding an aqueous core and they can therefore transport lipid-soluble toxins (lipopolysaccharide endotoxin) on their surface and protein toxins in their core. They release their content by fusing with the lipid bilayer of target cells. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<center>'''''The project :'''''</center><br />
<br />
We decided to''' improve bacterial communication''' thanks to the vesicles formation process. In this direction our engineered communication platform consists in '''controlling OMV production''' by destabilizing membrane integrity through over-expression of specific periplasmic proteins of the Tol/Pal system. The over-production of TolR (a major protein of the Tol/Pal system which ensure the membrane integrity) has to be controled to avoid the bacteria death. <br />
Another important key point of our project is to obtain a delay between the production of protein of interest and the vesicle formation, to be sure that the produced vesicles carried the different protein required for the recognition of the target bacteria and thus the one essential for the signal transduction.<br />
<br />
<br />
<font color=red>Producing the messenger :</font> <br />
<br />
In order to control and modulate message content, we used fusions with our protein of interest and OmpA signal sequence or the ClyA hemolysin as delivery tags. OmpA is a major protein of the external membrane of ''E.Coli'' and is also localize on OMVs. In this direction OmpA seems to be appropriate to deliver a specific protein to the outer membrane and, by consequence into vesicles. As OmpA, ClyA is an interesting way to explore to send protein to the external membrane.<br />
<br />
<br />
<font color=red>Addressing the message :</font> <br />
<br />
To own the communication between the donnor and the receiver a targeting system was developed. This system is based on the outer-membrane expression of Jun/Fos leucine zippers to control the vesicle flux between donor and recipient cells. Jun was mutated into its leucine zipper-motif to abolished the homodimer formation but to allow the development of heterodimer with Fos. To express these protein to the outer membrane of bacteria, they were merged with AIDA autotransporter. In this direction, the direction and the specificity of communication is controled.<br />
<br />
<br />
<font color=red>Receiving the message :</font><br />
<br />
Once received, the signal from incoming vesicles is transduced through a modified Fec pathway, whereby the receptor is provided by the OMV. Few ABC transporter such as FecABCD (iron transporter) are able to induce a response regardless of the tranlocation, due to the activity of FecA. Moreover some mutant can also have a constitutive expression of FecABCD .So, we would like to use FecA- mutant receiver and FecA+ mutant donor to transfert the constitutive FecA protein to the receiver to transmit to the target cell the message (which comes from vesicles (bubbles)). <br />
<br />
<br />
Computational models provided insight to all of the above steps and suggested directions for system improvement. '''Such reliable communications systems have wide biotechnological implications, ranging from targeted drugs delivery and detoxification to advanced division of labor or even cell-based computing.'''<br />
<br />
<br />
<br />
<br />
All of our constructions are just [[Team:Paris/constructions | here]]<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
<div id="paris_content_boxtop"><br />
</div><br />
<div id="paris_content"><br />
<br />
<br />
==='''A. Plasmid construction'''===<br />
<br />
The plasmid construction is divided into 2 functional modules :<br />
*'''The emission system''', which aims at producing vesicules.<br />
*'''The reception system''' of the signal sent via the vesicules.<br />
<br />
====A.1. The emission system ====<br />
<br />
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.<br />
<br />
<br />
<font color=red>Writing the message: production of signaling proteins </font><br><br />
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.<br />
<br />
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.<br />
<br />
<br />
<font color=red>Preparing the messenger: creation of the vesicles </font><br><br />
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). <br />
<br />
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.<br />
<br />
<br />
* In '''presence of Arabinose''', proteins of interest are created as well as vesicles :<br />
[[Image:Global_On.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
<br />
<br />
*In the '''absence of Arabinose''', the pBad promoter is repressed and there is no production of proteins nor vesicles :<br />
[[Image:Global_Off.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
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.<br />
<br />
====A.2. The reception system====<br />
<br />
To implement our vesicles reception 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.<br />
<br />
<br />
<font color=red>Giving the message: fusion of the vesicles with the receiver.</font><br><br />
To fusion the OMVs with the targeted bacteria. We have explored two differents methods : Jun/Fos dimere and G3P. <br>With Jun/Fos, after mutations into the leucine zipper motif of Jun, we fused it to AIDA to send them to the extern membrane of bacteria. <br>With G3P, we fuse it to the OmpA- Linker protein to target it at the surface of the vesicles.<br />
<br />
<br />
<br />
<font color=red>Transduction: decryption of the message.</font><br>P<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<html><br />
</div><br />
<div id="paris_content_boxtop"><br />
</div><br />
<div id="paris_content"><br />
</html><br />
<br />
<br />
{{Template:Paris2009_guided2|#top|/DryLab}}</div>Fanny.chttp://2009.igem.org/Team:Paris/ProjectTeam:Paris/Project2009-10-22T03:12:12Z<p>Fanny.c: /* A.2. The reception system */</p>
<hr />
<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris#top | Home]] > [[Team:Paris/Project#bottom | OMV Project]]<br />
{{Template:Paris2009}}<br />
{{Template:Paris2009_menu}}<br />
== '''Overall project:''' '''''Message in a bubble'''''==<br />
<br />
<center>'''Message in a Bubble: cell-cell communication using vesicles. '''</center><br />
<br />
<br />
<center>''Communication is a "two way" process. When you communicate you perceive the other persons responses and react with your own thoughts and feelings. It is only by paying attention to the other person that you have any idea about what to say or do next.''</center><br />
<br />
<br />
*''Bacterial communication:''<br />
<br />
Bacteria communicate with another one using chemical signal molecules. As in higher organisms, the information supplied by these molecules is critical for synchronizing the activities of large groups of cells. In bacteria, chemical communication involves producing, releasing, detecting, and responding to small hormone-like molecules<br />
(called acylhomoserine lactones, AHL). This process, also known as quorum sensing, allows bacteria to monitor the environment for other bacteria and to alter behavior on a population-wide scale in response to changes in the number and/or species present in a community. Nevertheless, AHL molecules are broken down by other bacteria, and some AHL signals are poorly soluble in water, so '''they cannot travel far in an aqueous environment (this factor limits their potential as a long communication signals)'''. <br />
<br />
<br />
*''Outer membrane vesicules in bacteria''<br />
Growing '''gram-negative bacteria (like ''E.Coli'' ) release vesicles from their outer membranes as a means of delivering toxins to host cells and other bacteria'''. This mecanism is conserved among Gram-negative bacteria. The vesicles consist of a lipid bilayer surrounding an aqueous core and they can therefore transport lipid-soluble toxins (lipopolysaccharide endotoxin) on their surface and protein toxins in their core. They release their content by fusing with the lipid bilayer of target cells. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<center>'''''The project :'''''</center><br />
<br />
We decided to''' improve bacterial communication''' thanks to the vesicles formation process. In this direction our engineered communication platform consists in '''controlling OMV production''' by destabilizing membrane integrity through over-expression of specific periplasmic proteins of the Tol/Pal system. The over-production of TolR (a major protein of the Tol/Pal system which ensure the membrane integrity) has to be controled to avoid the bacteria death. <br />
Another important key point of our project is to obtain a delay between the production of protein of interest and the vesicle formation, to be sure that the produced vesicles carried the different protein required for the recognition of the target bacteria and thus the one essential for the signal transduction.<br />
<br />
<br />
<font color=red>Producing the messenger :</font> <br />
<br />
In order to control and modulate message content, we used fusions with our protein of interest and OmpA signal sequence or the ClyA hemolysin as delivery tags. OmpA is a major protein of the external membrane of ''E.Coli'' and is also localize on OMVs. In this direction OmpA seems to be appropriate to deliver a specific protein to the outer membrane and, by consequence into vesicles. As OmpA, ClyA is an interesting way to explore to send protein to the external membrane.<br />
<br />
<br />
<font color=red>Addressing the message :</font> <br />
<br />
To own the communication between the donnor and the receiver a targeting system was developed. This system is based on the outer-membrane expression of Jun/Fos leucine zippers to control the vesicle flux between donor and recipient cells. Jun was mutated into its leucine zipper-motif to abolished the homodimer formation but to allow the development of heterodimer with Fos. To express these protein to the outer membrane of bacteria, they were merged with AIDA autotransporter. In this direction, the direction and the specificity of communication is controled.<br />
<br />
<br />
<font color=red>Receiving the message :</font><br />
<br />
Once received, the signal from incoming vesicles is transduced through a modified Fec pathway, whereby the receptor is provided by the OMV. Few ABC transporter such as FecABCD (iron transporter) are able to induce a response regardless of the tranlocation, due to the activity of FecA. Moreover some mutant can also have a constitutive expression of FecABCD .So, we would like to use FecA- mutant receiver and FecA+ mutant donor to transfert the constitutive FecA protein to the receiver to transmit to the target cell the message (which comes from vesicles (bubbles)). <br />
<br />
<br />
Computational models provided insight to all of the above steps and suggested directions for system improvement. '''Such reliable communications systems have wide biotechnological implications, ranging from targeted drugs delivery and detoxification to advanced division of labor or even cell-based computing.'''<br />
<br />
<br />
<br />
<br />
All of our constructions are just [[Team:Paris/constructions | here]]<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
<div id="paris_content_boxtop"><br />
</div><br />
<div id="paris_content"><br />
<br />
<br />
==='''A. Plasmid construction'''===<br />
<br />
The plasmid construction is divided into 2 functional modules :<br />
*'''The emission system''', which aims at producing vesicules.<br />
*'''The reception system''' of the signal sent via the vesicules.<br />
<br />
====A.1. The emission system ====<br />
<br />
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.<br />
<br />
<br />
<font color=red>Writing the message: production of signaling proteins </font><br><br />
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.<br />
<br />
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.<br />
<br />
<br />
<font color=red>Preparing the messenger: creation of the vesicles </font><br><br />
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). <br />
<br />
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.<br />
<br />
<br />
* In '''presence of Arabinose''', proteins of interest are created as well as vesicles :<br />
[[Image:Global_On.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
<br />
<br />
*In the '''absence of Arabinose''', the pBad promoter is repressed and there is no production of proteins nor vesicles :<br />
[[Image:Global_Off.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
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.<br />
<br />
====A.2. The reception system====<br />
<br />
To implement our vesicles reception 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.<br />
<br />
<br />
<font color=red>Giving the message: fusion of the vesicles with the receiver.</font><br><br />
To fusion the OMVs with the targeted bacteria. We have explored two differents methods : Jun/Fos dimere and G3P. With Jun/Fos, after mutations into the leucine zipper motif of Jun, we fused it to AIDA to send them to the extern membrane of bacteria. With G3P, we fuse it to the OmpA- Linker protein to target it at the surface of the vesicles.<br />
<br />
<br />
<br />
<font color=red>Transduction: decryption of the message.</font><br>P<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<html><br />
</div><br />
<div id="paris_content_boxtop"><br />
</div><br />
<div id="paris_content"><br />
</html><br />
<br />
<br />
{{Template:Paris2009_guided2|#top|/DryLab}}</div>Fanny.chttp://2009.igem.org/Team:Paris/ProjectTeam:Paris/Project2009-10-22T02:55:39Z<p>Fanny.c: /* A.2. The reception system */</p>
<hr />
<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris#top | Home]] > [[Team:Paris/Project#bottom | OMV Project]]<br />
{{Template:Paris2009}}<br />
{{Template:Paris2009_menu}}<br />
== '''Overall project:''' '''''Message in a bubble'''''==<br />
<br />
<center>'''Message in a Bubble: cell-cell communication using vesicles. '''</center><br />
<br />
<br />
<center>''Communication is a "two way" process. When you communicate you perceive the other persons responses and react with your own thoughts and feelings. It is only by paying attention to the other person that you have any idea about what to say or do next.''</center><br />
<br />
<br />
*''Bacterial communication:''<br />
<br />
Bacteria communicate with another one using chemical signal molecules. As in higher organisms, the information supplied by these molecules is critical for synchronizing the activities of large groups of cells. In bacteria, chemical communication involves producing, releasing, detecting, and responding to small hormone-like molecules<br />
(called acylhomoserine lactones, AHL). This process, also known as quorum sensing, allows bacteria to monitor the environment for other bacteria and to alter behavior on a population-wide scale in response to changes in the number and/or species present in a community. Nevertheless, AHL molecules are broken down by other bacteria, and some AHL signals are poorly soluble in water, so '''they cannot travel far in an aqueous environment (this factor limits their potential as a long communication signals)'''. <br />
<br />
<br />
*''Outer membrane vesicules in bacteria''<br />
Growing '''gram-negative bacteria (like ''E.Coli'' ) release vesicles from their outer membranes as a means of delivering toxins to host cells and other bacteria'''. This mecanism is conserved among Gram-negative bacteria. The vesicles consist of a lipid bilayer surrounding an aqueous core and they can therefore transport lipid-soluble toxins (lipopolysaccharide endotoxin) on their surface and protein toxins in their core. They release their content by fusing with the lipid bilayer of target cells. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<center>'''''The project :'''''</center><br />
<br />
We decided to''' improve bacterial communication''' thanks to the vesicles formation process. In this direction our engineered communication platform consists in '''controlling OMV production''' by destabilizing membrane integrity through over-expression of specific periplasmic proteins of the Tol/Pal system. The over-production of TolR (a major protein of the Tol/Pal system which ensure the membrane integrity) has to be controled to avoid the bacteria death. <br />
Another important key point of our project is to obtain a delay between the production of protein of interest and the vesicle formation, to be sure that the produced vesicles carried the different protein required for the recognition of the target bacteria and thus the one essential for the signal transduction.<br />
<br />
<br />
<font color=red>Producing the messenger :</font> <br />
<br />
In order to control and modulate message content, we used fusions with our protein of interest and OmpA signal sequence or the ClyA hemolysin as delivery tags. OmpA is a major protein of the external membrane of ''E.Coli'' and is also localize on OMVs. In this direction OmpA seems to be appropriate to deliver a specific protein to the outer membrane and, by consequence into vesicles. As OmpA, ClyA is an interesting way to explore to send protein to the external membrane.<br />
<br />
<br />
<font color=red>Addressing the message :</font> <br />
<br />
To own the communication between the donnor and the receiver a targeting system was developed. This system is based on the outer-membrane expression of Jun/Fos leucine zippers to control the vesicle flux between donor and recipient cells. Jun was mutated into its leucine zipper-motif to abolished the homodimer formation but to allow the development of heterodimer with Fos. To express these protein to the outer membrane of bacteria, they were merged with AIDA autotransporter. In this direction, the direction and the specificity of communication is controled.<br />
<br />
<br />
<font color=red>Receiving the message :</font><br />
<br />
Once received, the signal from incoming vesicles is transduced through a modified Fec pathway, whereby the receptor is provided by the OMV. Few ABC transporter such as FecABCD (iron transporter) are able to induce a response regardless of the tranlocation, due to the activity of FecA. Moreover some mutant can also have a constitutive expression of FecABCD .So, we would like to use FecA- mutant receiver and FecA+ mutant donor to transfert the constitutive FecA protein to the receiver to transmit to the target cell the message (which comes from vesicles (bubbles)). <br />
<br />
<br />
Computational models provided insight to all of the above steps and suggested directions for system improvement. '''Such reliable communications systems have wide biotechnological implications, ranging from targeted drugs delivery and detoxification to advanced division of labor or even cell-based computing.'''<br />
<br />
<br />
<br />
<br />
All of our constructions are just [[Team:Paris/constructions | here]]<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
<div id="paris_content_boxtop"><br />
</div><br />
<div id="paris_content"><br />
<br />
<br />
==='''A. Plasmid construction'''===<br />
<br />
The plasmid construction is divided into 2 functional modules :<br />
*'''The emission system''', which aims at producing vesicules.<br />
*'''The reception system''' of the signal sent via the vesicules.<br />
<br />
====A.1. The emission system ====<br />
<br />
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.<br />
<br />
<br />
<font color=red>Writing the message: production of signaling proteins </font><br><br />
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.<br />
<br />
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.<br />
<br />
<br />
<font color=red>Preparing the messenger: creation of the vesicles </font><br><br />
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). <br />
<br />
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.<br />
<br />
<br />
* In '''presence of Arabinose''', proteins of interest are created as well as vesicles :<br />
[[Image:Global_On.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
<br />
<br />
*In the '''absence of Arabinose''', the pBad promoter is repressed and there is no production of proteins nor vesicles :<br />
[[Image:Global_Off.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
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.<br />
<br />
====A.2. The reception system====<br />
<br />
To implement our vesicles reception 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.<br />
<br />
<br />
<font color=red>Giving the message: fusion of the vesicles with the receiver.</font><br><br />
<br />
<br />
<br />
<font color=red>Transduction: decryption of the message.</font><br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<html><br />
</div><br />
<div id="paris_content_boxtop"><br />
</div><br />
<div id="paris_content"><br />
</html><br />
<br />
<br />
{{Template:Paris2009_guided2|#top|/DryLab}}</div>Fanny.chttp://2009.igem.org/Team:Paris/ProjectTeam:Paris/Project2009-10-22T02:53:40Z<p>Fanny.c: /* A.2. The reception system */</p>
<hr />
<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris#top | Home]] > [[Team:Paris/Project#bottom | OMV Project]]<br />
{{Template:Paris2009}}<br />
{{Template:Paris2009_menu}}<br />
== '''Overall project:''' '''''Message in a bubble'''''==<br />
<br />
<center>'''Message in a Bubble: cell-cell communication using vesicles. '''</center><br />
<br />
<br />
<center>''Communication is a "two way" process. When you communicate you perceive the other persons responses and react with your own thoughts and feelings. It is only by paying attention to the other person that you have any idea about what to say or do next.''</center><br />
<br />
<br />
*''Bacterial communication:''<br />
<br />
Bacteria communicate with another one using chemical signal molecules. As in higher organisms, the information supplied by these molecules is critical for synchronizing the activities of large groups of cells. In bacteria, chemical communication involves producing, releasing, detecting, and responding to small hormone-like molecules<br />
(called acylhomoserine lactones, AHL). This process, also known as quorum sensing, allows bacteria to monitor the environment for other bacteria and to alter behavior on a population-wide scale in response to changes in the number and/or species present in a community. Nevertheless, AHL molecules are broken down by other bacteria, and some AHL signals are poorly soluble in water, so '''they cannot travel far in an aqueous environment (this factor limits their potential as a long communication signals)'''. <br />
<br />
<br />
*''Outer membrane vesicules in bacteria''<br />
Growing '''gram-negative bacteria (like ''E.Coli'' ) release vesicles from their outer membranes as a means of delivering toxins to host cells and other bacteria'''. This mecanism is conserved among Gram-negative bacteria. The vesicles consist of a lipid bilayer surrounding an aqueous core and they can therefore transport lipid-soluble toxins (lipopolysaccharide endotoxin) on their surface and protein toxins in their core. They release their content by fusing with the lipid bilayer of target cells. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<center>'''''The project :'''''</center><br />
<br />
We decided to''' improve bacterial communication''' thanks to the vesicles formation process. In this direction our engineered communication platform consists in '''controlling OMV production''' by destabilizing membrane integrity through over-expression of specific periplasmic proteins of the Tol/Pal system. The over-production of TolR (a major protein of the Tol/Pal system which ensure the membrane integrity) has to be controled to avoid the bacteria death. <br />
Another important key point of our project is to obtain a delay between the production of protein of interest and the vesicle formation, to be sure that the produced vesicles carried the different protein required for the recognition of the target bacteria and thus the one essential for the signal transduction.<br />
<br />
<br />
<font color=red>Producing the messenger :</font> <br />
<br />
In order to control and modulate message content, we used fusions with our protein of interest and OmpA signal sequence or the ClyA hemolysin as delivery tags. OmpA is a major protein of the external membrane of ''E.Coli'' and is also localize on OMVs. In this direction OmpA seems to be appropriate to deliver a specific protein to the outer membrane and, by consequence into vesicles. As OmpA, ClyA is an interesting way to explore to send protein to the external membrane.<br />
<br />
<br />
<font color=red>Addressing the message :</font> <br />
<br />
To own the communication between the donnor and the receiver a targeting system was developed. This system is based on the outer-membrane expression of Jun/Fos leucine zippers to control the vesicle flux between donor and recipient cells. Jun was mutated into its leucine zipper-motif to abolished the homodimer formation but to allow the development of heterodimer with Fos. To express these protein to the outer membrane of bacteria, they were merged with AIDA autotransporter. In this direction, the direction and the specificity of communication is controled.<br />
<br />
<br />
<font color=red>Receiving the message :</font><br />
<br />
Once received, the signal from incoming vesicles is transduced through a modified Fec pathway, whereby the receptor is provided by the OMV. Few ABC transporter such as FecABCD (iron transporter) are able to induce a response regardless of the tranlocation, due to the activity of FecA. Moreover some mutant can also have a constitutive expression of FecABCD .So, we would like to use FecA- mutant receiver and FecA+ mutant donor to transfert the constitutive FecA protein to the receiver to transmit to the target cell the message (which comes from vesicles (bubbles)). <br />
<br />
<br />
Computational models provided insight to all of the above steps and suggested directions for system improvement. '''Such reliable communications systems have wide biotechnological implications, ranging from targeted drugs delivery and detoxification to advanced division of labor or even cell-based computing.'''<br />
<br />
<br />
<br />
<br />
All of our constructions are just [[Team:Paris/constructions | here]]<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
<div id="paris_content_boxtop"><br />
</div><br />
<div id="paris_content"><br />
<br />
<br />
==='''A. Plasmid construction'''===<br />
<br />
The plasmid construction is divided into 2 functional modules :<br />
*'''The emission system''', which aims at producing vesicules.<br />
*'''The reception system''' of the signal sent via the vesicules.<br />
<br />
====A.1. The emission system ====<br />
<br />
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.<br />
<br />
<br />
<font color=red>Writing the message: production of signaling proteins </font><br><br />
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.<br />
<br />
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.<br />
<br />
<br />
<font color=red>Preparing the messenger: creation of the vesicles </font><br><br />
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). <br />
<br />
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.<br />
<br />
<br />
* In '''presence of Arabinose''', proteins of interest are created as well as vesicles :<br />
[[Image:Global_On.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
<br />
<br />
*In the '''absence of Arabinose''', the pBad promoter is repressed and there is no production of proteins nor vesicles :<br />
[[Image:Global_Off.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
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.<br />
<br />
====A.2. The reception system====<br />
<br />
To implement our vesicles reception 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.<br />
<br />
<br />
<font color=red>Giving the message: fusion of the vesicles with the receiver.</font><br><br />
<br />
<br />
<br />
<font color=red>Reading the message: transduction of the message.</font><br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<html><br />
</div><br />
<div id="paris_content_boxtop"><br />
</div><br />
<div id="paris_content"><br />
</html><br />
<br />
<br />
{{Template:Paris2009_guided2|#top|/DryLab}}</div>Fanny.chttp://2009.igem.org/Team:Paris/ProjectTeam:Paris/Project2009-10-22T02:50:10Z<p>Fanny.c: /* A.2. The reception system */</p>
<hr />
<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris#top | Home]] > [[Team:Paris/Project#bottom | OMV Project]]<br />
{{Template:Paris2009}}<br />
{{Template:Paris2009_menu}}<br />
== '''Overall project:''' '''''Message in a bubble'''''==<br />
<br />
<center>'''Message in a Bubble: cell-cell communication using vesicles. '''</center><br />
<br />
<br />
<center>''Communication is a "two way" process. When you communicate you perceive the other persons responses and react with your own thoughts and feelings. It is only by paying attention to the other person that you have any idea about what to say or do next.''</center><br />
<br />
<br />
*''Bacterial communication:''<br />
<br />
Bacteria communicate with another one using chemical signal molecules. As in higher organisms, the information supplied by these molecules is critical for synchronizing the activities of large groups of cells. In bacteria, chemical communication involves producing, releasing, detecting, and responding to small hormone-like molecules<br />
(called acylhomoserine lactones, AHL). This process, also known as quorum sensing, allows bacteria to monitor the environment for other bacteria and to alter behavior on a population-wide scale in response to changes in the number and/or species present in a community. Nevertheless, AHL molecules are broken down by other bacteria, and some AHL signals are poorly soluble in water, so '''they cannot travel far in an aqueous environment (this factor limits their potential as a long communication signals)'''. <br />
<br />
<br />
*''Outer membrane vesicules in bacteria''<br />
Growing '''gram-negative bacteria (like ''E.Coli'' ) release vesicles from their outer membranes as a means of delivering toxins to host cells and other bacteria'''. This mecanism is conserved among Gram-negative bacteria. The vesicles consist of a lipid bilayer surrounding an aqueous core and they can therefore transport lipid-soluble toxins (lipopolysaccharide endotoxin) on their surface and protein toxins in their core. They release their content by fusing with the lipid bilayer of target cells. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<center>'''''The project :'''''</center><br />
<br />
We decided to''' improve bacterial communication''' thanks to the vesicles formation process. In this direction our engineered communication platform consists in '''controlling OMV production''' by destabilizing membrane integrity through over-expression of specific periplasmic proteins of the Tol/Pal system. The over-production of TolR (a major protein of the Tol/Pal system which ensure the membrane integrity) has to be controled to avoid the bacteria death. <br />
Another important key point of our project is to obtain a delay between the production of protein of interest and the vesicle formation, to be sure that the produced vesicles carried the different protein required for the recognition of the target bacteria and thus the one essential for the signal transduction.<br />
