Team:INSA-Lyon/Project/Stage3/Strategy

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<h3>Medias ansd Public speaks about <em> SynBio </em></h3> <br>
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<p>In the way to see how much people know about SynBio, Eleonore Pauwels, research scholar working in the foresight and governance project at the Woodrow Wilson International Center for Scholars in Washington D.C., has made a public investigation for the “Ethical Aspects of Synthetic Biology” table-round, organized by the European union. It appears that SynBio just begin to be an integrative process. <br>
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<h2>Strategy</h2>
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The first way to introduce <em>SynBio</em> to people is the newspapers. During the last few years, the number of articles published in American or European press has been increasing. And Europe is more concerned than the United States: more than 100 articles published in European press in five years, whereas about 50 in the USA. In Europe, France and United Kingdom are the leaders.
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<p> We chose to follow two differents strategies to obtain our systems of regulation.<br /></p>
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However, they have a different approach to SynBio. The US articles refer more about benefits of SynBio, and the topics are focused on Energy and Health. The European vision is more prudent: they prefer focusing on the ratio benefits/risks, in environment and energetic domains. And these differences get higher when they evoke the type of risks. American Medias just focus on the biosecurity, while European ones write about biosafety, biosecurity and ethics.<br>
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<h3><font color="purple">Thermoregulation</font></h3>
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Despite the facts that newspapers are trying to make <em>SynBio</em> a popular knowledge, focusing on people’s preoccupations, this subject doesn’t seem to be attractive. In an investigation realized in the US, more than 70% of the interrogated persons had never heard about <em>SynBio</em>. Ignorance is one of the big problems of <em>SynBio</em> because when people don’t know about something, they’re scared and cautious about it. And their principal preoccupation is the ratio benefits/risks. . Moreover, people can give importance to a fact they hear in medias for example, and that is a prejudice for SynBio and genetic modification. People have been informed of some scientific failures and as they know the limits and the safety of SynBio, they stay very cautious. Currently, nobody really knows if <em>SynBio</em> is safe and can have limits… <br>
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In our opinion, people are afraid about scientific progress. Because it’s complicated, and it appears to have some catastrophic consequences, people don’t trust very much in the scientists, in spite of the communication in the newspapers or the conferences.<br>
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<p>For this system of regulation, we decided to synthesized directly the sequence of the curli promoter with the igem restriction sites. In the same time a part with the gene ompR234 is created by PCR, from the genome of the chassi PHL818. You can find <a href="https://2010.igem.org/Team:INSA-Lyon/Project/Stage3/Strategy/Designcurli">here</a> some details about the design of these sequences.<br /></p>
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<p>Then we ligate an igem reporter gene with the curli promoter and we wanted to make a double transformation with ompR234 to see if our parts work as we hoped. <br/></p><br/>
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<a href="http://lh5.ggpht.com/_Uc3bmii-yi0/TMg58qKi8EI/AAAAAAAAAms/Fmtlk3nJ144/s912/Curli_GFP.png">
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<img class="image"src="http://lh3.ggpht.com/_zZap34AU7o8/TMfUhgHZylI/AAAAAAAAABo/Uuvk6X8bV8I/s800/curli%2014K.png"
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alt="curli 14K ou curli 22B" title="Curli promoter and reporter gene ligation" />
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<img class="image2"src="http://lh5.ggpht.com/_Uc3bmii-yi0/TMg59gvCO0I/AAAAAAAAAm4/EWBiy2sfqwk/Curli%20GFP_finale.png" alt="construction finale" title="final contruction">
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<p style="font-size:0.9em; text-indent:0px; margin-left: 300px;text-align:left"> <br/><em>Curli promoter and reporter gene ligation </em><br/><br/></p>
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<a href="http://lh4.ggpht.com/_zZap34AU7o8/TMfUg5CxIeI/AAAAAAAAABg/GEPJb3PV4z0/s800/18A%20OmpR234.png">
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<img class="image"src="http://lh4.ggpht.com/_zZap34AU7o8/TMfUg5CxIeI/AAAAAAAAABg/GEPJb3PV4z0/s800/18A%20OmpR234.png"
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alt="Constitutive promoter and ompR234 gene ligation" title="Constitutive promoter and ompR234 gene ligation" />
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<img src="http://lh6.ggpht.com/_Uc3bmii-yi0/TMg9-ve_0mI/AAAAAAAAAnE/QsHQjo9Giz0/18A%20OmpR234.png" alt="construction finale" title="final construction"><br />
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<p style="font-size:0.9em; text-indent:0px; margin-left: 300px;text-align:left"><br /><br /><em>Constitutive promoter and ompR234 gene ligation </em><br/><br/></p><br /><br />
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<p>The final plasmid construction of the thermometer RNA will contain a constitutive promoter(BBa_J23119), the thermometer RNA, the PPI-protein fused and a terminator.
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<a href="http://lh3.ggpht.com/_Uc3bmii-yi0/TMg5863f6QI/AAAAAAAAAmw/h_PnBEphSx0/s512/18A%201N%20PPI.png">
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<img class="image"src="http://lh3.ggpht.com/_Uc3bmii-yi0/TMg5863f6QI/AAAAAAAAAmw/h_PnBEphSx0/s512/18A%201N%20PPI.png"
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alt="Phasin-intein construction" title="Phasin-intein construction" />
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<img class="image2"src="http://lh4.ggpht.com/_Uc3bmii-yi0/TMhFUQhiSLI/AAAAAAAAAnY/a75XZT3PTdM/18A%201N%20PPi_finale.png" alt="construction finale" title="final contruction">
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<p style="font-size:0.9em; text-indent:0px; margin-left: 300px;text-align:left"> <br/><em>Phasin-intein construction</em><br/><br/></p><br/><br/>
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<h3><font style="color:purple">Control under Arabinose</font></h3><br>
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Reading people interview, <em>SynBio</em> appears like the new madness of the scientists, who want to play God and don’t care about ethics and people. Man likes controlling everything, and be able to control life is certainly the most powerful capacity. And we know how much Man loves power… Not only scientists by the way, since SynBio and its consequences imply everybody, from Politicians to citizens.
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<p>In this part we decided to realize three different constructions to control the regulation.  
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That’s why ethical framework is important to reassure people and give back some trust in science, and especially biology. All the polemics about GM should be avoided if some ethical works had been realized. But now it’s time to have a new start, and make SynBio a way to develop an integrative science, with the help of the political community.<br>
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The first one consists in the constitutive production of LuxR and LuxI proteins to control the promoter LuxR/HSL.<br /></p> <br/>
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<p>The second one is the construction with the promoter LuxR/HSL to control the expression of the operon PhaCAB.
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To finish a construction with Pbad/araC, which is a promoter whose activity depends on addition of arabinose in medium. In this case, the promoter influences the synthesis of a cI repressor and a phasin-intein construction. The cI repressor is isolated from bacteriophage λ and negatively regulates the activity of the LuxR/HSL regulated promoter.
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You can find more details on the three constructions <a href="https://2010.igem.org/wiki/index.php?title=Team:INSA-Lyon/Project/Stage3/Strategy/construction1">here</a>.
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<p style="text-align:center;"><a href="#top">Top of Page</a></p>
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Latest revision as of 20:47, 27 October 2010





