Difference between revisions of "Team:Toulouse-INSA-UPS/Parts"

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       <th>State</th>
 
       <th>State</th>
 
     </tr>
 
     </tr>
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<h2 class="heavy">Parts deposed on the registry</h2>
+
<h2 class="heavy">Parts Deposited On the Registry</h2>
 
<p> Note that we delibatately chose to submit only our validated functional parts to the registry.
 
<p> Note that we delibatately chose to submit only our validated functional parts to the registry.
 
</p>
 
</p>
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     <tr>
 
     <tr>
 
       <td></html><partinfo>BBa_K2668010</partinfo><html></td>
 
       <td></html><partinfo>BBa_K2668010</partinfo><html></td>
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       <td>CERBERUS: binding of biotinylated compounds, binding to cellulose, can covalently bind Alkynes decorated molecules</td>
+
       <td>CERBERUS: binding of biotinylated compounds, binding to cellulose, can covalently bind alkyne decorated molecules</td>
 
       <td>basic </td>
 
       <td>basic </td>
 
       <td>working</td>
 
       <td>working</td>
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</div>
 
</div>
 
<p>
 
<p>
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This part results from the fusion of Scygonadin, an antimicrobial peptide from the mudcrab <i>Scylla paramamosain</i>, with an AviTag, a short peptidic sequence recognized by BirA in order to biotinylate proteins <i>in vivo</i>. It was used to prove the affinity of Cerberus to biotinylated compounds and to check if they keep their biological activity thereafter.
+
This part results in the fusion of scygonadin, an antimicrobial peptide from the mudcrab <i>Scylla paramamosain</i>, with an Avi-tag, a short peptidic sequence recognized by BirA in order to biotinylate proteins <i>in vivo</i>. It was used to prove the affinity of Cerberus to biotinylated compounds and to check if they keep their biological activity thereafter.
 
Scygonadin was planed to be produced in <i>Pichia pastoris</i> since as an eukaryotic, microorganism, the production of an antibacterial peptide should not harm it.  
 
Scygonadin was planed to be produced in <i>Pichia pastoris</i> since as an eukaryotic, microorganism, the production of an antibacterial peptide should not harm it.  
 
 
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<figure class="figure"  style="text-align:center;">
 
<figure class="figure"  style="text-align:center;">
 
<img style="width : 70%; heigth = auto;" src="https://static.igem.org/mediawiki/2018/f/f1/T--Toulouse-INSA-UPS--Parts--Youn--RFP.png" class="figure-img img-fluid rounded" alt="A generic square placeholder image with rounded corners in a figure.">
 
<img style="width : 70%; heigth = auto;" src="https://static.igem.org/mediawiki/2018/f/f1/T--Toulouse-INSA-UPS--Parts--Youn--RFP.png" class="figure-img img-fluid rounded" alt="A generic square placeholder image with rounded corners in a figure.">
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<figcaption class="figure-caption"><strong>Figure 2:</strong> <i>mRFP1 Construction</i></figcaption>
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<figcaption class="figure-caption"><strong>Figure 2:</strong> <i>mRFP1 construction</i></figcaption>
 
</figure>
 
</figure>
 
</div>
 
</div>
 
<p>
 
<p>
−
This part was used as a negative control for the affinity of Sirius towards cellulose. Its production was achieved under the exact same conditions as Sirius. The designed part was cloned into pET28 which includes an HisTag and can therefore allow us to purify it using Cobalt column. It is always a pleasure to produce red proteins as it is very beautiful!
+
This part was used as a negative control for the affinity of Sirius towards cellulose. Its production was achieved under the exact same conditions as Sirius. The designed part was cloned into pET28 which includes an His-tag and can therefore allow us to purify it using cobalt column. It is always a pleasure to produce red proteins as it is very beautiful!
 
</p>
 
</p>
 
<br/>
 
<br/>
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</div>
 
</div>
 
<p>
 
<p>
−
         BirA is the <i>E. coli</i> biotin ligase and has a high affinity for AviTag, a short peptidic sequence. It was co-expressed with mTagBFP and Scygonadin for <i>in vivo</i> biotinylation. We used the pETDuet-1 for its production in <i>E. coli</i>. We got inspired by this system for the design of a double protein expression plasmid in <i>Pichia pastoris</i> for that we used the pPICZalpha to clone BirA into it and cloned the function to be biotinylated into the pGAPZalpha. Then we cloned the pGAP expression cassette (containing the CDS fused to an AviTag, a promoter and a terminator)  
+
         BirA is the <i>E. coli</i> biotin ligase and has a high affinity for Avi-tag, a short peptidic sequence. It was co-expressed with mTagBFP and scygonadin for <i>in vivo</i> biotinylation. We used the pETDuet-1 for its production in <i>E. coli</i>. We got inspired by this system for the design of a double protein expression plasmid in <i>Pichia pastoris</i> for that we used the pPICZalpha to clone BirA into it and cloned the function to be biotinylated into the pGAPZalpha. Then we cloned the pGAP expression cassette (containing the CDS fused to an Avi-tag, a promoter and a terminator)  
 