<br />
<br />
<font color=red>Producing the messenger :</font> <br />
<br />
In order to control and modulate message content, we used fusions with our protein of interest and OmpA signal sequence or the ClyA hemolysin as delivery tags. OmpA is a major protein of the external membrane of ''E.Coli'' and is also localize on OMVs. In this direction OmpA seems to be appropriate to deliver a specific protein to the outer membrane and, by consequence into vesicles. As OmpA, ClyA is an interesting way to explore to send protein to the external membrane.<br />
<br />
<br />
<font color=red>Addressing the message :</font> <br />
<br />
To own the communication between the donnor and the receiver a targeting system was developed. This system is based on the outer-membrane expression of Jun/Fos leucine zippers to control the vesicle flux between donor and recipient cells. Jun was mutated into its leucine zipper-motif to abolished the homodimer formation but to allow the development of heterodimer with Fos. To express these protein to the outer membrane of bacteria, they were merged with AIDA autotransporter. In this direction, the direction and the specificity of communication is controled.<br />
<br />
<br />
<font color=red>Receiving the message :</font><br />
<br />
Once received, the signal from incoming vesicles is transduced through a modified Fec pathway, whereby the receptor is provided by the OMV. Few ABC transporter such as FecABCD (iron transporter) are able to induce a response regardless of the tranlocation, due to the activity of FecA. Moreover some mutant can also have a constitutive expression of FecABCD .So, we would like to use FecA- mutant receiver and FecA+ mutant donor to transfert the constitutive FecA protein to the receiver to transmit to the target cell the message (which comes from vesicles (bubbles)). <br />
<br />
<br />
Computational models provided insight to all of the above steps and suggested directions for system improvement. '''Such reliable communications systems have wide biotechnological implications, ranging from targeted drugs delivery and detoxification to advanced division of labor or even cell-based computing.'''<br />
<br />
<br />
<br />
<br />
All of our constructions are just [[Team:Paris/constructions | here]]<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
<div id="paris_content_boxtop"><br />
</div><br />
<div id="paris_content"><br />
<br />
<br />
==='''A. Plasmid construction'''===<br />
<br />
The plasmid construction is divided into 2 functional modules :<br />
*'''The emission system''', which aims at producing vesicules.<br />
*'''The reception system''' of the signal sent via the vesicules.<br />
<br />
====A.1. The emission system ====<br />
<br />
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.<br />
<br />
<br />
<font color=red>Writing the message: production of signaling proteins </font><br><br />
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.<br />
<br />
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.<br />
<br />
<br />
<font color=red>Preparing the messenger: creation of the vesicles </font><br><br />
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). <br />
<br />
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.<br />
<br />
<br />
* In '''presence of Arabinose''', proteins of interest are created as well as vesicles :<br />
[[Image:Global_On.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
<br />
<br />
*In the '''absence of Arabinose''', the pBad promoter is repressed and there is no production of proteins nor vesicles :<br />
[[Image:Global_Off.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
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.<br />
<br />
====A.2. The reception system====<br />
<br />
To implement our vesicles reception project, we had to take several constrains into account. To put into place all the functionalities we needed two kinds of experiments . <br />
<br />
<br />
Giving the message: fusion of the vesicles with the receiver.<br />
<br />
<br />
Lecture of the message: decryption of the message by the receiver.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<html><br />
</div><br />
<div id="paris_content_boxtop"><br />
</div><br />
<div id="paris_content"><br />
</html><br />
<br />
<br />
{{Template:Paris2009_guided2|#top|/DryLab}}</div>Fanny.chttp://2009.igem.org/Team:Paris/ProjectTeam:Paris/Project2009-10-22T02:47:21Z<p>Fanny.c: /* A.2. The reception system */</p>
<hr />
<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris#top | Home]] > [[Team:Paris/Project#bottom | OMV Project]]<br />
{{Template:Paris2009}}<br />
{{Template:Paris2009_menu}}<br />
== '''Overall project:''' '''''Message in a bubble'''''==<br />
<br />
<center>'''Message in a Bubble: cell-cell communication using vesicles. '''</center><br />
<br />
<br />
<center>''Communication is a "two way" process. When you communicate you perceive the other persons responses and react with your own thoughts and feelings. It is only by paying attention to the other person that you have any idea about what to say or do next.''</center><br />
<br />
<br />
*''Bacterial communication:''<br />
<br />
Bacteria communicate with another one using chemical signal molecules. As in higher organisms, the information supplied by these molecules is critical for synchronizing the activities of large groups of cells. In bacteria, chemical communication involves producing, releasing, detecting, and responding to small hormone-like molecules<br />
(called acylhomoserine lactones, AHL). This process, also known as quorum sensing, allows bacteria to monitor the environment for other bacteria and to alter behavior on a population-wide scale in response to changes in the number and/or species present in a community. Nevertheless, AHL molecules are broken down by other bacteria, and some AHL signals are poorly soluble in water, so '''they cannot travel far in an aqueous environment (this factor limits their potential as a long communication signals)'''. <br />
<br />
<br />
*''Outer membrane vesicules in bacteria''<br />
Growing '''gram-negative bacteria (like ''E.Coli'' ) release vesicles from their outer membranes as a means of delivering toxins to host cells and other bacteria'''. This mecanism is conserved among Gram-negative bacteria. The vesicles consist of a lipid bilayer surrounding an aqueous core and they can therefore transport lipid-soluble toxins (lipopolysaccharide endotoxin) on their surface and protein toxins in their core. They release their content by fusing with the lipid bilayer of target cells. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<center>'''''The project :'''''</center><br />
<br />
We decided to''' improve bacterial communication''' thanks to the vesicles formation process. In this direction our engineered communication platform consists in '''controlling OMV production''' by destabilizing membrane integrity through over-expression of specific periplasmic proteins of the Tol/Pal system. The over-production of TolR (a major protein of the Tol/Pal system which ensure the membrane integrity) has to be controled to avoid the bacteria death. <br />
Another important key point of our project is to obtain a delay between the production of protein of interest and the vesicle formation, to be sure that the produced vesicles carried the different protein required for the recognition of the target bacteria and thus the one essential for the signal transduction.<br />
<br />
<br />
<font color=red>Producing the messenger :</font> <br />
<br />
In order to control and modulate message content, we used fusions with our protein of interest and OmpA signal sequence or the ClyA hemolysin as delivery tags. OmpA is a major protein of the external membrane of ''E.Coli'' and is also localize on OMVs. In this direction OmpA seems to be appropriate to deliver a specific protein to the outer membrane and, by consequence into vesicles. As OmpA, ClyA is an interesting way to explore to send protein to the external membrane.<br />
<br />
<br />
<font color=red>Addressing the message :</font> <br />
<br />
To own the communication between the donnor and the receiver a targeting system was developed. This system is based on the outer-membrane expression of Jun/Fos leucine zippers to control the vesicle flux between donor and recipient cells. Jun was mutated into its leucine zipper-motif to abolished the homodimer formation but to allow the development of heterodimer with Fos. To express these protein to the outer membrane of bacteria, they were merged with AIDA autotransporter. In this direction, the direction and the specificity of communication is controled.<br />
<br />
<br />
<font color=red>Receiving the message :</font><br />
<br />
Once received, the signal from incoming vesicles is transduced through a modified Fec pathway, whereby the receptor is provided by the OMV. Few ABC transporter such as FecABCD (iron transporter) are able to induce a response regardless of the tranlocation, due to the activity of FecA. Moreover some mutant can also have a constitutive expression of FecABCD .So, we would like to use FecA- mutant receiver and FecA+ mutant donor to transfert the constitutive FecA protein to the receiver to transmit to the target cell the message (which comes from vesicles (bubbles)). <br />
<br />
<br />
Computational models provided insight to all of the above steps and suggested directions for system improvement. '''Such reliable communications systems have wide biotechnological implications, ranging from targeted drugs delivery and detoxification to advanced division of labor or even cell-based computing.'''<br />
<br />
<br />
<br />
<br />
All of our constructions are just [[Team:Paris/constructions | here]]<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
<div id="paris_content_boxtop"><br />
</div><br />
<div id="paris_content"><br />
<br />
<br />
==='''A. Plasmid construction'''===<br />
<br />
The plasmid construction is divided into 2 functional modules :<br />
*'''The emission system''', which aims at producing vesicules.<br />
*'''The reception system''' of the signal sent via the vesicules.<br />
<br />
====A.1. The emission system ====<br />
<br />
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.<br />
<br />
<br />
<font color=red>Writing the message: production of signaling proteins </font><br><br />
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.<br />
<br />
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.<br />
<br />
<br />
<font color=red>Preparing the messenger: creation of the vesicles </font><br><br />
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). <br />
<br />
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.<br />
<br />
<br />
* In '''presence of Arabinose''', proteins of interest are created as well as vesicles :<br />
[[Image:Global_On.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
<br />
<br />
*In the '''absence of Arabinose''', the pBad promoter is repressed and there is no production of proteins nor vesicles :<br />
[[Image:Global_Off.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
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.<br />
<br />
====A.2. The reception system====<br />
<br />
To implement our vesicles reception project, we had to take several constrains into account. To put into place all the functionalities we needed two kinds of experiments . <br />
<br />
<br />
Giving the message: fusion of the vesicles with the receiver.<br />
<br />
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Lecture of the message: Transduction by the system of the message.<br />
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{{Template:Paris2009_guided2|#top|/DryLab}}</div>Fanny.chttp://2009.igem.org/Team:Paris/ProjectTeam:Paris/Project2009-10-22T02:43:17Z<p>Fanny.c: /* A.2. The reception system */</p>
<hr />
<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris#top | Home]] > [[Team:Paris/Project#bottom | OMV Project]]<br />
{{Template:Paris2009}}<br />
{{Template:Paris2009_menu}}<br />
== '''Overall project:''' '''''Message in a bubble'''''==<br />
<br />
<center>'''Message in a Bubble: cell-cell communication using vesicles. '''</center><br />
<br />
<br />
<center>''Communication is a "two way" process. When you communicate you perceive the other persons responses and react with your own thoughts and feelings. It is only by paying attention to the other person that you have any idea about what to say or do next.''</center><br />
<br />
<br />
*''Bacterial communication:''<br />
<br />
Bacteria communicate with another one using chemical signal molecules. As in higher organisms, the information supplied by these molecules is critical for synchronizing the activities of large groups of cells. In bacteria, chemical communication involves producing, releasing, detecting, and responding to small hormone-like molecules<br />
(called acylhomoserine lactones, AHL). This process, also known as quorum sensing, allows bacteria to monitor the environment for other bacteria and to alter behavior on a population-wide scale in response to changes in the number and/or species present in a community. Nevertheless, AHL molecules are broken down by other bacteria, and some AHL signals are poorly soluble in water, so '''they cannot travel far in an aqueous environment (this factor limits their potential as a long communication signals)'''. <br />
<br />
<br />
*''Outer membrane vesicules in bacteria''<br />
Growing '''gram-negative bacteria (like ''E.Coli'' ) release vesicles from their outer membranes as a means of delivering toxins to host cells and other bacteria'''. This mecanism is conserved among Gram-negative bacteria. The vesicles consist of a lipid bilayer surrounding an aqueous core and they can therefore transport lipid-soluble toxins (lipopolysaccharide endotoxin) on their surface and protein toxins in their core. They release their content by fusing with the lipid bilayer of target cells. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<center>'''''The project :'''''</center><br />
<br />
We decided to''' improve bacterial communication''' thanks to the vesicles formation process. In this direction our engineered communication platform consists in '''controlling OMV production''' by destabilizing membrane integrity through over-expression of specific periplasmic proteins of the Tol/Pal system. The over-production of TolR (a major protein of the Tol/Pal system which ensure the membrane integrity) has to be controled to avoid the bacteria death. <br />
Another important key point of our project is to obtain a delay between the production of protein of interest and the vesicle formation, to be sure that the produced vesicles carried the different protein required for the recognition of the target bacteria and thus the one essential for the signal transduction.<br />
<br />
<br />
<font color=red>Producing the messenger :</font> <br />
<br />
In order to control and modulate message content, we used fusions with our protein of interest and OmpA signal sequence or the ClyA hemolysin as delivery tags. OmpA is a major protein of the external membrane of ''E.Coli'' and is also localize on OMVs. In this direction OmpA seems to be appropriate to deliver a specific protein to the outer membrane and, by consequence into vesicles. As OmpA, ClyA is an interesting way to explore to send protein to the external membrane.<br />
<br />
<br />
<font color=red>Addressing the message :</font> <br />
<br />
To own the communication between the donnor and the receiver a targeting system was developed. This system is based on the outer-membrane expression of Jun/Fos leucine zippers to control the vesicle flux between donor and recipient cells. Jun was mutated into its leucine zipper-motif to abolished the homodimer formation but to allow the development of heterodimer with Fos. To express these protein to the outer membrane of bacteria, they were merged with AIDA autotransporter. In this direction, the direction and the specificity of communication is controled.<br />
<br />
<br />
<font color=red>Receiving the message :</font><br />
<br />
Once received, the signal from incoming vesicles is transduced through a modified Fec pathway, whereby the receptor is provided by the OMV. Few ABC transporter such as FecABCD (iron transporter) are able to induce a response regardless of the tranlocation, due to the activity of FecA. Moreover some mutant can also have a constitutive expression of FecABCD .So, we would like to use FecA- mutant receiver and FecA+ mutant donor to transfert the constitutive FecA protein to the receiver to transmit to the target cell the message (which comes from vesicles (bubbles)). <br />
<br />
<br />
Computational models provided insight to all of the above steps and suggested directions for system improvement. '''Such reliable communications systems have wide biotechnological implications, ranging from targeted drugs delivery and detoxification to advanced division of labor or even cell-based computing.'''<br />
<br />
<br />
<br />
<br />
All of our constructions are just [[Team:Paris/constructions | here]]<br />
<br />
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<br />
==='''A. Plasmid construction'''===<br />
<br />
The plasmid construction is divided into 2 functional modules :<br />
*'''The emission system''', which aims at producing vesicules.<br />
*'''The reception system''' of the signal sent via the vesicules.<br />
<br />
====A.1. The emission system ====<br />
<br />
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.<br />
<br />
<br />
<font color=red>Writing the message: production of signaling proteins </font><br><br />
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.<br />
<br />
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.<br />
<br />
<br />
<font color=red>Preparing the messenger: creation of the vesicles </font><br><br />
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). <br />
<br />
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.<br />
<br />
<br />
* In '''presence of Arabinose''', proteins of interest are created as well as vesicles :<br />
[[Image:Global_On.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
<br />
<br />
*In the '''absence of Arabinose''', the pBad promoter is repressed and there is no production of proteins nor vesicles :<br />
[[Image:Global_Off.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
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.<br />
<br />
====A.2. The reception system====<br />
<br />
To implement our vesicles reception project, we had to take several constrains into account. To put into place all the functionalities we needed two kinds of experiments . <br />
<br />
<br />
transmission of the message: fusion of the vesicles that contain the message with the receiver.<br />
<br />
<br />
Lecture of the messenger: the system will be able de decypher the message.<br />
<br />
<br />
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{{Template:Paris2009_guided2|#top|/DryLab}}</div>Fanny.chttp://2009.igem.org/Team:Paris/ProjectTeam:Paris/Project2009-10-22T02:25:09Z<p>Fanny.c: /* The reception system */</p>
<hr />
<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris#top | Home]] > [[Team:Paris/Project#bottom | OMV Project]]<br />
{{Template:Paris2009}}<br />
{{Template:Paris2009_menu}}<br />
== '''Overall project:''' '''''Message in a bubble'''''==<br />
<br />
<center>'''Message in a Bubble: cell-cell communication using vesicles. '''</center><br />
<br />
<br />
<center>''Communication is a "two way" process. When you communicate you perceive the other persons responses and react with your own thoughts and feelings. It is only by paying attention to the other person that you have any idea about what to say or do next.''</center><br />
<br />
<br />
*''Bacterial communication:''<br />
<br />
Bacteria communicate with another one using chemical signal molecules. As in higher organisms, the information supplied by these molecules is critical for synchronizing the activities of large groups of cells. In bacteria, chemical communication involves producing, releasing, detecting, and responding to small hormone-like molecules<br />
(called acylhomoserine lactones, AHL). This process, also known as quorum sensing, allows bacteria to monitor the environment for other bacteria and to alter behavior on a population-wide scale in response to changes in the number and/or species present in a community. Nevertheless, AHL molecules are broken down by other bacteria, and some AHL signals are poorly soluble in water, so '''they cannot travel far in an aqueous environment (this factor limits their potential as a long communication signals)'''. <br />
<br />
<br />
*''Outer membrane vesicules in bacteria''<br />
Growing '''gram-negative bacteria (like ''E.Coli'' ) release vesicles from their outer membranes as a means of delivering toxins to host cells and other bacteria'''. This mecanism is conserved among Gram-negative bacteria. The vesicles consist of a lipid bilayer surrounding an aqueous core and they can therefore transport lipid-soluble toxins (lipopolysaccharide endotoxin) on their surface and protein toxins in their core. They release their content by fusing with the lipid bilayer of target cells. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<center>'''''The project :'''''</center><br />
<br />
We decided to''' improve bacterial communication''' thanks to the vesicles formation process. In this direction our engineered communication platform consists in '''controlling OMV production''' by destabilizing membrane integrity through over-expression of specific periplasmic proteins of the Tol/Pal system. The over-production of TolR (a major protein of the Tol/Pal system which ensure the membrane integrity) has to be controled to avoid the bacteria death. <br />
Another important key point of our project is to obtain a delay between the production of protein of interest and the vesicle formation, to be sure that the produced vesicles carried the different protein required for the recognition of the target bacteria and thus the one essential for the signal transduction.<br />
<br />
<br />
<font color=red>Producing the messenger :</font> <br />
<br />
In order to control and modulate message content, we used fusions with our protein of interest and OmpA signal sequence or the ClyA hemolysin as delivery tags. OmpA is a major protein of the external membrane of ''E.Coli'' and is also localize on OMVs. In this direction OmpA seems to be appropriate to deliver a specific protein to the outer membrane and, by consequence into vesicles. As OmpA, ClyA is an interesting way to explore to send protein to the external membrane.<br />
<br />
<br />
<font color=red>Addressing the message :</font> <br />
<br />
To own the communication between the donnor and the receiver a targeting system was developed. This system is based on the outer-membrane expression of Jun/Fos leucine zippers to control the vesicle flux between donor and recipient cells. Jun was mutated into its leucine zipper-motif to abolished the homodimer formation but to allow the development of heterodimer with Fos. To express these protein to the outer membrane of bacteria, they were merged with AIDA autotransporter. In this direction, the direction and the specificity of communication is controled.<br />
<br />
<br />
<font color=red>Receiving the message :</font><br />
<br />
Once received, the signal from incoming vesicles is transduced through a modified Fec pathway, whereby the receptor is provided by the OMV. Few ABC transporter such as FecABCD (iron transporter) are able to induce a response regardless of the tranlocation, due to the activity of FecA. Moreover some mutant can also have a constitutive expression of FecABCD .So, we would like to use FecA- mutant receiver and FecA+ mutant donor to transfert the constitutive FecA protein to the receiver to transmit to the target cell the message (which comes from vesicles (bubbles)). <br />
<br />
<br />
Computational models provided insight to all of the above steps. '''Such reliable communications systems have wide biotechnological implications, ranging from targeted drugs delivery and detoxification to advanced division of labor or even cell-based computing.'''<br />
<br />
<br />
<br />
<br />
All of our constructions are just [[Team:Paris/constructions | here]]<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
<div id="paris_content_boxtop"><br />
</div><br />
<div id="paris_content"><br />
<br />
<br />
==='''A. Plasmid construction'''===<br />
<br />
The plasmid construction is divided into 2 functional modules :<br />
*'''The emission system''', which aims at producing vesicules.<br />
*'''The reception system''' of the signal sent via the vesicules.<br />
<br />
====A.1. The emission system ====<br />
<br />
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.<br />
<br />
<br />
<font color=red>Writing the message: production of signaling proteins </font><br><br />
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.<br />
<br />
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.<br />
<br />
<br />
<font color=red>Preparing the messenger: creation of the vesicles </font><br><br />
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). <br />
<br />
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.<br />
<br />
<br />
* In '''presence of Arabinose''', proteins of interest are created as well as vesicles :<br />
[[Image:Global_On.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
<br />
<br />
*In the '''absence of Arabinose''', the pBad promoter is repressed and there is no production of proteins nor vesicles :<br />
[[Image:Global_Off.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
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.<br />
<br />
====A.2. The reception system====<br />
<br />
A FAIRE RAPIDEMENT<br />
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{{Template:Paris2009_guided2|#top|/DryLab}}</div>Fanny.chttp://2009.igem.org/Team:Paris/TeamTeam:Paris/Team2009-10-22T02:20:40Z<p>Fanny.c: /* Advisors */</p>
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<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris/Team#bottom | Team]]<br />
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== Where we are==<br />
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<img src="http://www.cri-paris.org/templates/Cri/images/logocri.jpg"><br />
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<div class="rightcolumn"><br />
Located 5 minutes walk from the Luxembourg Gardens at the Paris Descartes Medicine Faculty in Cochin, the Centre for Research and Interdisciplinarity <a href="www.cri-paris.org/en/cri">CRI</a> was founded in 2005 as a convivial place at the crossroad between Life Sciences and exact, natural, cognitive and social sciences.<br />
Our environment includes a fully-equipped seminar room, a meeting room, office space for visiting professors, a library and coffee rooms and a modelling as well as wet-lab space. The CRI supported its students over three years ago to form the first French team to the iGEM and the tradition is continuing... This is where all happened!<br />
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== Student Team==<br />
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<img src="https://static.igem.org/mediawiki/2009/a/ae/Colonel_Chabbert.jpg" height=120px width=170px><br />
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<br />
<div class="rightcolumn"><br />
<h3>Christophe Chabbert</h3><br />
<I> Mathematics, <a href="http://www.aiv-paris.org/fr/master-aiv/"> Mines Paris Tech</a> </I><br />
<br><br />
AKA "Colonel Chabbert", he is our specialist in modelling especially for an "easy/ordinary" delay system.<br />
He likes sciences (really ?? ), his iGEM co-workers ;-), pizzas, burger and ... wait for it .... Coca-cola<br />
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<img src="https://static.igem.org/mediawiki/2009/7/7a/Caroline_Loy.jpg" height=120px width=100px><br />
</div><br />
<br />
<div class="rightcolumn"><br />
<h3>Caroline Loy</h3><br />
<I> Biology, <a href="http://www.aiv-paris.org/fr/master-aiv/"> Master 1 AIV</a></I> <br><br />
After 3 months as a cow-girl in the middle of nowhere (Scotland), she decided to make animal experimentation on bacteria =D<br />
<br />
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<img src="https://static.igem.org/mediawiki/2009/e/ec/Romain_bodinier.jpg" height=120px width=180px><br />
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<br />
<div class="rightcolumn"><br />
<h3>Romain Bodinier</h3><br />
<I> Biology, <a href="http://www.utc.fr/">UTC</a> </I><br />
<br><br />
Still wondering why he is here ... (Is he looking for a new bacto-foot after the lost of his left one ???). But we may have a proposition of his participation at iGEM....finally we don't.<br />
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<img src="https://static.igem.org/mediawiki/2009/d/da/Soufiane.jpg" height=120px width=100px><br />
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<div class="rightcolumn"><br />
<h3>Soufiane Boumahdi</h3><br />
<I> Biochemistry, <a href="http://www.insa-lyon.fr/">Insa Lyon</a> </I><br />
<br><br />
Every team needs to have "panem et circenses", Soufifi is here for entertaining us. Be careful for the jamboree, The crazy singer is coming !!! stay close to him and you will understand.... hoo year you will.<br />
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<img src="https://static.igem.org/mediawiki/2009/7/70/Charlotte_olivier.jpg" height=120px width=130px><br />
</div><br />
<br />
<div class="rightcolumn"><br />
<h3>Charlotte Olivier</h3><br />
<I>Biology/genetics, <a href="http://www.univ-paris-diderot.fr/magisteregenet/"> European Magister of Genetic</a> and Management/marketing: Specialized Master in management and marketing for the pharmaceutical industry, <a href="http://www.escpeurope.eu/">ESCP-Europe</a> </I><br />
<br><br />
Our Obiwan Kenoby of the lab (without the laser saber)!!! <br><br />
Stoff's note : i may say that she is more like princess Leyla :D<br />
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<img src="https://static.igem.org/mediawiki/2009/0/00/Guillaume_B.jpg" height=120px width=90px><br />
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<div class="rightcolumn"><br />
<h3>Guillaume Beauclair</h3><br />
<I> Biology/Infectiology, <a href="http://www.univ-paris-diderot.fr/">Paris 7</a> </I><br />
<br><br />
Labholic --> working too hard could be dangerous for your health (too much vegetables also). Our 20hour/day lab worker<br />
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<img src="https://static.igem.org/mediawiki/2009/3/35/Sara_Aguiton.jpg" height=120px width=180px><br />
</div><br />
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<div class="rightcolumn"><br />
<h3>Sara Aguiton</h3><br />
<I> Social Studies of Science, <a href="http://www.ehess.fr/fr/"> EHESS</a> </I><br />
<br><br />
Please, use simple ethical words when you are talking to a scientist assembly ;-)<br />
<br />
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<img src="https://static.igem.org/mediawiki/2009/5/56/Christophe_Richard.jpg" height=120px width=180px><br />
</div><br />
<br />