Strategy


We chose to follow two differents strategies to obtain our systems of regulation.



Thermoregulation


For this system of regulation, we decided to synthesized directly the sequence of the curli promoter with the igem restriction sites. In the same time a part with the gene ompR234 is created by PCR, from the genome of the chassi PHL818. You can find here some details about the design of these sequences.

Then we ligate an igem reporter gene with the curli promoter and we wanted to make a double transformation with ompR234 to see if our parts work as we hoped.


curli 14K ou curli 22B construction finale


Curli promoter and reporter gene ligation



Constitutive promoter and ompR234 gene ligation construction finale



Constitutive promoter and ompR234 gene ligation



The final plasmid construction of the thermometer RNA will contain a constitutive promoter(BBa_J23119), the thermometer RNA, the PPI-protein fused and a terminator.



Phasin-intein construction construction finale


Phasin-intein construction





Control under Arabinose



In this part we decided to realize three different constructions to control the regulation. The first one consists in the constitutive production of LuxR and LuxI proteins to control the promoter LuxR/HSL.


The second one is the construction with the promoter LuxR/HSL to control the expression of the operon PhaCAB. To finish a construction with Pbad/araC, which is a promoter whose activity depends on addition of arabinose in medium. In this case, the promoter influences the synthesis of a cI repressor and a phasin-intein construction. The cI repressor is isolated from bacteriophage λ and negatively regulates the activity of the LuxR/HSL regulated promoter. You can find more details on the three constructions here.


Top of Page

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