         </p>
 
         </p>
 
<br/>
 
<br/>
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<figure class="figure"  style="text-align:center;">
 
<figure class="figure"  style="text-align:center;">
 
<img style="width : 70%; heigth = auto;" src="https://static.igem.org/mediawiki/2018/e/ef/T--Toulouse-INSA-UPS--Parts--Youn--CerbTetra.png" class="figure-img img-fluid rounded" alt="A generic square placeholder image with rounded corners in a figure.">
 
<img style="width : 70%; heigth = auto;" src="https://static.igem.org/mediawiki/2018/e/ef/T--Toulouse-INSA-UPS--Parts--Youn--CerbTetra.png" class="figure-img img-fluid rounded" alt="A generic square placeholder image with rounded corners in a figure.">
−
<figcaption class="figure-caption"><strong>Figure 4:</strong><i> Cerberus Tetrameric construction</i></figcaption>
+
<figcaption class="figure-caption"><strong>Figure 4:</strong><i> Cerberus tetrameric construction</i></figcaption>
 
</figure>
 
</figure>
 
</div>
 
</div>
 
<p>
 
<p>
−
Cerberus was initially designed to use the classic Streptavidin, but as this version is tetrameric the choice was made to design a monomeric version using an enginered version. It was a good choice because the tetrameric version did not worked...   
+
Cerberus was initially designed to use the classic tetrameric streptavidin, but we chose to also design a monomeric version using an engineered version. It was a good choice because the tetrameric version did not work...   
 
</p>
 
</p>
 
<br/>
 
<br/>

Revision as of 20:35, 16 October 2018

Parts

Parts Deposited On the Registry

Note that we delibatately chose to submit only our validated functional parts to the registry.

See our Basic Part page

Name Function Type State
<partinfo>BBa_K2668010</partinfo> CERBERUS: binding of biotinylated compounds, binding to cellulose, can covalently bind alkyne decorated molecules basic working
<partinfo>BBa_K2668020</partinfo> SIRIUS: strong binding to cellulose basic working
<partinfo>BBa_K2668070</partinfo> BFP-AviTag: fluorescence in the blue wavelength, can be biotinylated basic working

Parts used on the project but not deposited on the registry

Scygonadin

A generic square placeholder image with rounded corners in a figure.
Figure 1: Scygonadin construction

This part results in the fusion of scygonadin, an antimicrobial peptide from the mudcrab Scylla paramamosain, with an Avi-tag, a short peptidic sequence recognized by BirA in order to biotinylate proteins in vivo. It was used to prove the affinity of Cerberus to biotinylated compounds and to check if they keep their biological activity thereafter. Scygonadin was planed to be produced in Pichia pastoris since as an eukaryotic, microorganism, the production of an antibacterial peptide should not harm it.


mRFP1

A generic square placeholder image with rounded corners in a figure.
Figure 2: mRFP1 construction

This part was used as a negative control for the affinity of Sirius towards cellulose. Its production was achieved under the exact same conditions as Sirius. The designed part was cloned into pET28 which includes an His-tag and can therefore allow us to purify it using cobalt column. It is always a pleasure to produce red proteins as it is very beautiful!


BirA

A generic square placeholder image with rounded corners in a figure.
Figure 3: BirA contruction

BirA is the E. coli biotin ligase and has a high affinity for Avi-tag, a short peptidic sequence. It was co-expressed with mTagBFP and scygonadin for in vivo biotinylation. We used the pETDuet-1 for its production in E. coli. We got inspired by this system for the design of a double protein expression plasmid in Pichia pastoris for that we used the pPICZalpha to clone BirA into it and cloned the function to be biotinylated into the pGAPZalpha. Then we cloned the pGAP expression cassette (containing the CDS fused to an Avi-tag, a promoter and a terminator)


Cerberus Tetrameric

A generic square placeholder image with rounded corners in a figure.
Figure 4: Cerberus tetrameric construction

Cerberus was initially designed to use the classic tetrameric streptavidin, but we chose to also design a monomeric version using an engineered version. It was a good choice because the tetrameric version did not work...


No dogs were harmed over the course of this iGEM project.

INSA Toulouse UPS LISBP TWB Defi Adisseo Foundation ESF Catalyses


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