<div class="rightcolumn"><br />
<h3>Christophe Richard</h3><br />
<I> Biology/Informatics, <a href="http://www.aiv-paris.org/fr/master-aiv/"> Master AIW </a> </I><br />
<br><br />
AKA "Bibi", he can talk to computer (Respect !!!!) but not to iPhone ... what a shame for an iPhone developer <br />
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<div class="leftcolumn"><br />
<img src="https://static.igem.org/mediawiki/2009/9/92/Image_8.png" height=120px width=150px><br />
</div><br />
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<div class="rightcolumn"><br />
<h3>Luc Malfondet</h3><br />
<I>Physics/ Nanotechnology & Nanobioscience <a href="http://www.aiv-paris.org/fr/master-aiv/"> Master 2 AIV</a> </I><br />
<br><br />
Open-minded guy !!!!!<br />
<br />
<br />
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<img src="https://static.igem.org/mediawiki/2009/1/1b/Pierre.jpg" height=120px width=180px><br />
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<h3>Pierre Escamilla</h3><br />
<I> Mathematics, <a href="http://www.supelec.fr/"> Supelec </a></I><br />
<br><br />
Cafeino-man and modellingo-man.<br><br />
Spanisho-man and algorythmo-man.<br />
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<div class="leftcolumn"><br />
<img src="https://static.igem.org/mediawiki/2009/7/7c/2009-02_Sylvain_Helas-Othenin.jpg" height=120px width=100px><br />
</div><br />
<br />
<div class="rightcolumn"><br />
<h3>Sylvain Hélas-Othenin</h3><br />
<I> Chemistry, <a href="http://www.chimie.ens.fr/"> Ecole Normale Supérieure</a> , <a href="http://www.aiv-paris.org/fr/master-aiv/"> Master 2 AIV</a> <br></I><br />
Graduation in Chemistry at Ecole Normale Supérieure, Paris - Interdisciplinary Master in Life Sciences at Université Paris Descartes, Paris ... but currently lost in the Middle-East !<br />
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<img src="https://static.igem.org/mediawiki/2009/3/34/Vico.JPG" height=120px width=180px> <br />
</div><br />
<br />
<div class="rightcolumn"><br />
<h3>Vicard Du</h3><br />
<I> Biology, <a href="http://www.aiv-paris.org/fr/master-aiv/"> Master 1 AIV </I></a> <br />
<br><br />
Uncle Mc Donalds is his best friend<br />
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== Supervisors ==<br />
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<h3>Ariel Lindner</h3><br />
<br />
Co-initiator of the Centre for Research and Interdisciplinarity (cri-paris.org), Ariel is an INSERM tenured senior researcher and director of the Interdisciplinary Approaches to Life Sciences ('AIV') master program at the Paris Descartes and Diderot Universities. Ariel has graduated from the Hebrew University (Jerusalem, Israel) "Amirim" interdisciplinary program with major in Chemistry and received his M.Sc. and Ph.D. from the Weizmann Institute of Science (Rehovot, Israel) in Chemical Immunology for his work on catalytic antibodies as enzyme models, antibody conformational changes and directed evolution. After a research period at the Scripps Institute (California, USA), he received EMBO and Marie Curie fellowships to pursue postdoctoral work in Paris. His study interests evolve around applying Physical, Chemical and Biological approaches to study aging and variability between clonal individuals. Ariel has been leading the Paris iGEM team since its conception over three years ago and cannot wait to bring another trophy home... <br><br />
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<h3>Samuel Bottani</h3><br />
<I>Physicist</I><br />
<br><br />
Trained as a physicist, natural evolution made me shift from cosmology to molecular biology. I'm interested in a variety of scientific issues and their applications. I'm inspired by beauty and how forms, behaviors and ideas emerge and this drives me in my explorations of the wonders on Nature. Primarily how complex structures and behavior emerge and evolve. We are in an extraordinary period where never so many details have been known on living organisms. Whole genomes are known and extraordinary technological advances enable in vivo tracking and action on single cells and molecules. There is the feeling that a new level of understanding of Life is about to be gained, with fantastic potential for the evolution of humanity. I am eager to participate to endeavor and by my research and critical thinking.<br />
<br />
It is my opinion that scientists, as highly and costly educated citizens have social duty for the collectivity. Engagement in education is essential, ethical concern and involvement in technological and business matters for the benefit of all.<br />
<br />
I am co-director of the international Ph.D. program "Frontières du Vivant" (Frontiers in Life Sciences, www.fdv-paris.org) of the Universities Paris Descartes and Paris Diderot for interdisciplinary research programs on Life Sciences issues. Hosted at the Centre for Research and Interdisciplinarity (cri-paris.org) as the IGEM Paris team we support active approaches in education for interdisciplinary research.<br />
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<img src="http://lh3.ggpht.com/_DG1UEKxMdRA/SqZmSnFS8ZI/AAAAAAAAC8w/JXQW3WtZh4M/flefevre.jpg" width=200px height=130px><br />
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<h3>François Le Fèvre</h3><br />
<I>Biocomputational scientist</I><br />
<br><br />
My research interests lie in the intersection of bioinformatics, and applied software engineering. My approach to research involves an interplay between experimental and theoretical modeling techniques, and is focused towards identifying and elucidating new knowledge interesting for biologists.<br />
<br><br />
Genomic Institute : <a href="http://www.cns.fr/" >Genoscope</a><br />
<br><br />
French Atomic Energy Commission : <a href="http://www.cea.fr/" >CEA</a><br />
<br><br />
Personal home page : <a href="http://sites.google.com/site/synbiocamp/" >SynBioCamp</a><br />
<br />
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<h3>Gregory Batt</h3><br />
<I><a href="http://www.inria.fr/" >INRIA</a> Paris-Rocquencourt</I><br />
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== Advisors ==<br />
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<h3>Guillaume Cambray</h3><br />
<I><a href="http://www.pasteur.fr" > Pasteur Institute </a> Post Doctoral student</I><br />
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<h3>David Bikard</h3><br />
<I> 2nd year PhD student in Biology at <a href="http://www.pasteur.fr" > the Pasteur Institute</I></a><br />
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iGEM Paris 2007 team member<br />
<br><br />
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<h3>Fanny Caffin</h3><br />
<I> 1st year PhD student in molecular & cellular Biology at <a href="http://inserm-u769.cep.u-psud.fr/fr/index.html/" > Paris XI University</I></a><br />
<br><br />
iGEM Paris 2008 team member<br />
<br><br />
...Your flight attendant at your service...<br />
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<img src="https://static.igem.org/mediawiki/2008/a/ad/N764862654_816423_9815.jpg" width=120px height=140px><br />
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<h3>Benoît d'Hayer</h3><br />
<I>Pharmacy at the <a href="http://www.univ-paris5.fr/" > Paris Descartes University</a></I><br />
<br><br />
Master's degree <a href="http://www.univ-paris-diderot.fr/magisteregenet/"> of the European Master of Genetic</a><br />
<br><br />
iGEM Paris 2008 team member<br />
<br><br />
Where is Ploum?<br />
<br><br />
<br />
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<img src="https://static.igem.org/mediawiki/2009/0/06/YannLC.jpg" width=150px height=130px><br />
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<h3>Yann Le Cunff</h3><br />
<I>Applied Mathematics, <a href="http://www.supelec.fr/"> Supelec </a></I><br />
<br><br />
iGEM Paris 2008 team member<br />
<br><br />
Pierre's and Colonel's biggest fan EVER.<br />
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</html></div>Fanny.chttp://2009.igem.org/Team:Paris/TeamTeam:Paris/Team2009-10-22T02:20:00Z<p>Fanny.c: /* Advisors */</p>
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== Where we are==<br />
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Located 5 minutes walk from the Luxembourg Gardens at the Paris Descartes Medicine Faculty in Cochin, the Centre for Research and Interdisciplinarity <a href="www.cri-paris.org/en/cri">CRI</a> was founded in 2005 as a convivial place at the crossroad between Life Sciences and exact, natural, cognitive and social sciences.<br />
Our environment includes a fully-equipped seminar room, a meeting room, office space for visiting professors, a library and coffee rooms and a modelling as well as wet-lab space. The CRI supported its students over three years ago to form the first French team to the iGEM and the tradition is continuing... This is where all happened!<br />
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== Student Team==<br />
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<img src="https://static.igem.org/mediawiki/2009/a/ae/Colonel_Chabbert.jpg" height=120px width=170px><br />
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<h3>Christophe Chabbert</h3><br />
<I> Mathematics, <a href="http://www.aiv-paris.org/fr/master-aiv/"> Mines Paris Tech</a> </I><br />
<br><br />
AKA "Colonel Chabbert", he is our specialist in modelling especially for an "easy/ordinary" delay system.<br />
He likes sciences (really ?? ), his iGEM co-workers ;-), pizzas, burger and ... wait for it .... Coca-cola<br />
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<img src="https://static.igem.org/mediawiki/2009/7/7a/Caroline_Loy.jpg" height=120px width=100px><br />
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<h3>Caroline Loy</h3><br />
<I> Biology, <a href="http://www.aiv-paris.org/fr/master-aiv/"> Master 1 AIV</a></I> <br><br />
After 3 months as a cow-girl in the middle of nowhere (Scotland), she decided to make animal experimentation on bacteria =D<br />
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<img src="https://static.igem.org/mediawiki/2009/e/ec/Romain_bodinier.jpg" height=120px width=180px><br />
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<h3>Romain Bodinier</h3><br />
<I> Biology, <a href="http://www.utc.fr/">UTC</a> </I><br />
<br><br />
Still wondering why he is here ... (Is he looking for a new bacto-foot after the lost of his left one ???). But we may have a proposition of his participation at iGEM....finally we don't.<br />
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<img src="https://static.igem.org/mediawiki/2009/d/da/Soufiane.jpg" height=120px width=100px><br />
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<h3>Soufiane Boumahdi</h3><br />
<I> Biochemistry, <a href="http://www.insa-lyon.fr/">Insa Lyon</a> </I><br />
<br><br />
Every team needs to have "panem et circenses", Soufifi is here for entertaining us. Be careful for the jamboree, The crazy singer is coming !!! stay close to him and you will understand.... hoo year you will.<br />
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<img src="https://static.igem.org/mediawiki/2009/7/70/Charlotte_olivier.jpg" height=120px width=130px><br />
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<h3>Charlotte Olivier</h3><br />
<I>Biology/genetics, <a href="http://www.univ-paris-diderot.fr/magisteregenet/"> European Magister of Genetic</a> and Management/marketing: Specialized Master in management and marketing for the pharmaceutical industry, <a href="http://www.escpeurope.eu/">ESCP-Europe</a> </I><br />
<br><br />
Our Obiwan Kenoby of the lab (without the laser saber)!!! <br><br />
Stoff's note : i may say that she is more like princess Leyla :D<br />
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<img src="https://static.igem.org/mediawiki/2009/0/00/Guillaume_B.jpg" height=120px width=90px><br />
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<h3>Guillaume Beauclair</h3><br />
<I> Biology/Infectiology, <a href="http://www.univ-paris-diderot.fr/">Paris 7</a> </I><br />
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Labholic --> working too hard could be dangerous for your health (too much vegetables also). Our 20hour/day lab worker<br />
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<img src="https://static.igem.org/mediawiki/2009/3/35/Sara_Aguiton.jpg" height=120px width=180px><br />
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<h3>Sara Aguiton</h3><br />
<I> Social Studies of Science, <a href="http://www.ehess.fr/fr/"> EHESS</a> </I><br />
<br><br />
Please, use simple ethical words when you are talking to a scientist assembly ;-)<br />
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<img src="https://static.igem.org/mediawiki/2009/5/56/Christophe_Richard.jpg" height=120px width=180px><br />
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<h3>Christophe Richard</h3><br />
<I> Biology/Informatics, <a href="http://www.aiv-paris.org/fr/master-aiv/"> Master AIW </a> </I><br />
<br><br />
AKA "Bibi", he can talk to computer (Respect !!!!) but not to iPhone ... what a shame for an iPhone developer <br />
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<img src="https://static.igem.org/mediawiki/2009/9/92/Image_8.png" height=120px width=150px><br />
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<h3>Luc Malfondet</h3><br />
<I>Physics/ Nanotechnology & Nanobioscience <a href="http://www.aiv-paris.org/fr/master-aiv/"> Master 2 AIV</a> </I><br />
<br><br />
Open-minded guy !!!!!<br />
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<img src="https://static.igem.org/mediawiki/2009/1/1b/Pierre.jpg" height=120px width=180px><br />
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<h3>Pierre Escamilla</h3><br />
<I> Mathematics, <a href="http://www.supelec.fr/"> Supelec </a></I><br />
<br><br />
Cafeino-man and modellingo-man.<br><br />
Spanisho-man and algorythmo-man.<br />
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<img src="https://static.igem.org/mediawiki/2009/7/7c/2009-02_Sylvain_Helas-Othenin.jpg" height=120px width=100px><br />
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<h3>Sylvain Hélas-Othenin</h3><br />
<I> Chemistry, <a href="http://www.chimie.ens.fr/"> Ecole Normale Supérieure</a> , <a href="http://www.aiv-paris.org/fr/master-aiv/"> Master 2 AIV</a> <br></I><br />
Graduation in Chemistry at Ecole Normale Supérieure, Paris - Interdisciplinary Master in Life Sciences at Université Paris Descartes, Paris ... but currently lost in the Middle-East !<br />
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<img src="https://static.igem.org/mediawiki/2009/3/34/Vico.JPG" height=120px width=180px> <br />
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<h3>Vicard Du</h3><br />
<I> Biology, <a href="http://www.aiv-paris.org/fr/master-aiv/"> Master 1 AIV </I></a> <br />
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Uncle Mc Donalds is his best friend<br />
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== Supervisors ==<br />
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<img src="https://static.igem.org/mediawiki/2009/f/f3/Ariel_Lindner.jpg" width=200px height=180px><br />
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<h3>Ariel Lindner</h3><br />
<br />
Co-initiator of the Centre for Research and Interdisciplinarity (cri-paris.org), Ariel is an INSERM tenured senior researcher and director of the Interdisciplinary Approaches to Life Sciences ('AIV') master program at the Paris Descartes and Diderot Universities. Ariel has graduated from the Hebrew University (Jerusalem, Israel) "Amirim" interdisciplinary program with major in Chemistry and received his M.Sc. and Ph.D. from the Weizmann Institute of Science (Rehovot, Israel) in Chemical Immunology for his work on catalytic antibodies as enzyme models, antibody conformational changes and directed evolution. After a research period at the Scripps Institute (California, USA), he received EMBO and Marie Curie fellowships to pursue postdoctoral work in Paris. His study interests evolve around applying Physical, Chemical and Biological approaches to study aging and variability between clonal individuals. Ariel has been leading the Paris iGEM team since its conception over three years ago and cannot wait to bring another trophy home... <br><br />
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<img src="https://static.igem.org/mediawiki/2009/8/8b/SamuelBottani2.JPG" width=142px height=213px><br />
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<h3>Samuel Bottani</h3><br />
<I>Physicist</I><br />
<br><br />
Trained as a physicist, natural evolution made me shift from cosmology to molecular biology. I'm interested in a variety of scientific issues and their applications. I'm inspired by beauty and how forms, behaviors and ideas emerge and this drives me in my explorations of the wonders on Nature. Primarily how complex structures and behavior emerge and evolve. We are in an extraordinary period where never so many details have been known on living organisms. Whole genomes are known and extraordinary technological advances enable in vivo tracking and action on single cells and molecules. There is the feeling that a new level of understanding of Life is about to be gained, with fantastic potential for the evolution of humanity. I am eager to participate to endeavor and by my research and critical thinking.<br />
<br />
It is my opinion that scientists, as highly and costly educated citizens have social duty for the collectivity. Engagement in education is essential, ethical concern and involvement in technological and business matters for the benefit of all.<br />
<br />
I am co-director of the international Ph.D. program "Frontières du Vivant" (Frontiers in Life Sciences, www.fdv-paris.org) of the Universities Paris Descartes and Paris Diderot for interdisciplinary research programs on Life Sciences issues. Hosted at the Centre for Research and Interdisciplinarity (cri-paris.org) as the IGEM Paris team we support active approaches in education for interdisciplinary research.<br />
</div><br />
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<div class="leftcolumn"><br />
<img src="http://lh3.ggpht.com/_DG1UEKxMdRA/SqZmSnFS8ZI/AAAAAAAAC8w/JXQW3WtZh4M/flefevre.jpg" width=200px height=130px><br />
</div><br />
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<h3>François Le Fèvre</h3><br />
<I>Biocomputational scientist</I><br />
<br><br />
My research interests lie in the intersection of bioinformatics, and applied software engineering. My approach to research involves an interplay between experimental and theoretical modeling techniques, and is focused towards identifying and elucidating new knowledge interesting for biologists.<br />
<br><br />
Genomic Institute : <a href="http://www.cns.fr/" >Genoscope</a><br />
<br><br />
French Atomic Energy Commission : <a href="http://www.cea.fr/" >CEA</a><br />
<br><br />
Personal home page : <a href="http://sites.google.com/site/synbiocamp/" >SynBioCamp</a><br />
<br />
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<img src="https://static.igem.org/mediawiki/2009/9/9c/Gregory_Batt.jpg" width=120px height=153px><br />
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<h3>Gregory Batt</h3><br />
<I><a href="http://www.inria.fr/" >INRIA</a> Paris-Rocquencourt</I><br />
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== Advisors ==<br />
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<h3>Guillaume Cambray</h3><br />
<I><a href="http://www.pasteur.fr" > Pasteur Institute </a> Post Doctoral student</I><br />
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<img src="https://static.igem.org/mediawiki/2009/f/f1/David_Bikard.jpg" width=200px height=130px><br />
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<h3>David Bikard</h3><br />
<I> 2nd year PhD student in Biology at <a href="http://www.pasteur.fr" > the Pasteur Institute</I></a><br />
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iGEM Paris 2007 team member<br />
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<img src="https://static.igem.org/mediawiki/2009/d/d6/Fanny_Caffin.jpg" width=100px height=130px><br />
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<h3>Fanny Caffin</h3><br />
<I> 1st year PhD student in molecular & cellular Biology, <a href="http://inserm-u769.cep.u-psud.fr/fr/index.html/" > Paris XI University</I></a><br />
<br><br />
iGEM Paris 2008 team member<br />
<br><br />
...Your flight attendant at your service...<br />
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<br />
<div class="leftcolumn"><br />
<img src="https://static.igem.org/mediawiki/2008/a/ad/N764862654_816423_9815.jpg" width=120px height=140px><br />
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<div class="rightcolumn"><br />
<h3>Benoît d'Hayer</h3><br />
<I>Pharmacy at the <a href="http://www.univ-paris5.fr/" > Paris Descartes University</a></I><br />
<br><br />
Master's degree <a href="http://www.univ-paris-diderot.fr/magisteregenet/"> of the European Master of Genetic</a><br />
<br><br />
iGEM Paris 2008 team member<br />
<br><br />
Where is Ploum?<br />
<br><br />
<br />
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<img src="https://static.igem.org/mediawiki/2009/0/06/YannLC.jpg" width=150px height=130px><br />
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<h3>Yann Le Cunff</h3><br />
<I>Applied Mathematics, <a href="http://www.supelec.fr/"> Supelec </a></I><br />
<br><br />
iGEM Paris 2008 team member<br />
<br><br />
Pierre's and Colonel's biggest fan EVER.<br />
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</html></div>Fanny.chttp://2009.igem.org/Team:Paris/ProjectTeam:Paris/Project2009-10-22T01:29:59Z<p>Fanny.c: /* A.1. The emission system */</p>
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<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris#top | Home]] > [[Team:Paris/Project#bottom | OMV Project]]<br />
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== '''Overall project:''' '''''Message in a bubble'''''==<br />
<br />
<center>'''Message in a Bubble: cell-cell communication using vesicles. '''</center><br />
<br />
<br />
<center>''Communication is a "two way" process. When you communicate you perceive the other persons responses and react with your own thoughts and feelings. It is only by paying attention to the other person that you have any idea about what to say or do next.''</center><br />
<br />
<br />
*''Bacterial communication:''<br />
<br />
Bacteria communicate with another one using chemical signal molecules. As in higher organisms, the information supplied by these molecules is critical for synchronizing the activities of large groups of cells. In bacteria, chemical communication involves producing, releasing, detecting, and responding to small hormone-like molecules<br />
(called acylhomoserine lactones, AHL). This process, also known as quorum sensing, allows bacteria to monitor the environment for other bacteria and to alter behavior on a population-wide scale in response to changes in the number and/or species present in a community. Nevertheless, AHL molecules are broken down by other bacteria, and some AHL signals are poorly soluble in water, so '''they cannot travel far in an aqueous environment (this factor limits their potential as a long communication signals)'''. <br />
<br />
<br />
*''Outer membrane vesicules in bacteria''<br />
Growing '''gram-negative bacteria (like ''E.Coli'' ) release vesicles from their outer membranes as a means of delivering toxins to host cells and other bacteria'''. This mecanism is conserved among Gram-negative bacteria. The vesicles consist of a lipid bilayer surrounding an aqueous core and they can therefore transport lipid-soluble toxins (lipopolysaccharide endotoxin) on their surface and protein toxins in their core. They release their content by fusing with the lipid bilayer of target cells. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<center>'''''The project :'''''</center><br />
<br />
We decided to''' improve bacterial communication''' thanks to the vesicles formation process. In this direction our engineered communication platform consists in '''controlling OMV production''' by destabilizing membrane integrity through over-expression of specific periplasmic proteins of the Tol/Pal system. The over-production of TolR (a major protein of the Tol/Pal system which ensure the membrane integrity) has to be controled to avoid the bacteria death. <br />
Another important key point of our project is to obtain a delay between the production of protein of interest and the vesicle formation, to be sure that the produced vesicles carried the different protein required for the recognition of the target bacteria and thus the one essential for the signal transduction.<br />
<br />
<br />
<font color=red>Producing the messenger :</font> <br />
<br />
In order to control and modulate message content, we used fusions with our protein of interest and OmpA signal sequence or the ClyA hemolysin as delivery tags. OmpA is a major protein of the external membrane of ''E.Coli'' and is also localize on OMVs. In this direction OmpA seems to be appropriate to deliver a specific protein to the outer membrane and, by consequence into vesicles. As OmpA, ClyA is an interesting way to explore to send protein to the external membrane.<br />
<br />
<br />
<font color=red>Addressing the message :</font> <br />
<br />
To own the communication between the donnor and the receiver a targeting system was developed. This system is based on the outer-membrane expression of Jun/Fos leucine zippers to control the vesicle flux between donor and recipient cells. Jun was mutated into its leucine zipper-motif to abolished the homodimer formation but to allow the development of heterodimer with Fos. To express these protein to the outer membrane of bacteria, they were merged with AIDA autotransporter. In this direction, the direction and the specificity of communication is controled.<br />
<br />
<br />
<font color=red>Receiving the message :</font><br />
<br />
Once received, the signal from incoming vesicles is transduced through a modified Fec pathway, whereby the receptor is provided by the OMV. Few ABC transporter such as FecABCD (iron transporter) are able to induce a response regardless of the tranlocation, due to the activity of FecA. Moreover some mutant can also have a constitutive expression of FecABCD .So, we would like to use FecA- mutant receiver and FecA+ mutant donor to transfert the constitutive FecA protein to the receiver to transmit to the target cell the message (which comes from vesicles (bubbles)). <br />
<br />
<br />
Computational models provided insight to all of the above steps. '''Such reliable communications systems have wide biotechnological implications, ranging from targeted drugs delivery and detoxification to advanced division of labor or even cell-based computing.'''<br />
<br />
<br />
<br />
<br />
All of our constructions are just [[Team:Paris/constructions | here]]<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
<div id="paris_content_boxtop"><br />
</div><br />
<div id="paris_content"><br />
<br />
<br />
==='''A. Plasmid construction'''===<br />
<br />
The plasmid construction is divided into 2 functional modules :<br />
*'''The emission system''', which aims at producing vesicules.<br />
*'''The reception system''' of the signal sent via the vesicules.<br />
<br />
====A.1. The emission system ====<br />
<br />
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.<br />
<br />
<br />
<font color=red>Writing the message: production of signaling proteins </font><br><br />
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.<br />
<br />
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.<br />
<br />
<br />
<font color=red>Preparing the messenger: creation of the vesicles </font><br><br />
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). <br />
<br />
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.<br />
<br />
<br />
* In '''presence of Arabinose''', proteins of interest are created as well as vesicles :<br />
[[Image:Global_On.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
<br />
<br />
*In the '''absence of Arabinose''', the pBad promoter is repressed and there is no production of proteins nor vesicles :<br />
[[Image:Global_Off.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
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.<br />
<br />
====The reception system====<br />
<br />
A FAIRE RAPIDEMENT<br />
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{{Template:Paris2009_guided2|#top|/DryLab}}</div>Fanny.chttp://2009.igem.org/Team:Paris/ProjectTeam:Paris/Project2009-10-22T01:29:14Z<p>Fanny.c: /* A.1. The emission system */</p>
<hr />
<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris#top | Home]] > [[Team:Paris/Project#bottom | OMV Project]]<br />
{{Template:Paris2009}}<br />
{{Template:Paris2009_menu}}<br />
== '''Overall project:''' '''''Message in a bubble'''''==<br />
<br />
<center>'''Message in a Bubble: cell-cell communication using vesicles. '''</center><br />
<br />
<br />
<center>''Communication is a "two way" process. When you communicate you perceive the other persons responses and react with your own thoughts and feelings. It is only by paying attention to the other person that you have any idea about what to say or do next.''</center><br />
<br />
<br />
*''Bacterial communication:''<br />
<br />
Bacteria communicate with another one using chemical signal molecules. As in higher organisms, the information supplied by these molecules is critical for synchronizing the activities of large groups of cells. In bacteria, chemical communication involves producing, releasing, detecting, and responding to small hormone-like molecules<br />
(called acylhomoserine lactones, AHL). This process, also known as quorum sensing, allows bacteria to monitor the environment for other bacteria and to alter behavior on a population-wide scale in response to changes in the number and/or species present in a community. Nevertheless, AHL molecules are broken down by other bacteria, and some AHL signals are poorly soluble in water, so '''they cannot travel far in an aqueous environment (this factor limits their potential as a long communication signals)'''. <br />
<br />
<br />
*''Outer membrane vesicules in bacteria''<br />
Growing '''gram-negative bacteria (like ''E.Coli'' ) release vesicles from their outer membranes as a means of delivering toxins to host cells and other bacteria'''. This mecanism is conserved among Gram-negative bacteria. The vesicles consist of a lipid bilayer surrounding an aqueous core and they can therefore transport lipid-soluble toxins (lipopolysaccharide endotoxin) on their surface and protein toxins in their core. They release their content by fusing with the lipid bilayer of target cells. <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<center>'''''The project :'''''</center><br />
<br />
We decided to''' improve bacterial communication''' thanks to the vesicles formation process. In this direction our engineered communication platform consists in '''controlling OMV production''' by destabilizing membrane integrity through over-expression of specific periplasmic proteins of the Tol/Pal system. The over-production of TolR (a major protein of the Tol/Pal system which ensure the membrane integrity) has to be controled to avoid the bacteria death. <br />
Another important key point of our project is to obtain a delay between the production of protein of interest and the vesicle formation, to be sure that the produced vesicles carried the different protein required for the recognition of the target bacteria and thus the one essential for the signal transduction.<br />
<br />
<br />
<font color=red>Producing the messenger :</font> <br />
<br />
In order to control and modulate message content, we used fusions with our protein of interest and OmpA signal sequence or the ClyA hemolysin as delivery tags. OmpA is a major protein of the external membrane of ''E.Coli'' and is also localize on OMVs. In this direction OmpA seems to be appropriate to deliver a specific protein to the outer membrane and, by consequence into vesicles. As OmpA, ClyA is an interesting way to explore to send protein to the external membrane.<br />
<br />
<br />
<font color=red>Addressing the message :</font> <br />
<br />
To own the communication between the donnor and the receiver a targeting system was developed. This system is based on the outer-membrane expression of Jun/Fos leucine zippers to control the vesicle flux between donor and recipient cells. Jun was mutated into its leucine zipper-motif to abolished the homodimer formation but to allow the development of heterodimer with Fos. To express these protein to the outer membrane of bacteria, they were merged with AIDA autotransporter. In this direction, the direction and the specificity of communication is controled.<br />
<br />
<br />
<font color=red>Receiving the message :</font><br />
<br />
Once received, the signal from incoming vesicles is transduced through a modified Fec pathway, whereby the receptor is provided by the OMV. Few ABC transporter such as FecABCD (iron transporter) are able to induce a response regardless of the tranlocation, due to the activity of FecA. Moreover some mutant can also have a constitutive expression of FecABCD .So, we would like to use FecA- mutant receiver and FecA+ mutant donor to transfert the constitutive FecA protein to the receiver to transmit to the target cell the message (which comes from vesicles (bubbles)). <br />
<br />
<br />
Computational models provided insight to all of the above steps. '''Such reliable communications systems have wide biotechnological implications, ranging from targeted drugs delivery and detoxification to advanced division of labor or even cell-based computing.'''<br />
<br />
<br />
<br />
<br />
All of our constructions are just [[Team:Paris/constructions | here]]<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
<div id="paris_content_boxtop"><br />
</div><br />
<div id="paris_content"><br />
<br />
<br />
==='''A. Plasmid construction'''===<br />
<br />
The plasmid construction is divided into 2 functional modules :<br />
*'''The emission system''', which aims at producing vesicules.<br />
*'''The reception system''' of the signal sent via the vesicules.<br />
<br />
====A.1. The emission system ====<br />
<br />
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.<br />
<br />
<br />
<font color=red>Writing the message: production of signaling proteins </font><br><br />
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.<br />
<br />
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.<br />
<br />
<br />
<font color=red>Preparing the messenger: creation of the vesicles </font><br><br />
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). <br />
<br />
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.<br />
<br />
<br />
* In '''presence of Arabinose''', proteins of interest are created as well as vesicles :<br />
[[Image:Global_On.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
*In the '''absence of Arabinose''', the pBad promoter is repressed and there is no production of proteins nor vesicles :<br />
[[Image:Global_Off.jpg|800px|center| Plasmid construction of the emitting bacteria]]<br />
<br />
<br />
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.<br />
<br />
====The reception system====<br />
<br />
A FAIRE RAPIDEMENT<br />
<br />
<html><br />
</div><br />
<div id="paris_content_boxtop"><br />
</div><br />
<div id="paris_content"><br />
</html><br />
<br />
<br />
{{Template:Paris2009_guided2|#top|/DryLab}}</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview2Team:Paris/Addressing overview22009-10-22T01:14:51Z<p>Fanny.c: /* Bibliography : */</p>
<hr />
<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris/Addressing_overview#top | Adressing]] > [[Team:Paris/Addressing_overview#bottom | ClyA]]<br />
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==Addressing the message in the outer membrane : Main==<br />
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<a class="menu_sub_active"href="https://2009.igem.org/Team:Paris/Addressing_overview2#bottom"> Main </a>|<br />
<a class="menu_sub" href="https://2009.igem.org/Team:Paris/Addressing_overview3#bottom"> ClyA</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview4#bottom"> OmpA</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#bottom"> Our strategy</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview_Construction#bottom"> Construction</a><br />
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<br />
Bacterial pathogens display proteins on their surface that may interact with their hosts in order to mount successful infections. Although the primary function of the peptidoglycan is to provide a physical barrier for protection against both mechanical and osmotic stresses, it also serves as a scaffold to anchor external structures such as the outer cell membrane in ''Escherichia Coli''. Over the past 20 years, it has become apparent that Gram-negative bacteria have evolved a variety of mechanism by which proteins are displayed on the cell surface (Tat and Sec transporter for example, cf the section "to the periplasm, export system" for more information about them). <br />
Using these transport system, lot of protein are localized on the outer membrane, and one of the major protein of the bacterial membrane is OmpA. There is also an other protein, ClyA, but less known than OmpA which can be exported from cytoplasm to outer membrane, then by vesiculation interact with their host.<br />
<br />
<br />
<br />
<br />
'''ClyA : '''<br />
<br />
<br />
<br />
Pore-forming toxins (PFTs) are a class of potent virulence factors that convert from a soluble form to a membrane-integrated pore. They exhibit their toxic effect either by destruction of the membrane permeability barrier or by delivery of toxic components through the pores.<br />
<br />
Cytolysin A (ClyA, also known as HlyE), a PFT, is a cytolytic α-helical toxin responsible for the haemolytic phenotype of several ''E. coli''. [http://www.ncbi.nlm.nih.gov/pubmed/19421192[1]]<br />
<br />
<br />
<br />
Why did we decided to use ClyA?<br />
<br />
Althought the toxic effect of Cly A, using it is an interesting way to adress protein to the external membrane, because ClyA contain the signal peptide required to be exported form the cytoplasm to the outer membrane. In addition, it has been shown that certain membrane and/or soluble periplasmic proteins are enriched in vesicles while others are preferentially excluded. The majority of these enriched proteins happen to be virulence factors including cytolysin A of ''E. coli'' [http://www.ncbi.nlm.nih.gov/pubmed/14532000[2]].<br />
<br />
<br />
Previous studies demonstrated that genetic fusions between ClyA of ''E. coli'' and reporter proteins such as Bla and GFP were translocated across the cytoplasmic membrane [http://www.ncbi.nlm.nih.gov/pubmed/11731136[3]],[http://www.ncbi.nlm.nih.gov/pubmed/15557633[4]] and that localization was independent of the position (N or C terminus) of ClyA in the fusion protein. Moreover a recent article [http://www.ncbi.nlm.nih.gov/pubmed/18511069[5]] demonstrated that fusions to the C terminus of ClyA allow the transport of protein closer to the surface of outer membrane and to extend them into the extracellular environment.<br />
<br />
<br />
<br />
'''OmpA : '''<br />
<br />
<br />
<br />
OmpA is a major protein of the outer membrane of ''E.Coli''. Moreover Kesty and co-workers demonstrated that OmpA is an outer-membrane component of native vesicles [http://www.ncbi.nlm.nih.gov/pubmed/14578354[6]]. In this direction, our strategy is to fuse our protein of interest to OmpA to allow its transport to the outer membrane and to increase the probability of its integration into vesicles. <br />
We finally decided to focus our efforts on the ClyA strategy because of the previous work that was done on this protein especially the "proof" that it's possible to merge our protein of interest with ClyA without inhibiting both activities.<br />
<br />
<br />
<br />
<br />
<br />
====References====<br />
<ol class="references"><br />
<li> [[Team:Paris/Production_overview#1 | ]] The structure of a cytolytic a-helical toxin pore reveals its assembly mechanism, M.Mueller & N.Ban. 2009 - [http://www.ncbi.nlm.nih.gov/pubmed/19421192 | 19421192 ] <li> <br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/14532000[2]] S.N. Wai, B.Lindmark, T.Soderblom, A.Takade, M.Westermark, J.Oscarsson, et al. Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin, 2003, Cell, 115, 25–35.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11731136[3]] F.J del Castillo, F. Moreno. and I.del Castillo. Secretion of the Escherichia coli K-12 SheA hemolysin is independent of its cytolytic activity, 2001, FEMS Microbiol.Lett. 204, 281–285.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/15557633[4]] J.E.Galen, L.Zhao, M.Chinchilla, J.Y.Wang,M.F.Pasetti, J.Green and M.M. Levine. Adaptation of the endogenous Salmonella enterica serovar Typhi clyA-encoded hemolysin for antigen export enhances the immunogenicity of anthrax protective antigen domain 4 expressed by the attenuated live-vector vaccine strain CVD 908-htrA, 2004, Infect. Immun. 72, 7096–7106.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/18511069[5]] J.Y. Kim, A.M. Doody, D. J. Chen, G.H. Cremona, M.L. Shuler, D.Putnam,and M.P. DeLisa.Engineered. Bacterial Outer Membrane Vesicles with Enhanced Functionality, 2008, J. Mol. Biol. 380, 51–66. <br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/14578354[6]] N.C.Kesty, M.J.Kuehn. Incorporation of heterologous outer membrane and periplasmic proteins into Escherichia coli outer membrane vesicles, 2004, J Biol Chem. 279(3):2069-76.<br />
<br />
<br />
<br />
<br />
{{Template:Paris2009_guided|Addressing_overview#Our_strategy|Addressing_overview3#bottom}}</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overviewTeam:Paris/Addressing overview2009-10-22T00:55:28Z<p>Fanny.c: /* Adressing the message to the periplasm : Export system */</p>
<hr />
<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris/Addressing_overview#top | Adressing]] > [[Team:Paris/Addressing_overview_exportsystem#bottom | Export systems ]]<br />
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== Addressing the message to the periplasm : Export system ==<br />
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<a class="menu_sub_active" href="https://2009.igem.org/Team:Paris/Addressing_overview#bottom"> Export system</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview#Our_strategy"> Our Strategy</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_testing#bottom"> Construction</a><br />
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<br />
Export systems are important to allow the transport of proteins to the periplasm or to the outer membrane of the bacteria. <br />
<br />
In our project, we would like to address a specific message to the target bacteria. That's why, it’s important to address specific protein into vesicles to send a particular message to the recipient cell. <br />
Adressing protein to the periplasm is a key point to try to control the content of vesicles. If a protein has a high concentration in the periplasm, some of these proteins could be into the vesicles during their formation. To ensure a high periplasmic concentration, we need to better understand the translocation mechanism : therefore, we focus on Tat and Sec transporter. The second strategy is to fuse our protein of interest to a protein (or just a domain) which is inserted intà the outer membrane (for example, OmpA or ClyA). <br />
<br />
<br />
===Introduction===<br />
<br />
<br />
'''Tat and Sec Transporter : '''<br />
<br />
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The '''Tat''' (twin-arginine translocation) system is a bacterial protein export pathway with the remarkable ability to transport folded proteins across the cytoplasmic membrane. Preproteins are directed to the Tat pathway by signal peptides that bear a characteristic sequence motif, which includes consecutive arginine residues.<br />
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The most remarkable characteristic of the Tat pathway is that it apparently functions to transport folded proteins of variable dimensions across the cytoplasmic membrane, a feat that must be achieved without rendering the membrane freely permeable to protons and other ions.<br />
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In most cases, the substrates of this pathway are proteins that bind one of a range of cofactors in the cytoplasm and are thus folded before export. Some cofactorless proteins may also be transported by the Tat pathway, probably because they either require cytoplasmic factors for folding or fold too rapidly or tightly for transport by the Sec apparatus.<br />
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Recent work has shown that the bacterial Tat system is very closely related to the DeltapH-dependent protein import pathway of the plant chloroplast thylakoid membrane. The bacterial and plant systems do, however, differ in the treatment of precursors before the transport step because, in contrast to well-characterized delta pH-dependent pathway precursors, bacterial Tat substrates has to be co-ordinates with cofactor insertion.<br />
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The '''Sec''' machinery is composed of a membrane-embedded SecYEG translocation complex + an ATP-hydrolysing SecA protein. Major feature of the Sec mechanism is that proteins are translocated in an extended conformation and are often bound by SecB or other cytoplasmic chaperones to prevent folding before export.<br />
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===About signal peptides===<br />
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'''Sec peptide signal'''<br />
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Sec pathway signal peptides in Gram-negative bacteria are on average 24 amino acids in length and comprise three distinct regions : an N-terminal positively charged region (n-region), a hydrophobic alpha-helical region (h-region) and a c-domain that contains the site of cleavage by signal peptidase.<br />
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'''Tat peptide signal'''<br />
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Tat pathway signal peptides have a similar tripartite organization to Sec signal peptides but exhibit a number of disctinctive features, the most notable of which is a conserved (S/T)-R-R-x-F-L-K sequence motif at the n-region/h-region boundary, in which the consecutive arginine residues are invariant and the other motif residues occur at a frequency of more than 50%.<br />
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'''Tat vs Sec'''<br />
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We observe the high occurrence of proline residues at position -6 to the signal peptidase cleavage site. <br />
The c-region of Tat signal peptides also characteristically contains basic amino acids whereas Sec pathway precursors show a bias against positively charged residues in the vicinity of the signal peptidase cleavage site. <br />
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Bacterial Tat signal peptides are on average 14 amino acids longer than Sec signal peptides, with most of this additional length being caused by an extended n-region. Further the h-region of the at signal peptides is significantly less hydrophobic than that of Sec signal peptide due to a higher occurrence of the amino acids glycine and threonine and a signifivantly lower abundance of leucine residues.<br />
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'''Targeting to Tat and avoidance of Sec'''<br />
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Increasing the h-region hydrophobicity redirects the protein through the Sec translocon.<br />
Thus, co-translational Sec targeting not only overrides Tat targeting information but can also force an otherwise Sec-incompatible wild-type TorA signal sequence into the Sec translocon.<br />
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There are many features preventing Tat signals peptides to act as efficiently as Sec targerting signals.<br />
It has been shown that the signal peptide c-region basic residues block the Sec-dependent thylakoid import (althought not required for the DeltA pH pathway).<br />
The c-region basic residues of bacterial Tat signal peptides also interfere with Sec pathway export.<br />
This '''Sec avoidance characteristic''' might be connected to the long-recongnized phenomenon that basic residues are poorly tolerated in the vicinity of the signal peptidase cleavage site of Sec pathway precursors.<br />
If the h-region hydrophobicity is increased, the export defect caused by basic residues is suppressed.<br />
The cytoplasmic membrane potential inhibits insertion of precursors with such basic residues into the Sec translocon.<br />
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'''The twin-arginine consensus has no Sec-avoidance role.'''<br />
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No studies have so far addressed the role of the non-arginine amino-acids of the Tat consensus motif in the transport process.<br />
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===Translocation mechanism===<br />
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'''Sec Mechanism'''<br />
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In the classic Sec loop, the tip of the loop containing the signal peptidase cleavage site exposed at the periplasmic face of the membrane with the two arms of the loop being formed by the inverted signal peptide and approximately the first 20 amino acids of the mature protein. The mature protein arm is subsequently segmentally extruded by SecA insertion cycles.<br />
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'''Tat Mechanism'''<br />
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It has been suggested that, by analogy with protein transport by the Sec pathway and across the endoplasmic reticulum (ER), the Tat system may operate via loop mechanism in which both the N-terminus of the signal peptide and the bulk of the passengers protein remain at the cytoplasmic side of the membrane after the initial translocon-precursor interaction. Weak evidence is available that the N-terminus of the tat signal peptide does indeed remain at the cytoplasmic side of the membrane. The N-terminus of the precursor must remain on the stromal side of the membrane.<br />
It remains possible that the mature protein N-terminus does not attain its final conformation until after signal peptide cleavage.<br />
If the mature protein is fully folded at the translocon binding step, the signal peptide might pivot around the consensus motif binding site during substrate transport.<br />
Alternatively, at the start of transport, when the mature domain is at the cytoplasmic side of the membrane, the signal peptide alone could form a loop that would flex to a fully extended conformation across the membrane by the end of the translocation process.<br />
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''Folding and cofactors''<br />
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The cofactor insertion is assisted by dedicated cytoplasmic assembly factors that recognize and bind the precursor.<br />
A single signal peptide may even be capable of targeting a three-subunit enzyme though the Tat pathway.<br />
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''Does the transporter mechanistically require that the substrate be folded ?''<br />
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No, but maybe the Tat pathway contains elements that check the folding state.<br />
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Proofreading would not be a necessity for those Tat substrates that do not bind cofactors, if the reason such proteins are targeted to the Tat pathway is because Sec-dependent export is too slow to prevent the protein forming a Sec-incompatible structure.<br />
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A mechanism for checking the cofactor loading will be needed for : tat substrates that binds to cofactors + proteins without cofactors because they require cytosolic folding cofactors.<br />
This mechanisme should be able to look to the hydrophobic regions of the preprotein : via chaperonnes or via tat signal peptide.<br />
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In all above models, the signal peptide is '''bifunctionnal''' : acting both to '''direct export''' and to '''signal the cofactor status'''. Thus, in addition to the common features of twin arginine signal peptides required for the Tat targeting, signal peptides for the cofactor containing proteins should have distinct structural features allowing specific protein-protein interactions.<br />
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==Our strategy==<br />
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We thought of overexpressing the important proteins in the Tat pathway (that is to say TatABCE) in order to avoid the early saturation phenomenon that is likely to occur when we will overexpress proteins that will be targeted to the outer membrane or to the periplasm.<br />
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However, in our strategy, the only protein that needs to use the TAT pathway to translocate from the cytoplasm to the periplasm is clyA. Finally, we decided that the overexpression won't be necessary neither for the TAT pathway, nor for the SEC pathway, both of them constitutively expressed in E Coli K12.<br />
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{{Template:Paris2009_guided|Conclusion#top|Addressing_overview2#top}}</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview_ConstructionTeam:Paris/Addressing overview Construction2009-10-22T00:54:33Z<p>Fanny.c: /* Construction */</p>
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<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris/Addressing_overview2#top | ClyA]] > [[Team:Paris/Addressing_overview_Construction#bottom | Construction]]<br />
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==Addressing the message in the membrane : Construction ==<br />
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<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2#bottom"> Main </a>|<br />
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<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#bottom"> Our strategy</a>|<br />
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[[Image:Clya_construction.jpg|500px|center|Activation of our construction by Arabinose]]<br />
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Here the activation of our construction '''by arabinose''', in order to test the construction.<br />
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[[Image:Clya_construction2.jpg|500px|center|Inhibition of our construction by Glucose]]<br />
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Here the inhibition of our construction '''by glucose''', in order to test the construction.<br />
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[[Image:ClyA_construction_3.jpg|500px|center|Activation of our construction by Glucose]]<br />
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Here the activation of our construction '''by arabinose'''. <br />
<br> It is quite the same as the first construction, but in [http://www.ncbi.nlm.nih.gov/pubmed/18511069[1]] the RFP-Cter link to Nter-ClyA seems to have a better fluorescence. Because of our late about receiving the oligo for its synthesis in biobrick format, we used the first construction (ClyA-Cter link to Nter-RFP).<br />
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====Bibliography :====<br />
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[http://www.ncbi.nlm.nih.gov/pubmed/18511069[1]] Kim, J.-Y. & DeLisa, M.P. Engineered bacterial outer membrane vesicles with enhanced functionality J.Mol. Biol. (2008) 380, 51–66</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview2_strategyTeam:Paris/Addressing overview2 strategy2009-10-22T00:53:43Z<p>Fanny.c: /* Adressing the message in the membrane : Our strategy */</p>
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<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris/Addressing_overview2#top | ClyA]] > [[Team:Paris/Addressing_overview2_strategy#bottom | Our strategy]]<br />
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==Addressing the message in the membrane : Our strategy==<br />
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Our strategy is to use clyA to export a protein to the outer-membrane of the cell. The protein fused to clyA will be incorporated into the vesicle during the vesiculation process and it's also express on the surface. ClyA contain the signal peptide required for the exportation process from the cytoplasm to the periplasm. The overall idea is to fused the protein of interest to clyA. <br />
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So in a first time in order to see if our ClyA are localize in OMVs, we fused it we a RFP. For that we add a poly glycine linker to ClyA biobrick to improve ClyA-RFP fusion. Moreover if we put RFP before ClyA, it seem that there is more fluorescence than ClyA before RFP under [[http://www.ncbi.nlm.nih.gov/pubmed/18511069 1]]. You can see the contruction [https://2009.igem.org/Team:Paris/Addressing_overview_Construction#Overview here]<br />
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Then if this test work, we could replace the RFP by the protein of interest for signal transduction, moreover this system couple to fec system can transduct a signal from outer membran to the cytoplasm.<br />
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====Bibliography :====<br />
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[[http://www.ncbi.nlm.nih.gov/pubmed/18511069 1]]J.Y. Kim, A.M. Doody, D. J. Chen, G.H. Cremona, M.L. Shuler, D.Putnam,and M.P. DeLisa.Engineered. Bacterial Outer Membrane Vesicles with Enhanced Functionality, 2008, J. Mol. Biol. 380, 51–66. <br />
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{{Template:Paris2009_guided|Addressing_overview4#bottom|Production_overview#top}}</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview4Team:Paris/Addressing overview42009-10-22T00:53:18Z<p>Fanny.c: /* Adressing the message to the outer membrane : OmpA */</p>
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==Addressing the message to the outer membrane : OmpA==<br />
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Outer membrane protein A (OmpA) as previously been used in protein fusion to expose heterologuous protein domains to the surface of ''Escherichia coli''. As an outer membrane protein, ompA could be incorporated into vesicles and is similarly to clyA also a good candidate for adressing protein domains to vesicles. <br />
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[[Image:OmpA1.JPG|250px|left]][[Image:OmpA2.JPG|250px|right]]<br />
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OmpA is a major protein in the Escherichia coli outer membrane. OmpA plays a vital structural role in ''E. coli'', and suggested that a perfect β-barrel structure of OmpA is important for outer membrane stability[http://www.ncbi.nlm.nih.gov/pubmed/11906175[1]]. OmpA is the most well-studied outer membrane protein in ''E. coli''. This 325-residue protein was thought to contain two domains. The classic N-terminal domain, consisting of 171 amino acid residues, was shown to cross the membrane eight times in antiparallel β-strands with four relatively large and hydrophilic surface-exposed loops and short periplasmic turns[http://www.ncbi.nlm.nih.gov/pubmed/11276254[2]]. The C-terminal domain is located in the periplasm, and binds to the peptidoglycan thus connecting it to the outer membrane[http://www.ncbi.nlm.nih.gov/pubmed/8577259[3]]. The function of OmpA is thought to contribute to the structural integrity of the outer membrane<br />
along with murein lipoprotein[http://www.ncbi.nlm.nih.gov/pubmed/4261992[4]] and peptidoglycanassociated lipoprotein . In addition to its structural role, OmpA serves as a receptor of colicin and several phages[http://www.ncbi.nlm.nih.gov/pubmed/1574003[5]], and it's required in F-conjugation[http://www.ncbi.nlm.nih.gov/pubmed/321438[6]],[http://www.ncbi.nlm.nih.gov/pubmed/10368142[7]]<br />
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====Bibliography:====<br />
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[http://www.ncbi.nlm.nih.gov/pubmed/11906175[1]] Ying Wang, (2002) The Function of OmpA in Escherichia coli, Biochem Biophys Res Commun.292(2):396-401<br />
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[http://www.ncbi.nlm.nih.gov/pubmed/11276254[2]] Arora, A., Abildgaard, F., Bushweller, J. H., and Tamm, L. K. (2001) Structure of outer membrane protein A transmembrane domain by NMR spectroscopy. Nat. Struct. Biol 8, 334–338.<br />
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[http://www.ncbi.nlm.nih.gov/pubmed/8577259[3]] Koebnik, R. (1995) Proposal for a peptidoglycan associating alpha-helical motif in the C-terminal regions of some bacterial cell-surface proteins. Mol. Microbiol. 16, 1269–1270.<br />
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[http://www.ncbi.nlm.nih.gov/pubmed/4261992[4]] Braun, V., and Bosch, V. (1972) Sequence of the mureinlipoprotein and the attachment site of the lipid. Eur. J. Biochem. 28, 51–69.<br />
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[http://www.ncbi.nlm.nih.gov/pubmed/1574003[5]] Lazzaroni, J.-C., and Portalier, R. (1992) The excC gene of Escherichia coli K-12 required for cell envelope integrity encodes the peptidoglycan-associated lipoprotein. Mol. Microbiol. 6, 735–742.<br />
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[http://www.ncbi.nlm.nih.gov/pubmed/321438[6] Schweizer, M., and Henning, U. (1977) Action of major outer cell envelope membrane protein in conjugation of Escherichia coli K-12. J. Bacteriol. 129, 1651–1652.<br />
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[http://www.ncbi.nlm.nih.gov/pubmed/10368142[7]] Koebnik, R. (1999) Structural and functional roles of the surfaceexposed loops of the β-barrel membrane protein OmpA from Escherichia coli. J. Bacteriol. 181, 3688–3694.</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview3Team:Paris/Addressing overview32009-10-22T00:52:55Z<p>Fanny.c: /* Adressing the message in the outer membrane : ClyA */</p>
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==Addressing the message in the outer membrane : ClyA==<br />
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<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview4#bottom"> OmpA</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#bottom"> Our strategy</a>|<br />
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We work on the cell-cell communication using vesicle:<br />
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In this part we look into adressing a protein into the sender outer membrane that could be incoporated into outer membrane vesicles (OMVs). This protein would then be able to transmit a message after the fusion of OMVs with a receiver cell.<br />
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In this direction ClyA (the cytolysine A of E.Coli) seems to be a good candidate. ClyA is one of the proteins that has been previously detected into OMVs and is known to be specificly exported to the outer membrane [http://www.ncbi.nlm.nih.gov/pubmed/14532000 [3]]. ClyA is thus expressed on bacteria and OMVs surface. Moreover, when ClyA is overproduced, it is accumulated into the periplasmic space [http://www.ncbi.nlm.nih.gov/pubmed/14532000 [3]].<br />
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However there is an inconvenient to use this protein. ClyA is an alpha-Pore Forming Toxin (PFT). PFT are widely distributed proteins which form lesions in biological membranes. They exhibit their toxic effect in different manner. The first one is that ClyA allows the destruction of membrane permeability barrier. Furthermore, the toxic effect of ClyA could be explain by its capacity to deliver toxic component after the assembly of 8 or 13 of its subunits. PFTs can be subdivided into two classes; α-PFTs and β-PFTs, depending on the suspected mode of membrane integration, either by α-helical or β-sheet elements.[http://www.ncbi.nlm.nih.gov/pubmed/19421192 [2]]<br />
<br />
[[Image:Clya_simple.jpg|ClyA subunit|150px|left]] [[Image:Clya_structure.jpg|ClyA assembled|150px|right]] [[Image:ClyA.jpg|ClyA are assembling in outer membrane of a host cell|150px|center]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
Some article argue that E.Coli K12 use this ClyA to lyse other cell (specially mamalian cell or eurcaryote cell[http://www.ncbi.nlm.nih.gov/pubmed/14532000 [3]]). But E. coli cells expressing clyA do not lyse each other.<br />
<br />
<br />
<br />
Kim et al. have successfully fused clyA to GFP in order to observe vesicles [http://www.ncbi.nlm.nih.gov/pubmed/18511069 [1]], so we know that we can try to fuse clyA to a protein domain that would induce a signal transduction into the receiver cell. To see how we want to exploite clyA properties see [https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#Overview our strategy].<br />
<br />
<br />
<br />
<br />
<br />
====Bibliography :====<br />
<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/18511069 [1]]Kim, J.-Y. & DeLisa, M.P. Engineered bacterial outer membrane vesicles with enhanced functionality J.Mol. Biol. (2008) 380, 51–66<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/19421192 [2]]Muller, M. & Ban, N. The structure of a cytolytic a-helical toxin pore reveals its assembly mechanism Nature (4 June 2009) 459, 726-730 <br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/14532000 [3]]Wai, S.N. & Lindmark, B. Vesicle-Mediated Export and Assembly of Pore-Forming Oligomers of the Enterobacterial ClyA Cytotoxin Cell (October 2003), 115,25-35<br />
<br />
<br />
<br />
{{Template:Paris2009_guided|Addressing_overview2#bottom|Addressing_overview4#bottom}}</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview2Team:Paris/Addressing overview22009-10-22T00:34:08Z<p>Fanny.c: /* Addressing the message in the outer membrane : Main */</p>
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Bacterial pathogens display proteins on their surface that may interact with their hosts in order to mount successful infections. Although the primary function of the peptidoglycan is to provide a physical barrier for protection against both mechanical and osmotic stresses, it also serves as a scaffold to anchor external structures such as the outer cell membrane in ''Escherichia Coli''. Over the past 20 years, it has become apparent that Gram-negative bacteria have evolved a variety of mechanism by which proteins are displayed on the cell surface (Tat and Sec transporter for example, cf the section "to the periplasm, export system" for more information about them). <br />
Using these transport system, lot of protein are localized on the outer membrane, and one of the major protein of the bacterial membrane is OmpA. There is also an other protein, ClyA, but less known than OmpA which can be exported from cytoplasm to outer membrane, then by vesiculation interact with their host.<br />
<br />
<br />
<br />
<br />
'''ClyA : '''<br />
<br />
<br />
<br />
Pore-forming toxins (PFTs) are a class of potent virulence factors that convert from a soluble form to a membrane-integrated pore. They exhibit their toxic effect either by destruction of the membrane permeability barrier or by delivery of toxic components through the pores.<br />
<br />
Cytolysin A (ClyA, also known as HlyE), a PFT, is a cytolytic α-helical toxin responsible for the haemolytic phenotype of several ''E. coli''. [http://www.ncbi.nlm.nih.gov/pubmed/19421192[1]]<br />
<br />
<br />
<br />
Why did we decided to use ClyA?<br />
<br />
Althought the toxic effect of Cly A, using it is an interesting way to adress protein to the external membrane, because ClyA contain the signal peptide required to be exported form the cytoplasm to the outer membrane. In addition, it has been shown that certain membrane and/or soluble periplasmic proteins are enriched in vesicles while others are preferentially excluded. The majority of these enriched proteins happen to be virulence factors including cytolysin A of ''E. coli'' [http://www.ncbi.nlm.nih.gov/pubmed/14532000[2]].<br />
<br />
<br />
Previous studies demonstrated that genetic fusions between ClyA of ''E. coli'' and reporter proteins such as Bla and GFP were translocated across the cytoplasmic membrane [http://www.ncbi.nlm.nih.gov/pubmed/11731136[3]],[http://www.ncbi.nlm.nih.gov/pubmed/15557633[4]] and that localization was independent of the position (N or C terminus) of ClyA in the fusion protein. Moreover a recent article [http://www.ncbi.nlm.nih.gov/pubmed/18511069[5]] demonstrated that fusions to the C terminus of ClyA allow the transport of protein closer to the surface of outer membrane and to extend them into the extracellular environment.<br />
<br />
<br />
<br />
'''OmpA : '''<br />
<br />
<br />
<br />
OmpA is a major protein of the outer membrane of ''E.Coli''. Moreover Kesty and co-workers demonstrated that OmpA is an outer-membrane component of native vesicles [http://www.ncbi.nlm.nih.gov/pubmed/14578354[6]]. In this direction, our strategy is to fuse our protein of interest to OmpA to allow its transport to the outer membrane and to increase the probability of its integration into vesicles. <br />
We finally decided to focus our efforts on the ClyA strategy because of the previous work that was done on this protein especially the "proof" that it's possible to merge our protein of interest with ClyA without inhibiting both activities.<br />
<br />
<br />
<br />
<br />
<br />
====Bibliography :====<br />
<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/19421192[1]] M.Mueller, U.Grauschopf, T.Maier, R.Glockshuber1 & N.Ban, The structure of a cytolytic a-helical toxin pore reveals its assembly mechanism, 2009, Nature vol 459 , 726-731.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/14532000[2]] S.N. Wai, B.Lindmark, T.Soderblom, A.Takade, M.Westermark, J.Oscarsson, et al. Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin, 2003, Cell, 115, 25–35.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11731136[3]] F.J del Castillo, F. Moreno. and I.del Castillo. Secretion of the Escherichia coli K-12 SheA hemolysin is independent of its cytolytic activity, 2001, FEMS Microbiol.Lett. 204, 281–285.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/15557633[4]] J.E.Galen, L.Zhao, M.Chinchilla, J.Y.Wang,M.F.Pasetti, J.Green and M.M. Levine. Adaptation of the endogenous Salmonella enterica serovar Typhi clyA-encoded hemolysin for antigen export enhances the immunogenicity of anthrax protective antigen domain 4 expressed by the attenuated live-vector vaccine strain CVD 908-htrA, 2004, Infect. Immun. 72, 7096–7106.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/18511069[5]] J.Y. Kim, A.M. Doody, D. J. Chen, G.H. Cremona, M.L. Shuler, D.Putnam,and M.P. DeLisa.Engineered. Bacterial Outer Membrane Vesicles with Enhanced Functionality, 2008, J. Mol. Biol. 380, 51–66. <br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/14578354[6]] N.C.Kesty, M.J.Kuehn. Incorporation of heterologous outer membrane and periplasmic proteins into Escherichia coli outer membrane vesicles, 2004, J Biol Chem. 279(3):2069-76.<br />
<br />
<br />
<br />
<br />
{{Template:Paris2009_guided|Addressing_overview#Our_strategy|Addressing_overview3#bottom}}</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview2Team:Paris/Addressing overview22009-10-22T00:33:29Z<p>Fanny.c: /* Adressing the message in the outer membrane : Main */</p>
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<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris/Addressing_overview#top | Adressing]] > [[Team:Paris/Addressing_overview#bottom | ClyA]]<br />
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<br />
Bacterial pathogens display proteins on their surface that may interact with their hosts in order to mount successful infections. Although the primary function of the peptidoglycan is to provide a physical barrier for protection against both mechanical and osmotic stresses, it also serves as a scaffold to anchor external structures such as the outer cell membrane in ''Escherichia Coli''. Over the past 20 years, it has become apparent that Gram-negative bacteria have evolved a variety of mechanism by which proteins are displayed on the cell surface (Tat and Sec transporter for example, cf the section "to the periplasm, export system" for more information about them). <br />
Using these transport system, lot of protein are localized on the outer membrane, and one of the major protein of the bacterial membrane is OmpA. There is also an other protein, ClyA, but less known than OmpA which can be exported from cytoplasm to outer membrane, then by vesiculation interact with their host.<br />
<br />
<br />
<br />
<br />
'''ClyA : '''<br />
<br />
<br />
<br />
Pore-forming toxins (PFTs) are a class of potent virulence factors that convert from a soluble form to a membrane-integrated pore. They exhibit their toxic effect either by destruction of the membrane permeability barrier or by delivery of toxic components through the pores.<br />
<br />
Cytolysin A (ClyA, also known as HlyE), a PFT, is a cytolytic α-helical toxin responsible for the haemolytic phenotype of several Escherichia coli. [http://www.ncbi.nlm.nih.gov/pubmed/19421192[1]]<br />
<br />
<br />
<br />
Why did we decided to use ClyA?<br />
<br />
Althought the toxic effect of Cly A, using it is an interesting way to adress protein to the external membrane, because ClyA contain the signal peptide required to be exported form the cytoplasm to the outer membrane. In addition, it has been shown that certain membrane and/or soluble periplasmic proteins are enriched in vesicles while others are preferentially excluded. The majority of these enriched proteins happen to be virulence factors including cytolysin A of ''E. coli'' [http://www.ncbi.nlm.nih.gov/pubmed/14532000[2]].<br />
<br />
<br />
Previous studies demonstrated that genetic fusions between ClyA of ''E. coli'' and reporter proteins such as Bla and GFP were translocated across the cytoplasmic membrane [http://www.ncbi.nlm.nih.gov/pubmed/11731136[3]],[http://www.ncbi.nlm.nih.gov/pubmed/15557633[4]] and that localization was independent of the position (N or C terminus) of ClyA in the fusion protein. Moreover a recent article [http://www.ncbi.nlm.nih.gov/pubmed/18511069[5]] demonstrated that fusions to the C terminus of ClyA allow the transport of protein closer to the surface of outer membrane and to extend them into the extracellular environment.<br />
<br />
<br />
<br />
'''OmpA : '''<br />
<br />
<br />
<br />
OmpA is a major protein of the outer membrane of ''E.Coli''. Moreover Kesty and co-workers demonstrated that OmpA is an outer-membrane component of native vesicles [http://www.ncbi.nlm.nih.gov/pubmed/14578354[6]]. In this direction, our strategy is to fuse our protein of interest to OmpA to allow its transport to the outer membrane and to increase the probability of its integration into vesicles. <br />
We finally decided to focus our efforts on the ClyA strategy because of the previous work that was done on this protein especially the "proof" that it's possible to merge our protein of interest with ClyA without inhibiting both activities.<br />
<br />
<br />
<br />
<br />
<br />
====Bibliography :====<br />
<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/19421192[1]] M.Mueller, U.Grauschopf, T.Maier, R.Glockshuber1 & N.Ban, The structure of a cytolytic a-helical toxin pore reveals its assembly mechanism, 2009, Nature vol 459 , 726-731.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/14532000[2]] S.N. Wai, B.Lindmark, T.Soderblom, A.Takade, M.Westermark, J.Oscarsson, et al. Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin, 2003, Cell, 115, 25–35.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11731136[3]] F.J del Castillo, F. Moreno. and I.del Castillo. Secretion of the Escherichia coli K-12 SheA hemolysin is independent of its cytolytic activity, 2001, FEMS Microbiol.Lett. 204, 281–285.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/15557633[4]] J.E.Galen, L.Zhao, M.Chinchilla, J.Y.Wang,M.F.Pasetti, J.Green and M.M. Levine. Adaptation of the endogenous Salmonella enterica serovar Typhi clyA-encoded hemolysin for antigen export enhances the immunogenicity of anthrax protective antigen domain 4 expressed by the attenuated live-vector vaccine strain CVD 908-htrA, 2004, Infect. Immun. 72, 7096–7106.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/18511069[5]] J.Y. Kim, A.M. Doody, D. J. Chen, G.H. Cremona, M.L. Shuler, D.Putnam,and M.P. DeLisa.Engineered. Bacterial Outer Membrane Vesicles with Enhanced Functionality, 2008, J. Mol. Biol. 380, 51–66. <br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/14578354[6]] N.C.Kesty, M.J.Kuehn. Incorporation of heterologous outer membrane and periplasmic proteins into Escherichia coli outer membrane vesicles, 2004, J Biol Chem. 279(3):2069-76.<br />
<br />
<br />
<br />
<br />
{{Template:Paris2009_guided|Addressing_overview#Our_strategy|Addressing_overview3#bottom}}</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview4Team:Paris/Addressing overview42009-10-22T00:28:47Z<p>Fanny.c: /* Adressing the message to the outer membrane : OmpA */</p>
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<br />
Outer membrane protein A (OmpA) as previously been used in protein fusion to expose heterologuous protein domains to the surface of ''Escherichia coli''. As an outer membrane protein, ompA could be incorporated into vesicles and is similarly to clyA also a good candidate for adressing protein domains to vesicles. <br />
<br />
<br />
<br />
[[Image:OmpA1.JPG|250px|left]][[Image:OmpA2.JPG|250px|right]]<br />
<br />
OmpA is a major protein in the Escherichia coli outer membrane. OmpA plays a vital structural role in ''E. coli'', and suggested that a perfect β-barrel structure of OmpA is important for outer membrane stability[http://www.ncbi.nlm.nih.gov/pubmed/11906175[1]]. OmpA is the most well-studied outer membrane protein in ''E. coli''. This 325-residue protein was thought to contain two domains. The classic N-terminal domain, consisting of 171 amino acid residues, was shown to cross the membrane eight times in antiparallel β-strands with four relatively large and hydrophilic surface-exposed loops and short periplasmic turns[http://www.ncbi.nlm.nih.gov/pubmed/11276254[2]]. The C-terminal domain is located in the periplasm, and binds to the peptidoglycan thus connecting it to the outer membrane[http://www.ncbi.nlm.nih.gov/pubmed/8577259[3]]. The function of OmpA is thought to contribute to the structural integrity of the outer membrane<br />
along with murein lipoprotein[http://www.ncbi.nlm.nih.gov/pubmed/4261992[4]] and peptidoglycanassociated lipoprotein . In addition to its structural role, OmpA serves as a receptor of colicin and several phages[http://www.ncbi.nlm.nih.gov/pubmed/1574003[5]], and it's required in F-conjugation[http://www.ncbi.nlm.nih.gov/pubmed/321438[6]],[http://www.ncbi.nlm.nih.gov/pubmed/10368142[7]]<br />
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====Bibliography:====<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11906175[1]] Ying Wang, (2002) The Function of OmpA in Escherichia coli, Biochem Biophys Res Commun.292(2):396-401<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11276254[2]] Arora, A., Abildgaard, F., Bushweller, J. H., and Tamm, L. K. (2001) Structure of outer membrane protein A transmembrane domain by NMR spectroscopy. Nat. Struct. Biol 8, 334–338.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/8577259[3]] Koebnik, R. (1995) Proposal for a peptidoglycan associating alpha-helical motif in the C-terminal regions of some bacterial cell-surface proteins. Mol. Microbiol. 16, 1269–1270.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/4261992[4]] Braun, V., and Bosch, V. (1972) Sequence of the mureinlipoprotein and the attachment site of the lipid. Eur. J. Biochem. 28, 51–69.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/1574003[5]] Lazzaroni, J.-C., and Portalier, R. (1992) The excC gene of Escherichia coli K-12 required for cell envelope integrity encodes the peptidoglycan-associated lipoprotein. Mol. Microbiol. 6, 735–742.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/321438[6] Schweizer, M., and Henning, U. (1977) Action of major outer cell envelope membrane protein in conjugation of Escherichia coli K-12. J. Bacteriol. 129, 1651–1652.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/10368142[7]] Koebnik, R. (1999) Structural and functional roles of the surfaceexposed loops of the β-barrel membrane protein OmpA from Escherichia coli. J. Bacteriol. 181, 3688–3694.</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview4Team:Paris/Addressing overview42009-10-22T00:27:10Z<p>Fanny.c: /* Adressing the message to the outer membrane : OmpA */</p>
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Outer membrane protein A (OmpA) as previously been used in protein fusion to expose heterologuous protein domains to the surface of E. coli. As an outer membrane protein, ompA could be incorporated into vesicles and is similarly to clyA also a good candidate for adressing protein domains to vesicles. <br />
<br />
<br />
<br />
[[Image:OmpA1.JPG|250px|left]][[Image:OmpA2.JPG|250px|right]]<br />
<br />
OmpA is a major protein in the Escherichia coli outer membrane. OmpA plays a vital structural role in ''E. coli'', and suggested that a perfect β-barrel structure of OmpA is important for outer membrane stability[http://www.ncbi.nlm.nih.gov/pubmed/11906175[1]]. OmpA is the most well-studied outer membrane protein in ''Escherichia coli''. This 325-residue protein was thought to contain two domains. The classic N-terminal domain, consisting of 171 amino acid residues, was shown to cross the membrane eight times in antiparallel β-strands with four relatively large and hydrophilic surface-exposed loops and short periplasmic turns[http://www.ncbi.nlm.nih.gov/pubmed/11276254[2]]. The C-terminal domain is located in the periplasm, and binds to the peptidoglycan thus connecting it to the outer membrane[http://www.ncbi.nlm.nih.gov/pubmed/8577259[3]]. The function of OmpA is thought to contribute to the structural integrity of the outer membrane<br />
along with murein lipoprotein[http://www.ncbi.nlm.nih.gov/pubmed/4261992[4]] and peptidoglycanassociated lipoprotein . In addition to its structural role, OmpA serves as a receptor of colicin and several phages[http://www.ncbi.nlm.nih.gov/pubmed/1574003[5]], and it's required in F-conjugation[http://www.ncbi.nlm.nih.gov/pubmed/321438[6]],[http://www.ncbi.nlm.nih.gov/pubmed/10368142[7]]<br />
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<br />
{{Template:Paris2009_guided|Addressing_overview3#bottom|Addressing_overview2_strategy#bottom}}<br />
<br />
<br />
====Bibliography:====<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11906175[1]] Ying Wang, (2002) The Function of OmpA in Escherichia coli, Biochem Biophys Res Commun.292(2):396-401<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11276254[2]] Arora, A., Abildgaard, F., Bushweller, J. H., and Tamm, L. K. (2001) Structure of outer membrane protein A transmembrane domain by NMR spectroscopy. Nat. Struct. Biol 8, 334–338.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/8577259[3]] Koebnik, R. (1995) Proposal for a peptidoglycan associating alpha-helical motif in the C-terminal regions of some bacterial cell-surface proteins. Mol. Microbiol. 16, 1269–1270.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/4261992[4]] Braun, V., and Bosch, V. (1972) Sequence of the mureinlipoprotein and the attachment site of the lipid. Eur. J. Biochem. 28, 51–69.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/1574003[5]] Lazzaroni, J.-C., and Portalier, R. (1992) The excC gene of Escherichia coli K-12 required for cell envelope integrity encodes the peptidoglycan-associated lipoprotein. Mol. Microbiol. 6, 735–742.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/321438[6] Schweizer, M., and Henning, U. (1977) Action of major outer cell envelope membrane protein in conjugation of Escherichia coli K-12. J. Bacteriol. 129, 1651–1652.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/10368142[7]] Koebnik, R. (1999) Structural and functional roles of the surfaceexposed loops of the β-barrel membrane protein OmpA from Escherichia coli. J. Bacteriol. 181, 3688–3694.</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview4Team:Paris/Addressing overview42009-10-22T00:25:41Z<p>Fanny.c: /* Adressing the message to the outer membrane : OmpA */</p>
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<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#bottom"> Our strategy</a>|<br />
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<br />
<br />
Outer membrane protein A (OmpA) as previously been used in protein fusion to expose heterologuous protein domains to the surface of E. coli. As an outer membrane protein, ompA could be incorporated into vesicles and is similarly to clyA also a good candidate for adressing protein domains to vesicles. <br />
<br />
<br />
<br />
[[Image:OmpA1.JPG|260px|left]][[Image:OmpA2.JPG|240px|right]]<br />
<br />
OmpA is a major protein in the Escherichia coli outer membrane. OmpA plays a vital structural role in ''E. coli'', and suggested that a perfect β-barrel structure of OmpA is important for outer membrane stability[http://www.ncbi.nlm.nih.gov/pubmed/11906175[1]]. OmpA is the most well-studied outer membrane protein in ''Escherichia coli''. This 325-residue protein was thought to contain two domains. The classic N-terminal domain, consisting of 171 amino acid residues, was shown to cross the membrane eight times in antiparallel β-strands with four relatively large and hydrophilic surface-exposed loops and short periplasmic turns[http://www.ncbi.nlm.nih.gov/pubmed/11276254[2]]. The C-terminal domain is located in the periplasm, and binds to the peptidoglycan thus connecting it to the outer membrane[http://www.ncbi.nlm.nih.gov/pubmed/8577259[3]]. The function of OmpA is thought to contribute to the structural integrity of the outer membrane<br />
along with murein lipoprotein[http://www.ncbi.nlm.nih.gov/pubmed/4261992[4]] and peptidoglycanassociated lipoprotein . In addition to its structural role, OmpA serves as a receptor of colicin and several phages[http://www.ncbi.nlm.nih.gov/pubmed/1574003[5]], and it's required in F-conjugation[http://www.ncbi.nlm.nih.gov/pubmed/321438[6]],[http://www.ncbi.nlm.nih.gov/pubmed/10368142[7]]<br />
<br />
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<br />
<br />
<br />
<br />
{{Template:Paris2009_guided|Addressing_overview3#bottom|Addressing_overview2_strategy#bottom}}<br />
<br />
<br />
====Bibliography:====<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11906175[1]] Ying Wang, (2002) The Function of OmpA in Escherichia coli, Biochem Biophys Res Commun.292(2):396-401<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11276254[2]] Arora, A., Abildgaard, F., Bushweller, J. H., and Tamm, L. K. (2001) Structure of outer membrane protein A transmembrane domain by NMR spectroscopy. Nat. Struct. Biol 8, 334–338.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/8577259[3]] Koebnik, R. (1995) Proposal for a peptidoglycan associating alpha-helical motif in the C-terminal regions of some bacterial cell-surface proteins. Mol. Microbiol. 16, 1269–1270.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/4261992[4]] Braun, V., and Bosch, V. (1972) Sequence of the mureinlipoprotein and the attachment site of the lipid. Eur. J. Biochem. 28, 51–69.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/1574003[5]] Lazzaroni, J.-C., and Portalier, R. (1992) The excC gene of Escherichia coli K-12 required for cell envelope integrity encodes the peptidoglycan-associated lipoprotein. Mol. Microbiol. 6, 735–742.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/321438[6] Schweizer, M., and Henning, U. (1977) Action of major outer cell envelope membrane protein in conjugation of Escherichia coli K-12. J. Bacteriol. 129, 1651–1652.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/10368142[7]] Koebnik, R. (1999) Structural and functional roles of the surfaceexposed loops of the β-barrel membrane protein OmpA from Escherichia coli. J. Bacteriol. 181, 3688–3694.</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview4Team:Paris/Addressing overview42009-10-22T00:23:31Z<p>Fanny.c: /* Adressin the message in the outer membrane : OmpA */</p>
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<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#bottom"> Our strategy</a>|<br />
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<br />
<br />
OmpA as previously been used in protein fusion to expose heterologuous protein domains to the surface of E. coli. As an outer membrane protein, ompA could be incorporated into vesicles and is similarly to clyA also a good candidate for adressing protein domains to vesicles. <br />
<br />
<br />
<br />
[[Image:OmpA1.JPG|260px|left]][[Image:OmpA2.JPG|240px|right]]<br />
<br />
Outer membrane protein A (OmpA) is a major protein in the Escherichia coli outer membrane. OmpA plays a vital structural role in ''E. coli'', and suggested that a perfect β-barrel structure of OmpA is important for outer membrane stability[http://www.ncbi.nlm.nih.gov/pubmed/11906175[1]]. OmpA is the most well-studied outer membrane protein in ''Escherichia coli''. This 325-residue protein was thought to contain two domains. The classic N-terminal domain, consisting of 171 amino acid residues, was shown to cross the membrane eight times in antiparallel β-strands with four relatively large and hydrophilic surface-exposed loops and short periplasmic turns[http://www.ncbi.nlm.nih.gov/pubmed/11276254[2]]. The C-terminal domain is located in the periplasm, and binds to the peptidoglycan thus connecting it to the outer membrane[http://www.ncbi.nlm.nih.gov/pubmed/8577259[3]]. The function of OmpA is thought to contribute to the structural integrity of the outer membrane<br />
along with murein lipoprotein[http://www.ncbi.nlm.nih.gov/pubmed/4261992[4]] and peptidoglycanassociated lipoprotein . In addition to its structural role, OmpA serves as a receptor of colicin and several phages[http://www.ncbi.nlm.nih.gov/pubmed/1574003[5]], and it's required in F-conjugation[http://www.ncbi.nlm.nih.gov/pubmed/321438[6]],[http://www.ncbi.nlm.nih.gov/pubmed/10368142[7]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{Template:Paris2009_guided|Addressing_overview3#bottom|Addressing_overview2_strategy#bottom}}<br />
<br />
<br />
====Bibliography:====<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11906175[1]] Ying Wang, (2002) The Function of OmpA in Escherichia coli, Biochem Biophys Res Commun.292(2):396-401<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11276254[2]] Arora, A., Abildgaard, F., Bushweller, J. H., and Tamm, L. K. (2001) Structure of outer membrane protein A transmembrane domain by NMR spectroscopy. Nat. Struct. Biol 8, 334–338.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/8577259[3]] Koebnik, R. (1995) Proposal for a peptidoglycan associating alpha-helical motif in the C-terminal regions of some bacterial cell-surface proteins. Mol. Microbiol. 16, 1269–1270.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/4261992[4]] Braun, V., and Bosch, V. (1972) Sequence of the mureinlipoprotein and the attachment site of the lipid. Eur. J. Biochem. 28, 51–69.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/1574003[5]] Lazzaroni, J.-C., and Portalier, R. (1992) The excC gene of Escherichia coli K-12 required for cell envelope integrity encodes the peptidoglycan-associated lipoprotein. Mol. Microbiol. 6, 735–742.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/321438[6] Schweizer, M., and Henning, U. (1977) Action of major outer cell envelope membrane protein in conjugation of Escherichia coli K-12. J. Bacteriol. 129, 1651–1652.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/10368142[7]] Koebnik, R. (1999) Structural and functional roles of the surfaceexposed loops of the β-barrel membrane protein OmpA from Escherichia coli. J. Bacteriol. 181, 3688–3694.</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview2_strategyTeam:Paris/Addressing overview2 strategy2009-10-22T00:18:30Z<p>Fanny.c: /* Adressing the message in the membrane : Our strategy */</p>
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<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris/Addressing_overview2#top | ClyA]] > [[Team:Paris/Addressing_overview2_strategy#bottom | Our strategy]]<br />
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<br />
Our strategy is to use clyA to export a protein to the outer-membrane of the cell. The protein fused to clyA will be incorporated into the vesicle during the vesiculation process and it's also express on the surface. ClyA contain the signal peptide required for the exportation process from the cytoplasm to the periplasm. The overall idea is to fused the protein of interest to clyA. <br />
<br />
So in a first time in order to see if our ClyA are localize in OMVs, we fused it we a RFP. For that we add a poly glycine linker to ClyA biobrick to improve ClyA-RFP fusion. Moreover if we put RFP before ClyA, it seem that there is more fluorescence than ClyA before RFP under [[http://www.ncbi.nlm.nih.gov/pubmed/18511069 1]]. You can see the contruction [https://2009.igem.org/Team:Paris/Addressing_overview_Construction#Overview here]<br />
<br />
Then if this test work, we could replace the RFP by the protein of interest for signal transduction, moreover this system couple to fec system can transduct a signal from outer membran to the cytoplasm.<br />
<br />
<br />
====Bibliography :====<br />
<br />
[[http://www.ncbi.nlm.nih.gov/pubmed/18511069 1]]J.Y. Kim, A.M. Doody, D. J. Chen, G.H. Cremona, M.L. Shuler, D.Putnam,and M.P. DeLisa.Engineered. Bacterial Outer Membrane Vesicles with Enhanced Functionality, 2008, J. Mol. Biol. 380, 51–66. <br />
<br />
{{Template:Paris2009_guided|Addressing_overview4#bottom|Production_overview#top}}</div>Fanny.chttp://2009.igem.org/Team:Paris/TeamTeam:Paris/Team2009-10-22T00:03:11Z<p>Fanny.c: /* Advisors */</p>
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<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris/Team#bottom | Team]]<br />
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== Student Team==<br />
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<h3>Christophe Chabbert</h3><br />
<I> Mathematics, <a href="http://www.aiv-paris.org/fr/master-aiv/"> Mines Paris Tech</a> </I><br />
<br><br />
AKA "Colonel Chabbert", he is our specialist in modelling especially for an "easy/ordinary" delay system.<br />
He likes sciences (really ?? ), his iGEM co-workers ;-), pizzas, burger and ... wait for it .... Coca-cola<br />
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<img src="https://static.igem.org/mediawiki/2009/7/7a/Caroline_Loy.jpg" height=120px width=100px><br />
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<h3>Caroline Loy</h3><br />
<I> Biology, <a href="http://www.aiv-paris.org/fr/master-aiv/"> Master 1 AIV</a></I> <br><br />
After 3 months as a cow-girl in the middle of nowhere (Scotland), she decided to make animal experimentation on bacteria =D<br />
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<h3>Romain Bodinier</h3><br />
<I> Biology, <a href="http://www.utc.fr/">UTC</a> </I><br />
<br><br />
Still wondering why he is here ... (Is he looking for a new bacto-foot after the lost of his left one ???). But we may have a proposition of his participation at iGEM....finally we don't.<br />
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<h3>Soufiane Boumahdi</h3><br />
<I> Biochemistry, <a href="http://www.insa-lyon.fr/">Insa Lyon</a> </I><br />
<br><br />
Every team needs to have "panem et circenses", Soufifi is here for entertaining us. Be careful for the jamboree, The crazy singer is coming !!! stay close to him and you will understand.... hoo year you will.<br />
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<h3>Charlotte Olivier</h3><br />
<I>Biology/genetics, <a href="http://www.univ-paris-diderot.fr/magisteregenet/"> European Magister of Genetic</a> and Management/marketing: Specialized Master in management and marketing for the pharmaceutical industry, <a href="http://www.escpeurope.eu/">ESCP-Europe</a> </I><br />
<br><br />
Our Obiwan Kenoby of the lab (without the laser saber)!!! <br><br />
Stoff's note : i may say that she is more like princess Leyla :D<br />
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<img src="https://static.igem.org/mediawiki/2009/0/00/Guillaume_B.jpg" height=120px width=90px><br />
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<h3>Guillaume Beauclair</h3><br />
<I> Biology/Infectiology, <a href="http://www.univ-paris-diderot.fr/">Paris 7</a> </I><br />
<br><br />
Labholic --> working too hard could be dangerous for your health (too much vegetables also). Our 20hour/day lab worker<br />
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<h3>Sara Aguiton</h3><br />
<I> Social Studies of Science, <a href="http://www.ehess.fr/fr/"> EHESS</a> </I><br />
<br><br />
Please, use simple ethical words when you are talking to a scientist assembly ;-)<br />
<br />
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<img src="https://static.igem.org/mediawiki/2009/5/56/Christophe_Richard.jpg" height=120px width=180px><br />
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<h3>Christophe Richard</h3><br />
<I> Biology/Informatics, <a href="http://www.aiv-paris.org/fr/master-aiv/"> Master AIW </a> </I><br />
<br><br />
AKA "Bibi", he can talk to computer (Respect !!!!) but not to iPhone ... what a shame for an iPhone developer <br />
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<img src="https://static.igem.org/mediawiki/2009/9/92/Image_8.png" height=120px width=150px><br />
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<b>Malfondet Luc</b><br><br />
<I>Physics/ Nanotechnology & Nanobioscience <a href="http://www.aiv-paris.org/fr/master-aiv/"> Master 2 AIV</a> </I><br />
<br><br />
<br />
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<img src="https://static.igem.org/mediawiki/2009/1/1b/Pierre.jpg" height=120px width=180px><br />
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<b>Escamilla Pierre</b><br><br />
<I> Mathematics, <a href="http://www.supelec.fr/"> Supelec </a></I><br />
<br><br />
Cafeino-man and modellingo-man.<br><br />
Spanisho-man and algorythmo-man.<br />
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<img src="https://static.igem.org/mediawiki/2009/7/7c/2009-02_Sylvain_Helas-Othenin.jpg" height=120px width=100px><br />
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<b>Hélas-Othenin Sylvain</b><br><br />
<br />
<I> Chemistry, <a href="http://www.chimie.ens.fr/"> Ecole Normale Supérieure</a> , <a href="http://www.aiv-paris.org/fr/master-aiv/"> Master 2 AIV</a> <br></I><br />
Graduation in Chemistry at Ecole Normale Supérieure, Paris - Interdisciplinary Master in Life Sciences at Université Paris Descartes, Paris ... but currently lost in the Middle-East !<br />
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<img src="https://static.igem.org/mediawiki/2009/3/34/Vico.JPG" height=120px width=180px> <br />
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<b>Du Vicard</b><br><br />
<I> Biology, <a href="http://www.aiv-paris.org/fr/master-aiv/"> Master 1 AIV </I></a> <br />
<br><br />
Uncle Mc Donalds is his best friend<br />
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<h3>Ariel Lindner</h3><br />
<br />
Co-initiator of the Centre for Research and Interdisciplinarity (cri-paris.org), Ariel is an INSERM tenured senior researcher and director of the Interdisciplinary Approaches to Life Sciences ('AIV') master program at the Paris Descartes and Diderot Universities. Ariel has graduated from the Hebrew University (Jerusalem, Israel) "Amirim" interdisciplinary program with major in Chemistry and received his M.Sc. and Ph.D. from the Weizmann Institute of Science (Rehovot, Israel) in Chemical Immunology for his work on catalytic antibodies as enzyme models, antibody conformational changes and directed evolution. After a research period at the Scripps Institute (California, USA), he received EMBO and Marie Curie fellowships to pursue postdoctoral work in Paris. His study interests evolve around applying Physical, Chemical and Biological approaches to study aging and variability between clonal individuals. Ariel has been leading the Paris iGEM team since its conception over three years ago and cannot wait to bring another trophy home... <br><br />
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<h3>Samuel Bottani</h3><br />
<I>Physicist</I><br />
<br><br />
Trained as a physicist, natural evolution made me shift from cosmology to molecular biology. I'm interested in a variety of scientific issues and their applications. I'm inspired by beauty and how forms, behaviors and ideas emerge and this drives me in my explorations of the wonders on Nature. Primarily how complex structures and behavior emerge and evolve. We are in an extraordinary period where never so many details have been known on living organisms. Whole genomes are known and extraordinary technological advances enable in vivo tracking and action on single cells and molecules. There is the feeling that a new level of understanding of Life is about to be gained, with fantastic potential for the evolution of humanity. I am eager to participate to endeavor and by my research and critical thinking.<br />
<br />
It is my opinion that scientists, as highly and costly educated citizens have social duty for the collectivity. Engagement in education is essential, ethical concern and involvement in technological and business matters for the benefit of all.<br />
<br />
I am co-director of the international Ph.D. program "Frontières du Vivant" (Frontiers in Life Sciences, www.fdv-paris.org) of the Universities Paris Descartes and Paris Diderot for interdisciplinary research programs on Life Sciences issues. Hosted at the Centre for Research and Interdisciplinarity (cri-paris.org) as the IGEM Paris team we support active approaches in education for interdisciplinary research.<br />
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<img src="http://lh3.ggpht.com/_DG1UEKxMdRA/SqZmSnFS8ZI/AAAAAAAAC8w/JXQW3WtZh4M/flefevre.jpg" width=200px height=130px><br />
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<h3>François Le Fèvre</h3><br />
<I>Biocomputational scientist</I><br />
<br><br />
My research interests lie in the intersection of bioinformatics, and applied software engineering. My approach to research involves an interplay between experimental and theoretical modeling techniques, and is focused towards identifying and elucidating new knowledge interesting for biologists.<br />
<br><br />
Genomic Institute : <a href="http://www.cns.fr/" >Genoscope</a><br />
<br><br />
French Atomic Energy Commission : <a href="http://www.cea.fr/" >CEA</a><br />
<br><br />
Personal home page : <a href="http://sites.google.com/site/synbiocamp/" >SynBioCamp</a><br />
<br />
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<img src="https://static.igem.org/mediawiki/2009/9/9c/Gregory_Batt.jpg" width=120px height=153px><br />
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<h3>Gregory Batt</h3><br />
<I><a href="http://www.inria.fr/" >INRIA</a> Paris-Rocquencourt</I><br />
<br><br />
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<h3>Guillaume Cambray</h3><br />
<I><a href="http://www.pasteur.fr" > Pasteur Institute </a> Post Doctoral student</I><br />
<br><br />
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<img src="https://static.igem.org/mediawiki/2009/f/f1/David_Bikard.jpg" width=200px height=130px><br />
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<h3>David Bikard</h3><br />
<I> 2nd year PhD student in Biology at <a href="http://www.pasteur.fr" > the Pasteur Institute</I></a><br />
<br><br />
iGEM Paris 2007 team member<br />
<br><br />
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<img src="https://static.igem.org/mediawiki/2009/d/d6/Fanny_Caffin.jpg" width=100px height=130px><br />
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<h3>Fanny Caffin</h3><br />
<I>Biologist (molecular & cellular biology), <a href="http://inserm-u769.cep.u-psud.fr/fr/index.html/" > 1st year of PhD at Paris XI University</I></a><br />
<br><br />
iGEM Paris 2008 team member<br />
<br><br />
...Your flight attendant at your service...<br />
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<img src="https://static.igem.org/mediawiki/2008/a/ad/N764862654_816423_9815.jpg" width=120px height=130px><br />
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<h3>Benoît d'Hayer</h3><br />
<I>Pharmacy at the <a href="http://www.univ-paris5.fr/" > Paris Descartes University</a></I><br />
<br><br />
Master's degree <a href="http://www.univ-paris-diderot.fr/magisteregenet/"> of the European Master of Genetic</a><br />
<br><br />
iGEM Paris 2008 team member<br />
<br><br />
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<img src="https://static.igem.org/mediawiki/2009/0/06/YannLC.jpg" width=150px height=130px><br />
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<h3>Yann Le Cunff</h3><br />
<I>Applied Mathematics, <a href="http://www.supelec.fr/"> Supelec </a></I><br />
<br><br />
iGEM Paris 2008 team member<br />
<br><br />
Pierre's and Colonel's biggest fan EVER.<br />
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</html></div>Fanny.chttp://2009.igem.org/Team:Paris/TeamTeam:Paris/Team2009-10-22T00:00:01Z<p>Fanny.c: /* Advisors */</p>
<hr />
<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris/Team#bottom | Team]]<br />
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== Student Team==<br />
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<img src="https://static.igem.org/mediawiki/2009/a/ae/Colonel_Chabbert.jpg" height=120px width=170px><br />
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<h3>Christophe Chabbert</h3><br />
<I> Mathematics, <a href="http://www.aiv-paris.org/fr/master-aiv/"> Mines Paris Tech</a> </I><br />
<br><br />
AKA "Colonel Chabbert", he is our specialist in modelling especially for an "easy/ordinary" delay system.<br />
He likes sciences (really ?? ), his iGEM co-workers ;-), pizzas, burger and ... wait for it .... Coca-cola<br />
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<img src="https://static.igem.org/mediawiki/2009/7/7a/Caroline_Loy.jpg" height=120px width=100px><br />
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<h3>Caroline Loy</h3><br />
<I> Biology, <a href="http://www.aiv-paris.org/fr/master-aiv/"> Master 1 AIV</a></I> <br><br />
After 3 months as a cow-girl in the middle of nowhere (Scotland), she decided to make animal experimentation on bacteria =D<br />
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<img src="https://static.igem.org/mediawiki/2009/e/ec/Romain_bodinier.jpg" height=120px width=180px><br />
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<h3>Romain Bodinier</h3><br />
<I> Biology, <a href="http://www.utc.fr/">UTC</a> </I><br />
<br><br />
Still wondering why he is here ... (Is he looking for a new bacto-foot after the lost of his left one ???). But we may have a proposition of his participation at iGEM....finally we don't.<br />
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<img src="https://static.igem.org/mediawiki/2009/d/da/Soufiane.jpg" height=120px width=100px><br />
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<h3>Soufiane Boumahdi</h3><br />
<I> Biochemistry, <a href="http://www.insa-lyon.fr/">Insa Lyon</a> </I><br />
<br><br />
Every team needs to have "panem et circenses", Soufifi is here for entertaining us. Be careful for the jamboree, The crazy singer is coming !!! stay close to him and you will understand.... hoo year you will.<br />
</div><br />
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<div class="leftcolumn"><br />
<img src="https://static.igem.org/mediawiki/2009/7/70/Charlotte_olivier.jpg" height=120px width=130px><br />
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<h3>Charlotte Olivier</h3><br />
<I>Biology/genetics, <a href="http://www.univ-paris-diderot.fr/magisteregenet/"> European Magister of Genetic</a> and Management/marketing: Specialized Master in management and marketing for the pharmaceutical industry, <a href="http://www.escpeurope.eu/">ESCP-Europe</a> </I><br />
<br><br />
Our Obiwan Kenoby of the lab (without the laser saber)!!! <br><br />
Stoff's note : i may say that she is more like princess Leyla :D<br />
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<img src="https://static.igem.org/mediawiki/2009/0/00/Guillaume_B.jpg" height=120px width=90px><br />
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<h3>Guillaume Beauclair</h3><br />
<I> Biology/Infectiology, <a href="http://www.univ-paris-diderot.fr/">Paris 7</a> </I><br />
<br><br />
Labholic --> working too hard could be dangerous for your health (too much vegetables also). Our 20hour/day lab worker<br />
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<img src="https://static.igem.org/mediawiki/2009/3/35/Sara_Aguiton.jpg" height=120px width=180px><br />
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<h3>Sara Aguiton</h3><br />
<I> Social Studies of Science, <a href="http://www.ehess.fr/fr/"> EHESS</a> </I><br />
<br><br />
Please, use simple ethical words when you are talking to a scientist assembly ;-)<br />
<br />
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<img src="https://static.igem.org/mediawiki/2009/5/56/Christophe_Richard.jpg" height=120px width=180px><br />
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<h3>Christophe Richard</h3><br />
<I> Biology/Informatics, <a href="http://www.aiv-paris.org/fr/master-aiv/"> Master AIW </a> </I><br />
<br><br />
AKA "Bibi", he can talk to computer (Respect !!!!) but not to iPhone ... what a shame for an iPhone developer <br />
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<img src="https://static.igem.org/mediawiki/2009/9/92/Image_8.png" height=120px width=150px><br />
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<b>Malfondet Luc</b><br><br />
<I>Physics/ Nanotechnology & Nanobioscience <a href="http://www.aiv-paris.org/fr/master-aiv/"> Master 2 AIV</a> </I><br />
<br><br />
<br />
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<img src="https://static.igem.org/mediawiki/2009/1/1b/Pierre.jpg" height=120px width=180px><br />
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<b>Escamilla Pierre</b><br><br />
<I> Mathematics, <a href="http://www.supelec.fr/"> Supelec </a></I><br />
<br><br />
Cafeino-man and modellingo-man.<br><br />
Spanisho-man and algorythmo-man.<br />
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<img src="https://static.igem.org/mediawiki/2009/7/7c/2009-02_Sylvain_Helas-Othenin.jpg" height=120px width=100px><br />
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<br />
<div class="rightcolumn"><br />
<b>Hélas-Othenin Sylvain</b><br><br />
<br />
<I> Chemistry, <a href="http://www.chimie.ens.fr/"> Ecole Normale Supérieure</a> , <a href="http://www.aiv-paris.org/fr/master-aiv/"> Master 2 AIV</a> <br></I><br />
Graduation in Chemistry at Ecole Normale Supérieure, Paris - Interdisciplinary Master in Life Sciences at Université Paris Descartes, Paris ... but currently lost in the Middle-East !<br />
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<img src="https://static.igem.org/mediawiki/2009/3/34/Vico.JPG" height=120px width=180px> <br />
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<b>Du Vicard</b><br><br />
<I> Biology, <a href="http://www.aiv-paris.org/fr/master-aiv/"> Master 1 AIV </I></a> <br />
<br><br />
Uncle Mc Donalds is his best friend<br />
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<h3>Ariel Lindner</h3><br />
<br />
Co-initiator of the Centre for Research and Interdisciplinarity (cri-paris.org), Ariel is an INSERM tenured senior researcher and director of the Interdisciplinary Approaches to Life Sciences ('AIV') master program at the Paris Descartes and Diderot Universities. Ariel has graduated from the Hebrew University (Jerusalem, Israel) "Amirim" interdisciplinary program with major in Chemistry and received his M.Sc. and Ph.D. from the Weizmann Institute of Science (Rehovot, Israel) in Chemical Immunology for his work on catalytic antibodies as enzyme models, antibody conformational changes and directed evolution. After a research period at the Scripps Institute (California, USA), he received EMBO and Marie Curie fellowships to pursue postdoctoral work in Paris. His study interests evolve around applying Physical, Chemical and Biological approaches to study aging and variability between clonal individuals. Ariel has been leading the Paris iGEM team since its conception over three years ago and cannot wait to bring another trophy home... <br><br />
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<img src="https://static.igem.org/mediawiki/2009/8/8b/SamuelBottani2.JPG" width=142px height=213px><br />
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<h3>Samuel Bottani</h3><br />
<I>Physicist</I><br />
<br><br />
Trained as a physicist, natural evolution made me shift from cosmology to molecular biology. I'm interested in a variety of scientific issues and their applications. I'm inspired by beauty and how forms, behaviors and ideas emerge and this drives me in my explorations of the wonders on Nature. Primarily how complex structures and behavior emerge and evolve. We are in an extraordinary period where never so many details have been known on living organisms. Whole genomes are known and extraordinary technological advances enable in vivo tracking and action on single cells and molecules. There is the feeling that a new level of understanding of Life is about to be gained, with fantastic potential for the evolution of humanity. I am eager to participate to endeavor and by my research and critical thinking.<br />
<br />
It is my opinion that scientists, as highly and costly educated citizens have social duty for the collectivity. Engagement in education is essential, ethical concern and involvement in technological and business matters for the benefit of all.<br />
<br />
I am co-director of the international Ph.D. program "Frontières du Vivant" (Frontiers in Life Sciences, www.fdv-paris.org) of the Universities Paris Descartes and Paris Diderot for interdisciplinary research programs on Life Sciences issues. Hosted at the Centre for Research and Interdisciplinarity (cri-paris.org) as the IGEM Paris team we support active approaches in education for interdisciplinary research.<br />
</div><br />
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<img src="http://lh3.ggpht.com/_DG1UEKxMdRA/SqZmSnFS8ZI/AAAAAAAAC8w/JXQW3WtZh4M/flefevre.jpg" width=200px height=130px><br />
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<h3>François Le Fèvre</h3><br />
<I>Biocomputational scientist</I><br />
<br><br />
My research interests lie in the intersection of bioinformatics, and applied software engineering. My approach to research involves an interplay between experimental and theoretical modeling techniques, and is focused towards identifying and elucidating new knowledge interesting for biologists.<br />
<br><br />
Genomic Institute : <a href="http://www.cns.fr/" >Genoscope</a><br />
<br><br />
French Atomic Energy Commission : <a href="http://www.cea.fr/" >CEA</a><br />
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Personal home page : <a href="http://sites.google.com/site/synbiocamp/" >SynBioCamp</a><br />
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<img src="https://static.igem.org/mediawiki/2009/9/9c/Gregory_Batt.jpg" width=120px height=153px><br />
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<h3>Gregory Batt</h3><br />
<I><a href="http://www.inria.fr/" >INRIA</a> Paris-Rocquencourt</I><br />
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== Advisors ==<br />
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<img src="https://static.igem.org/mediawiki/2009/6/68/Guillaume_Cambray.JPG" width=150px height=180px><br />
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<h3>Guillaume Cambray</h3><br />
<I><a href="http://www.pasteur.fr" > Pasteur Institute </a> Post Doctoral student</I><br />
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<img src="https://static.igem.org/mediawiki/2009/f/f1/David_Bikard.jpg" width=200px height=130px><br />
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<h3>David Bikard</h3><br />
<I> 2nd year PhD student in Biology at <a href="http://www.pasteur.fr" > the Pasteur Institute</I></a><br />
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iGEM Paris 2007 team member<br />
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<img src="https://static.igem.org/mediawiki/2009/d/d6/Fanny_Caffin.jpg" width=100px height=130px><br />
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<h3>Fanny Caffin</h3><br />
<I>Biologist (molecular & cellular biology)</I> <a href="http://inserm-u769.cep.u-psud.fr/fr/index.html/" > 1st year of PhD at Paris XI University</a><br />
<br><br />
iGEM Paris 2008 team member<br />
<br><br />
...Your flight attendant at your service...<br />
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<img src="https://static.igem.org/mediawiki/2008/a/ad/N764862654_816423_9815.jpg" width=120px height=130px><br />
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<div class="rightcolumn"><br />
<h3>Benoît d'Hayer</h3><br />
<I>Pharmacy at the <a href="http://www.univ-paris5.fr/" > Paris Descartes University</a></I><br />
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Master's degree <a href="http://www.univ-paris-diderot.fr/magisteregenet/"> of the European Master of Genetic</a><br />
<br><br />
iGEM Paris 2008 team member<br />
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<img src="https://static.igem.org/mediawiki/2009/0/06/YannLC.jpg" width=150px height=130px><br />
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<h3>Yann Le Cunff</h3><br />
<I>Applied Mathematics, <a href="http://www.supelec.fr/"> Supelec </a></I><br />
<br><br />
iGEM Paris 2008 team member<br />
<br><br />
Pierre's and Colonel's biggest fan EVER.<br />
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<div id="paris_content"><br />
</html></div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview3Team:Paris/Addressing overview32009-10-21T23:41:27Z<p>Fanny.c: /* Adressing the message in the outer membrane : ClyA */</p>
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<div>{{Template:Paris2009}}<br />
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==Adressing the message in the outer membrane : ClyA==<br />
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<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2#bottom"> Main </a>|<br />
<a class="menu_sub_active" href="https://2009.igem.org/Team:Paris/Addressing_overview3#bottom"> ClyA</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview4#bottom"> OmpA</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#bottom"> Our strategy</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview_Construction#bottom"> Construction</a><br />
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We work on the cell-cell communication using vesicle:<br />
<br><br />
In this part we look into adressing a protein into the sender outer membrane that could be incoporated into outer membrane vesicles (OMVs). This protein would then be able to transmit a message after the fusion of OMVs with a receiver cell.<br />
<br />
In this direction ClyA (the cytolysine A of E.Coli) seems to be a good candidate. ClyA is one of the proteins that has been previously detected into OMVs and is known to be specificly exported to the outer membrane [http://www.ncbi.nlm.nih.gov/pubmed/14532000 [3]]. ClyA is thus expressed on bacteria and OMVs surface. Moreover, when ClyA is overproduced, it is accumulated into the periplasmic space [http://www.ncbi.nlm.nih.gov/pubmed/14532000 [3]].<br />
<br />
<br />
However there is an inconvenient to use this protein. ClyA is an alpha-Pore Forming Toxin (PFT). PFT are widely distributed proteins which form lesions in biological membranes. They exhibit their toxic effect in different manner. The first one is that ClyA allows the destruction of membrane permeability barrier. Furthermore, the toxic effect of ClyA could be explain by its capacity to deliver toxic component after the assembly of 8 or 13 of its subunits. PFTs can be subdivided into two classes; α-PFTs and β-PFTs, depending on the suspected mode of membrane integration, either by α-helical or β-sheet elements.[http://www.ncbi.nlm.nih.gov/pubmed/19421192 [2]]<br />
<br />
[[Image:Clya_simple.jpg|ClyA subunit|150px|left]] [[Image:Clya_structure2.jpg|ClyA assembled|100px|right]][[Image:ClyA.jpg|ClyA are assembling in outer membrane of a host cell|150px|center]] [[Image:Clya_structure.jpg|ClyA assembled|150px|center]]<br />
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<br />
Some article argue that E.Coli K12 use this ClyA to lyse other cell (specially mamalian cell or eurcaryote cell[http://www.ncbi.nlm.nih.gov/pubmed/14532000 [3]]). But E. coli cells expressing clyA do not lyse each other.<br />
<br />
<br />
<br />
Kim et al. have successfully fused clyA to GFP in order to observe vesicles [http://www.ncbi.nlm.nih.gov/pubmed/18511069 [1]], so we know that we can try to fuse clyA to a protein domain that would induce a signal transduction into the receiver cell. To see how we want to exploite clyA properties see [https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#Overview our strategy].<br />
<br />
<br />
<br />
<br />
<br />
====Bibliography :====<br />
<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/18511069 [1]]Kim, J.-Y. & DeLisa, M.P. Engineered bacterial outer membrane vesicles with enhanced functionality J.Mol. Biol. (2008) 380, 51–66<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/19421192 [2]]Muller, M. & Ban, N. The structure of a cytolytic a-helical toxin pore reveals its assembly mechanism Nature (4 June 2009) 459, 726-730 <br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/14532000 [3]]Wai, S.N. & Lindmark, B. Vesicle-Mediated Export and Assembly of Pore-Forming Oligomers of the Enterobacterial ClyA Cytotoxin Cell (October 2003), 115,25-35<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/10383763 [4]] Oscarsson, J. & Uhlin, B.E. Molecular analysis of the cytolytic protein ClyA (SheA) from Escherichia coli Molecular Microbiology (1999) 32(6), 1226–1238<br />
<br />
[http://jb.asm.org/cgi/content/abstract/182/22/6347 [5]] Westermark, M. & Uhlin, B.E. Silencing and Activation of ClyA Cytotoxin Expression in Escherichia coli Journalof bacteriology,(Nov. 2000), Vol. 182, No. 22 6347–6357<br />
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{{Template:Paris2009_guided|Addressing_overview2#bottom|Addressing_overview4#bottom}}</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview_ConstructionTeam:Paris/Addressing overview Construction2009-10-21T23:29:57Z<p>Fanny.c: /* Construction */</p>
<hr />
<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris/Addressing_overview2#top | ClyA]] > [[Team:Paris/Addressing_overview_Construction#bottom | Construction]]<br />
{{Template:Paris2009}}<br />
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==Construction ==<br />
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<div id="middle-side"><center><br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2#bottom"> Main </a>|<br />
<a class="menu_sub" href="https://2009.igem.org/Team:Paris/Addressing_overview3#bottom"> ClyA</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview4#bottom"> OmpA</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#bottom"> Our strategy</a>|<br />
<a class="menu_sub_active"href="https://2009.igem.org/Team:Paris/Addressing_overview_Construction#bottom"> Construction</a><br />
</center><br />
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<br />
[[Image:Clya_construction.jpg|500px|center|Activation of our construction by Arabinose]]<br />
<br />
Here the activation of our construction '''by arabinose''', in order to test the construction.<br />
<br><br />
<br><br />
<br><br />
[[Image:Clya_construction2.jpg|500px|center|Inhibition of our construction by Glucose]]<br />
<br />
Here the inhibition of our construction '''by glucose''', in order to test the construction.<br />
<br><br />
<br><br />
<br><br />
[[Image:ClyA_construction_3.jpg|500px|center|Activation of our construction by Glucose]]<br />
<br />
Here the activation of our construction '''by arabinose'''. <br />
<br> It is quite the same as the first construction, but in [http://www.ncbi.nlm.nih.gov/pubmed/18511069[1]] the RFP-Cter link to Nter-ClyA seems to have a better fluorescence. Because of our late about receiving the oligo for its synthesis in biobrick format, we used the first construction (ClyA-Cter link to Nter-RFP).<br />
<br />
<br />
<br />
====Bibliography :====<br />
<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/18511069[1]] Kim, J.-Y. & DeLisa, M.P. Engineered bacterial outer membrane vesicles with enhanced functionality J.Mol. Biol. (2008) 380, 51–66</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview_ConstructionTeam:Paris/Addressing overview Construction2009-10-21T23:26:12Z<p>Fanny.c: /* Construction */</p>
<hr />
<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris/Addressing_overview2#top | ClyA]] > [[Team:Paris/Addressing_overview_Construction#bottom | Construction]]<br />
{{Template:Paris2009}}<br />
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==Construction ==<br />
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height: 23px;<br />
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top: 0px;<br />
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margin-top:10px;<br />
padding-top: 7px;<br />
background: url(https://static.igem.org/mediawiki/2009/1/1b/Left_menu_pari.png);<br />
z-index:4;<br />
}<br />
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#middle-side {<br />
height: 25px;<br />
width: 380px;<br />
position: absolute;<br />
top: 0px;<br />
left: 170px;<br />
margin-top:10px;<br />
padding-top: 5px;<br />
background: #dadada;<br />
z-index:5;<br />
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position: absolute;<br />
height: 23px;<br />
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<div id="left-side"></div><br />
<div id="middle-side"><center><br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2#bottom"> Main </a>|<br />
<a class="menu_sub" href="https://2009.igem.org/Team:Paris/Addressing_overview3#bottom"> ClyA</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview4#bottom"> OmpA</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#bottom"> Our strategy</a>|<br />
<a class="menu_sub_active"href="https://2009.igem.org/Team:Paris/Addressing_overview_Construction#bottom"> Construction</a><br />
</center><br />
</div><br />
<div id="right-side"></div><br />
</html><br />
<br />
[[Image:Clya_construction.jpg|500px|center|Activation of our construction by Arabinose]]<br />
<br />
Here the activation of our construction '''by arabinose''', in order to test the construction.<br />
<br><br />
<br><br />
<br><br />
[[Image:Clya_construction2.jpg|500px|center|Inhibition of our construction by Glucose]]<br />
<br />
Here the inhibition of our construction '''by glucose''', in order to test the construction.<br />
<br><br />
<br><br />
<br><br />
[[Image:ClyA_construction_3.jpg|500px|center|Activation of our construction by Glucose]]<br />
<br />
Here the activation of our construction '''by arabinose'''. <br />
<br> It is quite the same as the first construction but in [http://www.ncbi.nlm.nih.gov/pubmed/18511069[1]], the RFP-Cter link to Nter-ClyA seems to have better fluorescence. Because of our late about receiving the oligo for its synthesis in biobrick format we used the first construction (ClyA-Cter link to Nter-RFP).<br />
<br />
<br />
<br />
====Bibliography :====<br />
<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/18511069[1]] Kim, J.-Y. & DeLisa, M.P. Engineered bacterial outer membrane vesicles with enhanced functionality J.Mol. Biol. (2008) 380, 51–66</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview_ConstructionTeam:Paris/Addressing overview Construction2009-10-21T23:24:12Z<p>Fanny.c: /* Construction */</p>
<hr />
<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris/Addressing_overview2#top | ClyA]] > [[Team:Paris/Addressing_overview_Construction#bottom | Construction]]<br />
{{Template:Paris2009}}<br />
{{Template:Paris2009_menu3}}<br />
<br />
<br />
==Construction ==<br />
<html><br />
<style type="text/css"><br />
#left-side {<br />
position: absolute;<br />
height: 23px;<br />
width: 30px;<br />
top: 0px;<br />
left: 160px;<br />
margin-top:10px;<br />
padding-top: 7px;<br />
background: url(https://static.igem.org/mediawiki/2009/1/1b/Left_menu_pari.png);<br />
z-index:4;<br />
}<br />
<br />
#middle-side {<br />
height: 25px;<br />
width: 380px;<br />
position: absolute;<br />
top: 0px;<br />
left: 170px;<br />
margin-top:10px;<br />
padding-top: 5px;<br />
background: #dadada;<br />
z-index:5;<br />
}<br />
<br />
#right-side {<br />
position: absolute;<br />
height: 23px;<br />
width: 30px;<br />
margin-top:10px;<br />
padding-top: 7px;<br />
top: 0px;<br />
left: 530px;<br />
background: url(https://static.igem.org/mediawiki/2009/4/40/Right_menu_paris.png);<br />
z-index:4;<br />
}<br />
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a.menu_sub {<br />
padding-left: 7px;<br />
padding-right: 7px;<br />
}<br />
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a.menu_sub_active {<br />
padding-left: 7px;<br />
padding-right: 7px;<br />
color:#b0310e;<br />
font-weight:bold;<br />
}<br />
</style><br />
<div id="left-side"></div><br />
<div id="middle-side"><center><br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2#bottom"> Main </a>|<br />
<a class="menu_sub" href="https://2009.igem.org/Team:Paris/Addressing_overview3#bottom"> ClyA</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview4#bottom"> OmpA</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#bottom"> Our strategy</a>|<br />
<a class="menu_sub_active"href="https://2009.igem.org/Team:Paris/Addressing_overview_Construction#bottom"> Construction</a><br />
</center><br />
</div><br />
<div id="right-side"></div><br />
</html><br />
<br />
[[Image:Clya_construction.jpg|500px|center|Activation of our construction by Arabinose]]<br />
<br />
Here the activation of our construction '''by arabinose''', in order to test the construction.<br />
<br><br />
[[Image:Clya_construction2.jpg|500px|center|Inhibition of our construction by Glucose]]<br />
<br />
Here the inhibition of our construction '''by glucose''', in order to test the construction.<br />
<br><br />
[[Image:ClyA_construction_3.jpg|500px|center|Activation of our construction by Glucose]]<br />
<br />
Here the activation of our construction '''by arabinose'''. It is quite the same as the first construction but in [http://www.ncbi.nlm.nih.gov/pubmed/18511069[1]], the RFP-Cter link to Nter-ClyA seems to have better fluorescence. Because of our late about receiving the oligo for its synthesis in biobrick format we used the first construction (ClyA-Cter link to Nter-RFP).<br />
<br />
<br />
<br />
====Bibliography :====<br />
<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/18511069[1]] Kim, J.-Y. & DeLisa, M.P. Engineered bacterial outer membrane vesicles with enhanced functionality J.Mol. Biol. (2008) 380, 51–66</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview2_strategyTeam:Paris/Addressing overview2 strategy2009-10-21T23:13:58Z<p>Fanny.c: /* Adressing the message in the membrane : Our strategy */</p>
<hr />
<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris/Addressing_overview2#top | ClyA]] > [[Team:Paris/Addressing_overview2_strategy#bottom | Our strategy]]<br />
{{Template:Paris2009}}<br />
{{Template:Paris2009_menu3}}<br />
<br />
<br />
==Adressing the message in the membrane : Our strategy==<br />
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<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2#bottom"> Main </a>|<br />
<a class="menu_sub" href="https://2009.igem.org/Team:Paris/Addressing_overview3#bottom"> ClyA</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview4#bottom"> OmpA</a>|<br />
<a class="menu_sub_active"href="https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#bottom"> Our strategy</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview_Construction#bottom"> Construction</a><br />
</center><br />
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<br />
Our strategy is to use clyA to export a protein to the outer-membrane of the cell. The protein fused to clyA will be incorporated into the vesicle during the vesiculation process and it's also express on the surface. ClyA contain the signal peptide required for the exportation process from the cytoplasm to the periplasm. The overall idea is to fused the protein of interest to clyA. <br />
<br />
So in a first time in order to see if our ClyA are localize in OMVs, we fused it we a RFP. For that we add a poly glycine linker to ClyA biobrick to improve ClyA-RFP fusion. Moreover if we put RFP before ClyA, it seem that there is more fluorescence than ClyA before RFP under [[http://www.ncbi.nlm.nih.gov/pubmed/18511069 1]]. You can see the contruction [[https://2009.igem.org/Team:Paris/Addressing_overview_Construction#Overview here]]<br />
<br />
Then if this test work, we could replace the RFP by the protein of interest for signal transduction,moreover this system couple to fec system can transduct a signal from outer membran to the cytoplasm.<br />
<br />
{{Template:Paris2009_guided|Addressing_overview4#bottom|Production_overview#top}}</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview_ConstructionTeam:Paris/Addressing overview Construction2009-10-21T23:04:46Z<p>Fanny.c: /* Construction */</p>
<hr />
<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris/Addressing_overview2#top | ClyA]] > [[Team:Paris/Addressing_overview_Construction#bottom | Construction]]<br />
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==Construction ==<br />
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<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2#bottom"> Main </a>|<br />
<a class="menu_sub" href="https://2009.igem.org/Team:Paris/Addressing_overview3#bottom"> ClyA</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview4#bottom"> OmpA</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#bottom"> Our strategy</a>|<br />
<a class="menu_sub_active"href="https://2009.igem.org/Team:Paris/Addressing_overview_Construction#bottom"> Construction</a><br />
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<br />
[[Image:Clya_construction.jpg|500px|center|Activation of our construction by Arabinose]]<br />
<br />
Here the activation of our construction '''by arabinose''', in order to test the construction.<br />
<br><br />
[[Image:Clya_construction2.jpg|500px|center|Inhibition of our construction by Glucose]]<br />
<br />
Here the inhibition of our construction '''by glucose''', in order to test the construction.<br />
<br><br />
[[Image:ClyA_construction_3.jpg|500px|center|Activation of our construction by Glucose]]<br />
<br />
Here the activation of our construction '''by arabinose'''. It is quite the same as the first construction but in [article 1], the RFP-Cter link to Nter-ClyA seems to have better fluorescence. Because of our late about receiving the oligo for its synthesis in biobrick format we used the first construction (ClyA-Cter link to Nter-RFP).</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview_ConstructionTeam:Paris/Addressing overview Construction2009-10-21T22:57:46Z<p>Fanny.c: /* Construction */</p>
<hr />
<div><span/ id="bottom">[https://2009.igem.org/ iGEM ] > [[Team:Paris#top | Paris]] > [[Team:Paris/Addressing_overview2#top | ClyA]] > [[Team:Paris/Addressing_overview_Construction#bottom | Construction]]<br />
{{Template:Paris2009}}<br />
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<br />
<br />
==Construction ==<br />
<html><br />
<style type="text/css"><br />
#left-side {<br />
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background: #dadada;<br />
z-index:5;<br />
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<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2#bottom"> Main </a>|<br />
<a class="menu_sub" href="https://2009.igem.org/Team:Paris/Addressing_overview3#bottom"> ClyA</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview4#bottom"> OmpA</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#bottom"> Our strategy</a>|<br />
<a class="menu_sub_active"href="https://2009.igem.org/Team:Paris/Addressing_overview_Construction#bottom"> Construction</a><br />
</center><br />
</div><br />
<div id="right-side"></div><br />
</html><br />
<br />
[[Image:Clya_construction.jpg|500px|center|Activation of our construction by Arabinose]]<br />
<br />
Here the activation of our construction '''by arabinose''', in order to test the construction.<br />
<br><br />
[[Image:Clya_construction2.jpg|500px|center|Inhibition of our construction by Glucose]]<br />
<br />
Here the inhibition of our construction '''by glucose''', in order to test the construction.<br />
<br><br />
[[Image:ClyA_construction_3.jpg|500px|center|Activation of our construction by Glucose]]<br />
<br />
Here the activation of our construction by arabinose. It is quite the same as the first construction but in [article 1], the RFP-Cter link to Nter-ClyA seems to have better fluorescence. Because of our late about receiving the oligo for its synthesis in biobrick format we used the first construction (ClyA-Cter link to Nter-RFP).</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview4Team:Paris/Addressing overview42009-10-21T22:15:50Z<p>Fanny.c: /* Adressin the message in the outer membrane : OmpA */</p>
<hr />
<div>{{Template:Paris2009}}<br />
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==Adressin the message in the outer membrane : OmpA==<br />
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<div id="left-side"></div><br />
<div id="middle-side"><center><br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2#bottom"> Main </a>|<br />
<a class="menu_sub" href="https://2009.igem.org/Team:Paris/Addressing_overview3#bottom"> ClyA</a>|<br />
<a class="menu_sub_active"href="https://2009.igem.org/Team:Paris/Addressing_overview4#bottom"> OmpA</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#bottom"> Our strategy</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview_Construction#bottom"> Construction</a><br />
</center><br />
</div><br />
<div id="right-side"></div><br />
</html><br />
<br />
<br />
<br />
[[Image:OmpA1.JPG|250px|left]][[Image:OmpA2.JPG|230px|right]]<br />
<br />
OmpA as previously been used in protein fusion to expose heterologuous protein domains to the surface of E. coli. As an outer membrane protein, ompA could be incorporated into vesicles and is similarly to clyA also a good candidate for adressing protein domains to vesicles. <br />
<br />
<br />
Outer membrane protein A (OmpA) is a major protein in the Escherichia coli outer membrane. OmpA plays a vital structural role in ''E. coli'', and suggested that a perfect β-barrel structure of OmpA is important for outer membrane stability[http://www.ncbi.nlm.nih.gov/pubmed/11906175[1]]. OmpA is the most well-studied outer membrane protein in ''Escherichia coli''. This 325-residue protein was thought to contain two domains. The classic N-terminal domain, consisting of 171 amino acid residues, was shown to cross the membrane eight times in antiparallel β-strands with four relatively large and hydrophilic surface-exposed loops and short periplasmic turns[http://www.ncbi.nlm.nih.gov/pubmed/11276254[2]]. The C-terminal domain is located in the periplasm, and binds to the peptidoglycan thus connecting it to the outer membrane[http://www.ncbi.nlm.nih.gov/pubmed/8577259[3]]. The function of OmpA is thought to contribute to the structural integrity of the outer membrane<br />
along with murein lipoprotein[http://www.ncbi.nlm.nih.gov/pubmed/4261992[4]] and peptidoglycanassociated lipoprotein . In addition to its structural role, OmpA serves as a receptor of colicin and several phages[http://www.ncbi.nlm.nih.gov/pubmed/1574003[5]], and it's required in F-conjugation[http://www.ncbi.nlm.nih.gov/pubmed/321438[6]],[http://www.ncbi.nlm.nih.gov/pubmed/10368142[7]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{Template:Paris2009_guided|Addressing_overview3#bottom|Addressing_overview2_strategy#bottom}}<br />
<br />
<br />
====Bibliography:====<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11906175[1]] Ying Wang, (2002) The Function of OmpA in Escherichia coli, Biochem Biophys Res Commun.292(2):396-401<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11276254[2]] Arora, A., Abildgaard, F., Bushweller, J. H., and Tamm, L. K. (2001) Structure of outer membrane protein A transmembrane domain by NMR spectroscopy. Nat. Struct. Biol 8, 334–338.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/8577259[3]] Koebnik, R. (1995) Proposal for a peptidoglycan associating alpha-helical motif in the C-terminal regions of some bacterial cell-surface proteins. Mol. Microbiol. 16, 1269–1270.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/4261992[4]] Braun, V., and Bosch, V. (1972) Sequence of the mureinlipoprotein and the attachment site of the lipid. Eur. J. Biochem. 28, 51–69.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/1574003[5]] Lazzaroni, J.-C., and Portalier, R. (1992) The excC gene of Escherichia coli K-12 required for cell envelope integrity encodes the peptidoglycan-associated lipoprotein. Mol. Microbiol. 6, 735–742.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/321438[6] Schweizer, M., and Henning, U. (1977) Action of major outer cell envelope membrane protein in conjugation of Escherichia coli K-12. J. Bacteriol. 129, 1651–1652.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/10368142[7]] Koebnik, R. (1999) Structural and functional roles of the surfaceexposed loops of the β-barrel membrane protein OmpA from Escherichia coli. J. Bacteriol. 181, 3688–3694.</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview4Team:Paris/Addressing overview42009-10-21T22:15:19Z<p>Fanny.c: /* Adressin the message in the outer membrane : OmpA */</p>
<hr />
<div>{{Template:Paris2009}}<br />
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<br />
==Adressin the message in the outer membrane : OmpA==<br />
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<div id="left-side"></div><br />
<div id="middle-side"><center><br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2#bottom"> Main </a>|<br />
<a class="menu_sub" href="https://2009.igem.org/Team:Paris/Addressing_overview3#bottom"> ClyA</a>|<br />
<a class="menu_sub_active"href="https://2009.igem.org/Team:Paris/Addressing_overview4#bottom"> OmpA</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#bottom"> Our strategy</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview_Construction#bottom"> Construction</a><br />
</center><br />
</div><br />
<div id="right-side"></div><br />
</html><br />
<br />
<br />
<br />
[[Image:OmpA1.JPG|250px|left]][[Image:OmpA2.JPG|200px|right]]<br />
<br />
OmpA as previously been used in protein fusion to expose heterologuous protein domains to the surface of E. coli. As an outer membrane protein, ompA could be incorporated into vesicles and is similarly to clyA also a good candidate for adressing protein domains to vesicles. <br />
<br />
<br />
Outer membrane protein A (OmpA) is a major protein in the Escherichia coli outer membrane. OmpA plays a vital structural role in ''E. coli'', and suggested that a perfect β-barrel structure of OmpA is important for outer membrane stability[http://www.ncbi.nlm.nih.gov/pubmed/11906175[1]]. OmpA is the most well-studied outer membrane protein in ''Escherichia coli''. This 325-residue protein was thought to contain two domains. The classic N-terminal domain, consisting of 171 amino acid residues, was shown to cross the membrane eight times in antiparallel β-strands with four relatively large and hydrophilic surface-exposed loops and short periplasmic turns[http://www.ncbi.nlm.nih.gov/pubmed/11276254[2]]. The C-terminal domain is located in the periplasm, and binds to the peptidoglycan thus connecting it to the outer membrane[http://www.ncbi.nlm.nih.gov/pubmed/8577259[3]]. The function of OmpA is thought to contribute to the structural integrity of the outer membrane<br />
along with murein lipoprotein[http://www.ncbi.nlm.nih.gov/pubmed/4261992[4]] and peptidoglycanassociated lipoprotein . In addition to its structural role, OmpA serves as a receptor of colicin and several phages[http://www.ncbi.nlm.nih.gov/pubmed/1574003[5]], and it's required in F-conjugation[http://www.ncbi.nlm.nih.gov/pubmed/321438[6]],[http://www.ncbi.nlm.nih.gov/pubmed/10368142[7]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{Template:Paris2009_guided|Addressing_overview3#bottom|Addressing_overview2_strategy#bottom}}<br />
<br />
<br />
====Bibliography:====<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11906175[1]] Ying Wang, (2002) The Function of OmpA in Escherichia coli, Biochem Biophys Res Commun.292(2):396-401<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11276254[2]] Arora, A., Abildgaard, F., Bushweller, J. H., and Tamm, L. K. (2001) Structure of outer membrane protein A transmembrane domain by NMR spectroscopy. Nat. Struct. Biol 8, 334–338.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/8577259[3]] Koebnik, R. (1995) Proposal for a peptidoglycan associating alpha-helical motif in the C-terminal regions of some bacterial cell-surface proteins. Mol. Microbiol. 16, 1269–1270.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/4261992[4]] Braun, V., and Bosch, V. (1972) Sequence of the mureinlipoprotein and the attachment site of the lipid. Eur. J. Biochem. 28, 51–69.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/1574003[5]] Lazzaroni, J.-C., and Portalier, R. (1992) The excC gene of Escherichia coli K-12 required for cell envelope integrity encodes the peptidoglycan-associated lipoprotein. Mol. Microbiol. 6, 735–742.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/321438[6] Schweizer, M., and Henning, U. (1977) Action of major outer cell envelope membrane protein in conjugation of Escherichia coli K-12. J. Bacteriol. 129, 1651–1652.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/10368142[7]] Koebnik, R. (1999) Structural and functional roles of the surfaceexposed loops of the β-barrel membrane protein OmpA from Escherichia coli. J. Bacteriol. 181, 3688–3694.</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview4Team:Paris/Addressing overview42009-10-21T22:14:54Z<p>Fanny.c: /* Adressin the message in the outer membrane : OmpA */</p>
<hr />
<div>{{Template:Paris2009}}<br />
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==Adressin the message in the outer membrane : OmpA==<br />
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}<br />
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[[Image:OmpA1.JPG|200px|left]][[Image:OmpA2.JPG|200px|right]]<br />
<br />
OmpA as previously been used in protein fusion to expose heterologuous protein domains to the surface of E. coli. As an outer membrane protein, ompA could be incorporated into vesicles and is similarly to clyA also a good candidate for adressing protein domains to vesicles. <br />
<br />
<br />
Outer membrane protein A (OmpA) is a major protein in the Escherichia coli outer membrane. OmpA plays a vital structural role in ''E. coli'', and suggested that a perfect β-barrel structure of OmpA is important for outer membrane stability[http://www.ncbi.nlm.nih.gov/pubmed/11906175[1]]. OmpA is the most well-studied outer membrane protein in ''Escherichia coli''. This 325-residue protein was thought to contain two domains. The classic N-terminal domain, consisting of 171 amino acid residues, was shown to cross the membrane eight times in antiparallel β-strands with four relatively large and hydrophilic surface-exposed loops and short periplasmic turns[http://www.ncbi.nlm.nih.gov/pubmed/11276254[2]]. The C-terminal domain is located in the periplasm, and binds to the peptidoglycan thus connecting it to the outer membrane[http://www.ncbi.nlm.nih.gov/pubmed/8577259[3]]. The function of OmpA is thought to contribute to the structural integrity of the outer membrane<br />
along with murein lipoprotein[http://www.ncbi.nlm.nih.gov/pubmed/4261992[4]] and peptidoglycanassociated lipoprotein . In addition to its structural role, OmpA serves as a receptor of colicin and several phages[http://www.ncbi.nlm.nih.gov/pubmed/1574003[5]], and it's required in F-conjugation[http://www.ncbi.nlm.nih.gov/pubmed/321438[6]],[http://www.ncbi.nlm.nih.gov/pubmed/10368142[7]]<br />
<br />
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<br />
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<br />
<br />
{{Template:Paris2009_guided|Addressing_overview3#bottom|Addressing_overview2_strategy#bottom}}<br />
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<br />
====Bibliography:====<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11906175[1]] Ying Wang, (2002) The Function of OmpA in Escherichia coli, Biochem Biophys Res Commun.292(2):396-401<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11276254[2]] Arora, A., Abildgaard, F., Bushweller, J. H., and Tamm, L. K. (2001) Structure of outer membrane protein A transmembrane domain by NMR spectroscopy. Nat. Struct. Biol 8, 334–338.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/8577259[3]] Koebnik, R. (1995) Proposal for a peptidoglycan associating alpha-helical motif in the C-terminal regions of some bacterial cell-surface proteins. Mol. Microbiol. 16, 1269–1270.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/4261992[4]] Braun, V., and Bosch, V. (1972) Sequence of the mureinlipoprotein and the attachment site of the lipid. Eur. J. Biochem. 28, 51–69.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/1574003[5]] Lazzaroni, J.-C., and Portalier, R. (1992) The excC gene of Escherichia coli K-12 required for cell envelope integrity encodes the peptidoglycan-associated lipoprotein. Mol. Microbiol. 6, 735–742.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/321438[6] Schweizer, M., and Henning, U. (1977) Action of major outer cell envelope membrane protein in conjugation of Escherichia coli K-12. J. Bacteriol. 129, 1651–1652.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/10368142[7]] Koebnik, R. (1999) Structural and functional roles of the surfaceexposed loops of the β-barrel membrane protein OmpA from Escherichia coli. J. Bacteriol. 181, 3688–3694.</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview4Team:Paris/Addressing overview42009-10-21T22:13:56Z<p>Fanny.c: /* Adressin the message in the outer membrane : OmpA */</p>
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<a class="menu_sub_active"href="https://2009.igem.org/Team:Paris/Addressing_overview4#bottom"> OmpA</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#bottom"> Our strategy</a>|<br />
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[[Image:OmpA1.JPG|200px|left]][[Image:OmpA2.JPGpx|200px|right]]<br />
<br />
OmpA as previously been used in protein fusion to expose heterologuous protein domains to the surface of E. coli. As an outer membrane protein, ompA could be incorporated into vesicles and is similarly to clyA also a good candidate for adressing protein domains to vesicles. <br />
<br />
<br />
Outer membrane protein A (OmpA) is a major protein in the Escherichia coli outer membrane. OmpA plays a vital structural role in ''E. coli'', and suggested that a perfect β-barrel structure of OmpA is important for outer membrane stability[http://www.ncbi.nlm.nih.gov/pubmed/11906175[1]]. OmpA is the most well-studied outer membrane protein in ''Escherichia coli''. This 325-residue protein was thought to contain two domains. The classic N-terminal domain, consisting of 171 amino acid residues, was shown to cross the membrane eight times in antiparallel β-strands with four relatively large and hydrophilic surface-exposed loops and short periplasmic turns[http://www.ncbi.nlm.nih.gov/pubmed/11276254[2]]. The C-terminal domain is located in the periplasm, and binds to the peptidoglycan thus connecting it to the outer membrane[http://www.ncbi.nlm.nih.gov/pubmed/8577259[3]]. The function of OmpA is thought to contribute to the structural integrity of the outer membrane<br />
along with murein lipoprotein[http://www.ncbi.nlm.nih.gov/pubmed/4261992[4]] and peptidoglycanassociated lipoprotein . In addition to its structural role, OmpA serves as a receptor of colicin and several phages[http://www.ncbi.nlm.nih.gov/pubmed/1574003[5]], and it's required in F-conjugation[http://www.ncbi.nlm.nih.gov/pubmed/321438[6]],[http://www.ncbi.nlm.nih.gov/pubmed/10368142[7]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{Template:Paris2009_guided|Addressing_overview3#bottom|Addressing_overview2_strategy#bottom}}<br />
<br />
<br />
====Bibliography:====<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11906175[1]] Ying Wang, (2002) The Function of OmpA in Escherichia coli, Biochem Biophys Res Commun.292(2):396-401<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11276254[2]] Arora, A., Abildgaard, F., Bushweller, J. H., and Tamm, L. K. (2001) Structure of outer membrane protein A transmembrane domain by NMR spectroscopy. Nat. Struct. Biol 8, 334–338.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/8577259[3]] Koebnik, R. (1995) Proposal for a peptidoglycan associating alpha-helical motif in the C-terminal regions of some bacterial cell-surface proteins. Mol. Microbiol. 16, 1269–1270.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/4261992[4]] Braun, V., and Bosch, V. (1972) Sequence of the mureinlipoprotein and the attachment site of the lipid. Eur. J. Biochem. 28, 51–69.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/1574003[5]] Lazzaroni, J.-C., and Portalier, R. (1992) The excC gene of Escherichia coli K-12 required for cell envelope integrity encodes the peptidoglycan-associated lipoprotein. Mol. Microbiol. 6, 735–742.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/321438[6] Schweizer, M., and Henning, U. (1977) Action of major outer cell envelope membrane protein in conjugation of Escherichia coli K-12. J. Bacteriol. 129, 1651–1652.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/10368142[7]] Koebnik, R. (1999) Structural and functional roles of the surfaceexposed loops of the β-barrel membrane protein OmpA from Escherichia coli. J. Bacteriol. 181, 3688–3694.</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview4Team:Paris/Addressing overview42009-10-21T22:13:10Z<p>Fanny.c: /* Adressin the message in the outer membrane : OmpA */</p>
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<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#bottom"> Our strategy</a>|<br />
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<br />
<br />
<br />
[[Image:OmpA1.JPG|300px|left]][[Image:OmpA2.JPG|300px|right]]<br />
<br />
OmpA as previously been used in protein fusion to expose heterologuous protein domains to the surface of E. coli. As an outer membrane protein, ompA could be incorporated into vesicles and is similarly to clyA also a good candidate for adressing protein domains to vesicles. <br />
<br />
<br />
Outer membrane protein A (OmpA) is a major protein in the Escherichia coli outer membrane. OmpA plays a vital structural role in ''E. coli'', and suggested that a perfect β-barrel structure of OmpA is important for outer membrane stability[http://www.ncbi.nlm.nih.gov/pubmed/11906175[1]]. OmpA is the most well-studied outer membrane protein in ''Escherichia coli''. This 325-residue protein was thought to contain two domains. The classic N-terminal domain, consisting of 171 amino acid residues, was shown to cross the membrane eight times in antiparallel β-strands with four relatively large and hydrophilic surface-exposed loops and short periplasmic turns[http://www.ncbi.nlm.nih.gov/pubmed/11276254[2]]. The C-terminal domain is located in the periplasm, and binds to the peptidoglycan thus connecting it to the outer membrane[http://www.ncbi.nlm.nih.gov/pubmed/8577259[3]]. The function of OmpA is thought to contribute to the structural integrity of the outer membrane<br />
along with murein lipoprotein[http://www.ncbi.nlm.nih.gov/pubmed/4261992[4]] and peptidoglycanassociated lipoprotein . In addition to its structural role, OmpA serves as a receptor of colicin and several phages[http://www.ncbi.nlm.nih.gov/pubmed/1574003[5]], and it's required in F-conjugation[http://www.ncbi.nlm.nih.gov/pubmed/321438[6]],[http://www.ncbi.nlm.nih.gov/pubmed/10368142[7]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{Template:Paris2009_guided|Addressing_overview3#bottom|Addressing_overview2_strategy#bottom}}<br />
<br />
<br />
====Bibliography:====<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11906175[1]] Ying Wang, (2002) The Function of OmpA in Escherichia coli, Biochem Biophys Res Commun.292(2):396-401<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11276254[2]] Arora, A., Abildgaard, F., Bushweller, J. H., and Tamm, L. K. (2001) Structure of outer membrane protein A transmembrane domain by NMR spectroscopy. Nat. Struct. Biol 8, 334–338.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/8577259[3]] Koebnik, R. (1995) Proposal for a peptidoglycan associating alpha-helical motif in the C-terminal regions of some bacterial cell-surface proteins. Mol. Microbiol. 16, 1269–1270.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/4261992[4]] Braun, V., and Bosch, V. (1972) Sequence of the mureinlipoprotein and the attachment site of the lipid. Eur. J. Biochem. 28, 51–69.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/1574003[5]] Lazzaroni, J.-C., and Portalier, R. (1992) The excC gene of Escherichia coli K-12 required for cell envelope integrity encodes the peptidoglycan-associated lipoprotein. Mol. Microbiol. 6, 735–742.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/321438[6] Schweizer, M., and Henning, U. (1977) Action of major outer cell envelope membrane protein in conjugation of Escherichia coli K-12. J. Bacteriol. 129, 1651–1652.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/10368142[7]] Koebnik, R. (1999) Structural and functional roles of the surfaceexposed loops of the β-barrel membrane protein OmpA from Escherichia coli. J. Bacteriol. 181, 3688–3694.</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview4Team:Paris/Addressing overview42009-10-21T22:12:19Z<p>Fanny.c: /* Adressin the message in the outer membrane : OmpA */</p>
<hr />
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<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#bottom"> Our strategy</a>|<br />
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<br />
<br />
<br />
[[Image:OmpA1.JPG|240px|left]][[Image:OmpA2.JPG|240px|right]]<br />
<br />
OmpA as previously been used in protein fusion to expose heterologuous protein domains to the surface of E. coli. As an outer membrane protein, ompA could be incorporated into vesicles and is similarly to clyA also a good candidate for adressing protein domains to vesicles. <br />
<br />
<br />
Outer membrane protein A (OmpA) is a major protein in the Escherichia coli outer membrane. OmpA plays a vital structural role in ''E. coli'', and suggested that a perfect β-barrel structure of OmpA is important for outer membrane stability[http://www.ncbi.nlm.nih.gov/pubmed/11906175[1]]. OmpA is the most well-studied outer membrane protein in ''Escherichia coli''. This 325-residue protein was thought to contain two domains. The classic N-terminal domain, consisting of 171 amino acid residues, was shown to cross the membrane eight times in antiparallel β-strands with four relatively large and hydrophilic surface-exposed loops and short periplasmic turns[http://www.ncbi.nlm.nih.gov/pubmed/11276254[2]]. The C-terminal domain is located in the periplasm, and binds to the peptidoglycan thus connecting it to the outer membrane[http://www.ncbi.nlm.nih.gov/pubmed/8577259[3]]. The function of OmpA is thought to contribute to the structural integrity of the outer membrane<br />
along with murein lipoprotein[http://www.ncbi.nlm.nih.gov/pubmed/4261992[4]] and peptidoglycanassociated lipoprotein . In addition to its structural role, OmpA serves as a receptor of colicin and several phages[http://www.ncbi.nlm.nih.gov/pubmed/1574003[5]], and it's required in F-conjugation[http://www.ncbi.nlm.nih.gov/pubmed/321438[6]],[http://www.ncbi.nlm.nih.gov/pubmed/10368142[7]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{Template:Paris2009_guided|Addressing_overview3#bottom|Addressing_overview2_strategy#bottom}}<br />
<br />
<br />
====Bibliography:====<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11906175[1]] Ying Wang, (2002) The Function of OmpA in Escherichia coli, Biochem Biophys Res Commun.292(2):396-401<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11276254[2]] Arora, A., Abildgaard, F., Bushweller, J. H., and Tamm, L. K. (2001) Structure of outer membrane protein A transmembrane domain by NMR spectroscopy. Nat. Struct. Biol 8, 334–338.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/8577259[3]] Koebnik, R. (1995) Proposal for a peptidoglycan associating alpha-helical motif in the C-terminal regions of some bacterial cell-surface proteins. Mol. Microbiol. 16, 1269–1270.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/4261992[4]] Braun, V., and Bosch, V. (1972) Sequence of the mureinlipoprotein and the attachment site of the lipid. Eur. J. Biochem. 28, 51–69.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/1574003[5]] Lazzaroni, J.-C., and Portalier, R. (1992) The excC gene of Escherichia coli K-12 required for cell envelope integrity encodes the peptidoglycan-associated lipoprotein. Mol. Microbiol. 6, 735–742.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/321438[6] Schweizer, M., and Henning, U. (1977) Action of major outer cell envelope membrane protein in conjugation of Escherichia coli K-12. J. Bacteriol. 129, 1651–1652.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/10368142[7]] Koebnik, R. (1999) Structural and functional roles of the surfaceexposed loops of the β-barrel membrane protein OmpA from Escherichia coli. J. Bacteriol. 181, 3688–3694.</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview4Team:Paris/Addressing overview42009-10-21T21:56:25Z<p>Fanny.c: /* Adressin the message in the outer membrane : OmpA */</p>
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[[Image:OmpA1.JPG|left]][[Image:OmpA2.JPG|right]]<br />
<br />
OmpA as previously been used in protein fusion to expose heterologuous protein domains to the surface of E. coli. As an outer membrane protein, ompA could be incorporated into vesicles and is similarly to clyA also a good candidate for adressing protein domains to vesicles. <br />
<br />
<br />
<br />
Outer membrane protein A (OmpA) is a major protein in the Escherichia coli outer membrane. OmpA plays a vital structural role in ''E. coli'', and suggested that a perfect β-barrel structure of OmpA is important for outer membrane stability[http://www.ncbi.nlm.nih.gov/pubmed/11906175[1]]. OmpA is the most well-studied outer membrane protein in ''Escherichia coli''. This 325-residue protein was thought to contain two domains. The classic N-terminal domain, consisting of 171 amino acid residues, was shown to cross the membrane eight times in antiparallel β-strands with four relatively large and hydrophilic surface-exposed loops and short periplasmic turns[http://www.ncbi.nlm.nih.gov/pubmed/11276254[2]]. The C-terminal domain is located in the periplasm, and binds to the peptidoglycan thus connecting it to the outer membrane[http://www.ncbi.nlm.nih.gov/pubmed/8577259[3]]. The function of OmpA is thought to contribute to the structural integrity of the outer membrane<br />
along with murein lipoprotein[http://www.ncbi.nlm.nih.gov/pubmed/4261992[4]] and peptidoglycanassociated lipoprotein . In addition to its structural role, OmpA serves as a receptor of colicin and several phages[http://www.ncbi.nlm.nih.gov/pubmed/1574003[5]], and it's required in F-conjugation[http://www.ncbi.nlm.nih.gov/pubmed/321438[6]],[http://www.ncbi.nlm.nih.gov/pubmed/10368142[7]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{Template:Paris2009_guided|Addressing_overview3#bottom|Addressing_overview2_strategy#bottom}}<br />
<br />
<br />
====Bibliography:====<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11906175[1]] Ying Wang, (2002) The Function of OmpA in Escherichia coli, Biochem Biophys Res Commun.292(2):396-401<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11276254[2]] Arora, A., Abildgaard, F., Bushweller, J. H., and Tamm, L. K. (2001) Structure of outer membrane protein A transmembrane domain by NMR spectroscopy. Nat. Struct. Biol 8, 334–338.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/8577259[3]] Koebnik, R. (1995) Proposal for a peptidoglycan associating alpha-helical motif in the C-terminal regions of some bacterial cell-surface proteins. Mol. Microbiol. 16, 1269–1270.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/4261992[4]] Braun, V., and Bosch, V. (1972) Sequence of the mureinlipoprotein and the attachment site of the lipid. Eur. J. Biochem. 28, 51–69.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/1574003[5]] Lazzaroni, J.-C., and Portalier, R. (1992) The excC gene of Escherichia coli K-12 required for cell envelope integrity encodes the peptidoglycan-associated lipoprotein. Mol. Microbiol. 6, 735–742.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/321438[6] Schweizer, M., and Henning, U. (1977) Action of major outer cell envelope membrane protein in conjugation of Escherichia coli K-12. J. Bacteriol. 129, 1651–1652.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/10368142[7]] Koebnik, R. (1999) Structural and functional roles of the surfaceexposed loops of the β-barrel membrane protein OmpA from Escherichia coli. J. Bacteriol. 181, 3688–3694.</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview4Team:Paris/Addressing overview42009-10-21T21:56:01Z<p>Fanny.c: /* Adressin the message in the outer membrane : OmpA */</p>
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<br />
<br />
<br />
[[Image:OmpA1.JPG|left]][[Image:OmpA2.JPG|right]]<br />
<br />
OmpA as previously been used in protein fusion to expose heterologuous protein domains to the surface of E. coli. As an outer membrane protein, ompA could be incorporated into vesicles and is similarly to clyA also a good candidate for adressing protein domains to vesicles. <br />
<br />
Outer membrane protein A (OmpA) is a major protein in the Escherichia coli outer membrane. OmpA plays a vital structural role in ''E. coli'', and suggested that a perfect β-barrel structure of OmpA is important for outer membrane stability[http://www.ncbi.nlm.nih.gov/pubmed/11906175[1]]. OmpA is the most well-studied outer membrane protein in ''Escherichia coli''. This 325-residue protein was thought to contain two domains. The classic N-terminal domain, consisting of 171 amino acid residues, was shown to cross the membrane eight times in antiparallel β-strands with four relatively large and hydrophilic surface-exposed loops and short periplasmic turns[http://www.ncbi.nlm.nih.gov/pubmed/11276254[2]]. The C-terminal domain is located in the periplasm, and binds to the peptidoglycan thus connecting it to the outer membrane[http://www.ncbi.nlm.nih.gov/pubmed/8577259[3]]. The function of OmpA is thought to contribute to the structural integrity of the outer membrane<br />
along with murein lipoprotein[http://www.ncbi.nlm.nih.gov/pubmed/4261992[4]] and peptidoglycanassociated lipoprotein . In addition to its structural role, OmpA serves as a receptor of colicin and several phages[http://www.ncbi.nlm.nih.gov/pubmed/1574003[5]], and it's required in F-conjugation[http://www.ncbi.nlm.nih.gov/pubmed/321438[6]],[http://www.ncbi.nlm.nih.gov/pubmed/10368142[7]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{Template:Paris2009_guided|Addressing_overview3#bottom|Addressing_overview2_strategy#bottom}}<br />
<br />
<br />
====Bibliography:====<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11906175[1]] Ying Wang, (2002) The Function of OmpA in Escherichia coli, Biochem Biophys Res Commun.292(2):396-401<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/11276254[2]] Arora, A., Abildgaard, F., Bushweller, J. H., and Tamm, L. K. (2001) Structure of outer membrane protein A transmembrane domain by NMR spectroscopy. Nat. Struct. Biol 8, 334–338.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/8577259[3]] Koebnik, R. (1995) Proposal for a peptidoglycan associating alpha-helical motif in the C-terminal regions of some bacterial cell-surface proteins. Mol. Microbiol. 16, 1269–1270.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/4261992[4]] Braun, V., and Bosch, V. (1972) Sequence of the mureinlipoprotein and the attachment site of the lipid. Eur. J. Biochem. 28, 51–69.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/1574003[5]] Lazzaroni, J.-C., and Portalier, R. (1992) The excC gene of Escherichia coli K-12 required for cell envelope integrity encodes the peptidoglycan-associated lipoprotein. Mol. Microbiol. 6, 735–742.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/321438[6] Schweizer, M., and Henning, U. (1977) Action of major outer cell envelope membrane protein in conjugation of Escherichia coli K-12. J. Bacteriol. 129, 1651–1652.<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/10368142[7]] Koebnik, R. (1999) Structural and functional roles of the surfaceexposed loops of the β-barrel membrane protein OmpA from Escherichia coli. J. Bacteriol. 181, 3688–3694.</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview3Team:Paris/Addressing overview32009-10-21T21:52:29Z<p>Fanny.c: /* Adressing the message in the outer membrane : ClyA */</p>
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<a class="menu_sub_active" href="https://2009.igem.org/Team:Paris/Addressing_overview3#bottom"> ClyA</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview4#bottom"> OmpA</a>|<br />
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<br />
We work on the cell-cell communication using vesicle:<br />
<br><br />
In this part we look into adressing a protein into the sender outer membrane that could be incoporated into outer membrane vesicles (OMVs). This protein would then be able to transmit a message after the fusion of OMVs with a receiver cell.<br />
<br />
In this direction ClyA (the cytolysine A of E.Coli) seems to be a good candidate. ClyA is one of the proteins that has been previously detected into OMVs and is known to be specificly exported to the outer membrane [[http://www.ncbi.nlm.nih.gov/pubmed/14532000 [3]]]. ClyA is thus expressed on bacteria and OMVs surface. Moreover, when ClyA is overproduced, it is accumulated into the periplasmic space [[http://www.ncbi.nlm.nih.gov/pubmed/14532000 [3]]].<br />
<br />
<br />
However there is an inconvenient to use this protein. ClyA is an alpha-Pore Forming Toxin (PFT). PFT are widely distributed proteins which form lesions in biological membranes. They exhibit their toxic effect in different manner. The first one is that ClyA allows the destruction of membrane permeability barrier. Furthermore, the toxic effect of ClyA could be explain by its capacity to deliver toxic component after the assembly of 8 or 13 of its subunits. PFTs can be subdivided into two classes; α-PFTs and β-PFTs, depending on the suspected mode of membrane integration, either by α-helical or β-sheet elements.[[http://www.ncbi.nlm.nih.gov/pubmed/19421192 [2]]]<br />
<br />
[[Image:Clya_simple.jpg|ClyA subunit|150px|left]] [[Image:Clya_structure2.jpg|ClyA assembled|100px|right]][[Image:ClyA.jpg|ClyA are assembling in outer membrane of a host cell|150px|center]] [[Image:Clya_structure.jpg|ClyA assembled|150px|center]]<br />
<br />
<br />
<br />
<br />
Some article argue that E.Coli K12 use this ClyA to lyse other cell (specially mamalian cell or eurcaryote cell). But E. coli cells expressing clyA do not lyse each other.<br />
<br />
<br />
<br />
Kim et al. have successfully fused clyA to GFP in order to observe vesicles [[http://www.ncbi.nlm.nih.gov/pubmed/18511069 [1]]], so we know that we can try to fuse clyA to a protein domain that would induce a signal transduction into the receiver cell. To see how we want to exploite clyA properties see [https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#Overview our strategy].<br />
<br />
<br />
'''Source:'''<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/18511069 1-]Kim, J.-Y. & DeLisa, M.P. Engineered bacterial outer membrane vesicles with enhanced functionality J.Mol. Biol. (2008) 380, 51–66<br />
<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/19421192 2-]Muller, M. & Ban, N. The structure of a cytolytic a-helical toxin pore reveals its assembly mechanism Nature (4 June 2009) 459, 726-730 <br />
<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/14532000 3-]Wai, S.N. & Lindmark, B. Vesicle-Mediated Export and Assembly of Pore-Forming Oligomers of the Enterobacterial ClyA Cytotoxin Cell (October 2003), 115,25-35<br />
<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/10383763 4-] Oscarsson, J. & Uhlin, B.E. Molecular analysis of the cytolytic protein ClyA (SheA) from Escherichia coli Molecular Microbiology (1999) 32(6), 1226–1238<br />
<br />
<br />
[http://jb.asm.org/cgi/content/abstract/182/22/6347 5-] Westermark, M. & Uhlin, B.E. Silencing and Activation of ClyA Cytotoxin Expression in Escherichia coli Journal of bacteriology,(Nov. 2000), Vol. 182, No. 22 6347–6357<br />
<br />
<br />
{{Template:Paris2009_guided|Addressing_overview2#bottom|Addressing_overview4#bottom}}</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview3Team:Paris/Addressing overview32009-10-21T21:13:05Z<p>Fanny.c: /* Adressing the message in the outer membrane : ClyA */</p>
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==Adressing the message in the outer membrane : ClyA==<br />
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<a class="menu_sub_active" href="https://2009.igem.org/Team:Paris/Addressing_overview3#bottom"> ClyA</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview4#bottom"> OmpA</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#bottom"> Our strategy</a>|<br />
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<br />
We work on the cell-cell communication using vesicle:<br />
<br><br />
To begin we need to adress a signal to the outer membrane, then exported into the outer membrane vesicles (OMVs). And finally, this protein will be able to transmit a message via the properties to fusion with the outer membrane of the target cell.<br />
<br />
In this direction ClyA (the cytolysine A of E.Coli) seems to have an interesting ways to correspond. Actually, ClyA is one of the proteins that has a high expression into OMVs, thanks to the type I pathway. This "pathways is one-step mechanisms by which the secreted proteins cross directly from the cytoplasm to the bacterial surface."[[http://www.ncbi.nlm.nih.gov/pubmed/14532000 [3]]]. Thanks to this mechanism : ClyA is expressed on bacteria and OMVs surface. Moreover, when ClyA is overproduced, it could be accumulated into the periplasmic space[[http://www.ncbi.nlm.nih.gov/pubmed/14532000 [3]]].<br />
<br />
<br />
However there are some inconvenient using this protein because ClyA is an alpha-PFT for Pore Forming Toxins. PFTs are potent virulence factors class starting in a soluble form to an outer membrane-integrated pore. They exhibit their toxic effect either by membrane permeability barrier destruction or by toxic components delivery through the pores which forming by several assembly 8 or 13 ClyA subunits. PFTs can be subdivided into two classes; α-PFTs and β-PFTs, depending on the suspected mode of membrane integration, either by α-helical or β-sheet elements.[[http://www.ncbi.nlm.nih.gov/pubmed/19421192 [2]]]<br />
[[Image:Clya_simple.jpg|ClyA subunit|150px|left]] [[Image:Clya_structure2.jpg|ClyA assembled|100px|right]][[Image:ClyA.jpg|ClyA are assembling in outer membrane of a host cell|150px|center]] [[Image:Clya_structure.jpg|ClyA assembled|150px|center]]<br />
<br />
<br />
<br />
<br />
So some article show that E.Coli K12 using this ClyA to lyse other cell (specially mamalian cell or eurcaryote cell). But this virulence was not show in same strain. <br />
<br />
<br />
<br />
<br />
In some article, it’s fused to GFP in order to observed the vesicle[[http://www.ncbi.nlm.nih.gov/pubmed/18511069 [1]]], so we think the fusion of ClyA with a peptide signal can induct the receptor when the vesicle fusion to its cell target liberate the Cly A in the target cell, or when ClyA on the OMVs interact with outer membrane receptor of the receiver cell. And it's this information which we will exploit for [https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#Overview our strategy] <br />
<br />
'''AVANTAGE'''<br />
<br />
- ClyA can be used to co-localize fully functional heterologous proteins directly in bacterial OMVs<br />
<br />
-We can fuse GFP to the C or N term of Cly A, to track OMVs easily.<br />
<br />
-ClyA is capable of co-localizing a variety of structurally diverse fusion partners to the surface of E. coli and their released vesicles, but only when the periplasmic disulfide bond-forming machinery was present ,it’s makes OMVs an ideal structure to transport hydrophobic compounds like membrane proteins into the host.<br />
<br />
-Cly A confers vesicle binding to and invasion of host cells.[[http://www.ncbi.nlm.nih.gov/pubmed/18511069 [1]]] <br />
<br />
-ClyA was significantly enriched in OMVs relative to other lumenal and membrane bound OMV proteins.<br />
<br />
<br />
'''DRAWBACK'''<br />
<br />
-Cly A is a alpha-PFT; it can form pore in cell target. But we find ClyA is virulent for mammalian cell or erythrocytes only[[http://www.ncbi.nlm.nih.gov/pubmed/14532000 [3]]], because of its strong interaction with choloesterol which constitute mammalian cell membrane. For the virulence in bacteria cell we think that it’s not possible because there is no cholesterol in the bacteria membrane. <br />
<br />
<br />
'''INTERESTING QUOTATIONS:'''<br />
<br />
- "unfused ClyA accumulated in the cytoplasm, periplasm and OMV fractions."[[http://www.ncbi.nlm.nih.gov/pubmed/18511069|[1]]]<br />
<br />
<br />
<br />
-"It may also be possible to use this molecule as a model system to develop predictive rules that will aid in understanding of molecular events that govern related cellular processes such as membrane fusion of cellular compartments and viral membrane fusion."[[http://www.ncbi.nlm.nih.gov/pubmed/19421192|[2]]]<br />
<br />
<br />
'''Source:'''<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/18511069 1-]Kim, J.-Y. & DeLisa, M.P. Engineered bacterial outer membrane vesicles with enhanced functionality J.Mol. Biol. (2008) 380, 51–66<br />
<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/19421192 2-]Muller, M. & Ban, N. The structure of a cytolytic a-helical toxin pore reveals its assembly mechanism Nature (4 June 2009) 459, 726-730 <br />
<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/14532000 3-]Wai, S.N. & Lindmark, B. Vesicle-Mediated Export and Assembly of Pore-Forming Oligomers of the Enterobacterial ClyA Cytotoxin Cell (October 2003), 115,25-35<br />
<br />
<br />
[http://www.ncbi.nlm.nih.gov/pubmed/10383763 4-] Oscarsson, J. & Uhlin, B.E. Molecular analysis of the cytolytic protein ClyA (SheA) from Escherichia coli Molecular Microbiology (1999) 32(6), 1226–1238<br />
<br />
<br />
[http://jb.asm.org/cgi/content/abstract/182/22/6347 5-] Westermark, M. & Uhlin, B.E. Silencing and Activation of ClyA Cytotoxin Expression in Escherichia coli Journal of bacteriology,(Nov. 2000), Vol. 182, No. 22 6347–6357<br />
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{{Template:Paris2009_guided|Addressing_overview2#bottom|Addressing_overview4#bottom}}</div>Fanny.chttp://2009.igem.org/Team:Paris/Addressing_overview3Team:Paris/Addressing overview32009-10-21T20:40:12Z<p>Fanny.c: /* Adressing the message in the outer membrane : ClyA */</p>
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==Adressing the message in the outer membrane : ClyA==<br />
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<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2#bottom"> Main </a>|<br />
<a class="menu_sub_active" href="https://2009.igem.org/Team:Paris/Addressing_overview3#bottom"> ClyA</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview4#bottom"> OmpA</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#bottom"> Our strategy</a>|<br />
<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Addressing_overview_Construction#bottom"> Construction</a><br />
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We work on the cell-cell communication using vesicle:<br />
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Firstly we need to adress a signal to the outer membrane, then exported into vesicles. And finally, this protein will be able to transmit a message via the properties to fusion with the outer membrane of the target cell.<br />
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So ClyA (of ''E.Coli'') seems to have an interesting ways to succeed. Actually, ClyA has a high expression into OMVs, and it’s one of the proteins that can pass the cytoplasm to the outer membrane and integrated to vesicle easily utilizing the type I pathway( this "pathways is one-step mechanisms by which the secreted proteins cross directly from the cytoplasm to the bacterial surface."[[http://www.ncbi.nlm.nih.gov/pubmed/14532000 [3]]]). Thus ClyA is expressed on bacteria and OMVs surface. However when ClyA is overproduced, it could accumulate in the periplasmic space[[http://www.ncbi.nlm.nih.gov/pubmed/14532000 [3]]].<br />
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However there are some inconvenient using this protein because ClyA is an alpha-PFT for Pore Forming Toxins. PFTs are potent virulence factors class starting in a soluble form to an outer membrane-integrated pore. They exhibit their toxic effect either by membrane permeability barrier destruction or by toxic components delivery through the pores which forming by several assembly 8 or 13 ClyA subunits. PFTs can be subdivided into two classes; α-PFTs and β-PFTs, depending on the suspected mode of membrane integration, either by α-helical or β-sheet elements.[[http://www.ncbi.nlm.nih.gov/pubmed/19421192 [2]]]<br />
[[Image:Clya_simple.jpg|ClyA subunit|150px|left]] [[Image:Clya_structure2.jpg|ClyA assembled|100px|right]][[Image:ClyA.jpg|ClyA are assembling in outer membrane of a host cell|150px|center]] [[Image:Clya_structure.jpg|ClyA assembled|150px|center]]<br />
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So some article show that E.Coli K12 using this ClyA to lyse other cell (specially mamalian cell or eurcaryote cell). But this virulence was not show in same strain. <br />
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In some article, it’s fused to GFP in order to observed the vesicle[[http://www.ncbi.nlm.nih.gov/pubmed/18511069 [1]]], so we think the fusion of ClyA with a peptide signal can induct the receptor when the vesicle fusion to its cell target liberate the Cly A in the target cell, or when ClyA on the OMVs interact with outer membrane receptor of the receiver cell. And it's this information which we will exploit for [https://2009.igem.org/Team:Paris/Addressing_overview2_strategy#Overview our strategy] <br />
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'''AVANTAGE'''<br />
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- ClyA can be used to co-localize fully functional heterologous proteins directly in bacterial OMVs<br />
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-We can fuse GFP to the C or N term of Cly A, to track OMVs easily.<br />
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-ClyA is capable of co-localizing a variety of structurally diverse fusion partners to the surface of E. coli and their released vesicles, but only when the periplasmic disulfide bond-forming machinery was present ,it’s makes OMVs an ideal structure to transport hydrophobic compounds like membrane proteins into the host.<br />
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-Cly A confers vesicle binding to and invasion of host cells.[[http://www.ncbi.nlm.nih.gov/pubmed/18511069 [1]]] <br />
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-ClyA was significantly enriched in OMVs relative to other lumenal and membrane bound OMV proteins.<br />
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'''DRAWBACK'''<br />
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-Cly A is a alpha-PFT; it can form pore in cell target. But we find ClyA is virulent for mammalian cell or erythrocytes only[[http://www.ncbi.nlm.nih.gov/pubmed/14532000 [3]]], because of its strong interaction with choloesterol which constitute mammalian cell membrane. For the virulence in bacteria cell we think that it’s not possible because there is no cholesterol in the bacteria membrane. <br />
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'''INTERESTING QUOTATIONS:'''<br />
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- "unfused ClyA accumulated in the cytoplasm, periplasm and OMV fractions."[[http://www.ncbi.nlm.nih.gov/pubmed/18511069|[1]]]<br />
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-"It may also be possible to use this molecule as a model system to develop predictive rules that will aid in understanding of molecular events that govern related cellular processes such as membrane fusion of cellular compartments and viral membrane fusion."[[http://www.ncbi.nlm.nih.gov/pubmed/19421192|[2]]]<br />
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'''Source:'''<br />
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[http://www.ncbi.nlm.nih.gov/pubmed/18511069 1-]Kim, J.-Y. & DeLisa, M.P. Engineered bacterial outer membrane vesicles with enhanced functionality J.Mol. Biol. (2008) 380, 51–66<br />
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[http://www.ncbi.nlm.nih.gov/pubmed/19421192 2-]Muller, M. & Ban, N. The structure of a cytolytic a-helical toxin pore reveals its assembly mechanism Nature (4 June 2009) 459, 726-730 <br />
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[http://www.ncbi.nlm.nih.gov/pubmed/14532000 3-]Wai, S.N. & Lindmark, B. Vesicle-Mediated Export and Assembly of Pore-Forming Oligomers of the Enterobacterial ClyA Cytotoxin Cell (October 2003), 115,25-35<br />
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[http://www.ncbi.nlm.nih.gov/pubmed/10383763 4-] Oscarsson, J. & Uhlin, B.E. Molecular analysis of the cytolytic protein ClyA (SheA) from Escherichia coli Molecular Microbiology (1999) 32(6), 1226–1238<br />
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[http://jb.asm.org/cgi/content/abstract/182/22/6347 5-] Westermark, M. & Uhlin, B.E. Silencing and Activation of ClyA Cytotoxin Expression in Escherichia coli Journal of bacteriology,(Nov. 2000), Vol. 182, No. 22 6347–6357<br />
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