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

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<section>
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    <h1>Existing Parts we have contributed to characterized</h1>
  
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   <h2>Vh1: First part of <i>Vibrio harveyi</i> gene circuit</h2>
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   <h2></html><partinfo>BBa_J04450</partinfo><html>: RFP coding device </h2>
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  <figure>
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    <figure>
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       <img src="https://static.igem.org/mediawiki/parts/1/14/T--INSA-UPS_France--J04450.png
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      <img src="https://static.igem.org/mediawiki/parts/1/1e/T--INSA-UPS_France--Vh1.png" alt="">
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      <figcaption>
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      fig Vh1: first part of <i>Vibrio harveyi</i> gene circuit
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      </figcaption>
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    </figure>
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    <p>
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    This part contains the first half of <i>Vibrio harveyi</i> quorum CqsS receptor which is able to detect CAI-1 family molecule. The receptor is driven by a constitutive promoter.
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This part has been designed due to IDT gBlocks synthesis requirements. The ordered gBlocks size should not exceed 3kb (the total length of the <i>Vibrio harveyi</i> circuit is 6929 bp). The XhoI restriction site naturally present in the gene was used reassemble the <i>cqsS*</i> sequence. Therefore, it was impossible to use pSB1C3 vector because of the presence of a XhoI restriction on the vector. This part was then subcloned in pBR322.
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    </p>
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+
−
    <p>
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      <b>To learn more: </b> see our <a href="https://2017.igem.org/Team:INSA-UPS_France/Design">design</a>, and our<a href="https://2017.igem.org/Team:INSA-UPS_France/Results"> results</a>
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  <h2>Vh 2 : Second part of <i>Vibrio harveyi</i> gene circuit.
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  </h2>
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    <figure>
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       <img src="https://static.igem.org/mediawiki/parts/9/9f/T--INSA-UPS_France--Vh2.png
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" alt="">
 
" alt="">
 
       <figcaption>
 
       <figcaption>
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    Vh2 : second part of <i>Vibrio harveyi</i> gene circuit
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        BBa_J04450 biobrick conjugated in <i>Vibrio harveyi</i>
 
       </figcaption>
 
       </figcaption>
 
     </figure>
 
     </figure>
  
 
     <p>
 
     <p>
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    This part contains the second half of <i>Vibrio harveyi</i> quorum CqsS* receptor which is able to detect CAI-1 family molecule. It also contains the tetR repressor under the control of pQRR4 promoter.
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      BBa_J04450 was tested in the <i>Vibrio harveyi</i> background. BBa_J04450 biobrick was cloned in a broad host range plasmid (pBBR1MCS-4) and conjugated into <i>Vibrio harveyi</i> to demonstrate the production of RFP in this chassis.  
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This part has been designed due to IDT gBlocks synthesis requirements. The ordered gBlocks size should not exceed 3kb (the total length of the <i>Vibrio harveyi</i> circuit is 6929 bp). The ApaI (not present on pSB1C3 vector) and XhoI restriction (naturally present in the <i>cqsS*</i> gene) were used to reassemble the synthetic circuit.  
+
 
     </p>
 
     </p>
  
 
     <p>
 
     <p>
−
       <b>To learn more: </b> </html><partinfo>BBa_K431009</partinfo><html>,<a href="https://2017.igem.org/Team:INSA-UPS_France/Results">results</a>
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       <b>To learn more: </b> </html><partinfo>BBa_J04450</partinfo><html>, <a href="https://2017.igem.org/Team:INSA-UPS_France/Results">results</a>
 +
</p>
  
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  <h2></html><partinfo>BBa_K431009</partinfo><html>: glyceraldehyde 3-phosphate dehydrogenase promoter (pGAP)</h2>
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<h2>Vh 1+2: <i>Vibrio harveyi</i> invertor system </h2>
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     <figure>
 
     <figure>
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       <img src="https://static.igem.org/mediawiki/parts/a/ac/T--INSA-UPS_France--harveyi1%2B2.pn" alt="">
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       <img src="https://static.igem.org/mediawiki/parts/7/77/T--INSA-UPS_France--pGAP-D-NY15.png" alt="">
 
       <figcaption>
 
       <figcaption>
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      fig: Vh 1+2: <i>Vibrio harveyi</i> invertor system
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      Figure 2: construction to characterized BBa_K431009
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+
 
       </figcaption>
 
       </figcaption>
 
     </figure>
 
     </figure>
  
 
     <p>
 
     <p>
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      This part includes the complete <i>cqsS*</i> gene driven by a constitutive promoter and the tetracyclin repressor under the control of pQRR4 promoter which activation depends on CqsS* detection of quorum sensing inducers.
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    The pGAP promoter was used as a constitutive promoter and as a homology region to make easier the genomic recombination of our synthetic construction in <i>Pichia pastoris</i> genome.  
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Vh1+2 is the assembly of Vh2 in Vh1. This part was cloned in pBR322.  
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The promoter was characterized by RT-qPCR using <i>d-ny15</i> as reporter gene.  
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     </p>
 
     </p>
  
 
     <p>
 
     <p>
−
       <b>see our <a href="https://2017.igem.org/Team:INSA-UPS_France/Design">design</a>, and our<a href="https://2017.igem.org/Team:INSA-UPS_France/Results"> results</a>
+
       <b>To learn more: </b> </html><partinfo>BBa_K431009</partinfo><html> , <a href="https://2017.igem.org/Team:INSA-UPS_France/Results">results</a>
  
  
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<h2><i>Vibrio harveyi</i> complete gene circuit: </h2>
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  <h2></html><partinfo>BBa_K1800001</partinfo><html>: alpha factor secretion signal </h2>
  
 
     <figure>
 
     <figure>
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       <img src="https://static.igem.org/mediawiki/parts/3/37/T--INSA-UPS_France--harveyi1%2B2%2B3.png
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       <img src="https://static.igem.org/mediawiki/parts/1/19/T--INSA-UPS_France--K1800001.png" alt="">
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" alt="">
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       <figcaption>
 
       <figcaption>
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      fig : <i>Vibrio harveyi</i> complete gene circuit:
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      fig 1 : construction to characterized BBa_K1800001
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+
 
       </figcaption>
 
       </figcaption>
 
     </figure>
 
     </figure>
  
 
     <p>
 
     <p>
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      This part includes the complete <i>cqsS</i> gene driven by a constitutive promoter and the tetracycline repressor (TetR) under the control of pQRR4 promoter which activation depends on CqsS* recognition of quorum sensing inducers. The <i>als</i> gene, implied in the diacetyl production pathway, is under the control of pTet which can be repressed by TetR
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The α-factor sequence contains a kozak region and a signal sequence to secrete the produced peptides. The functionality of the signal factor was investigated by demonstrating that AMP are present in the supernatant through a toxicity assay.  
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This part is the assemblage of Vh1, Vh2 and BBa_K2278001. It allows to produce diacetyl is produced upon detection of V. cholerae quorum sensing molecule CAI-1.
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The <i>als</i> gene is flanked with two restriction enzyme sites to clone a reporter gene such as <i>rfp</i>.  
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The assembly of this part was not released due to technical and time issues.
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     </p>
 
     </p>
  
 
     <p>
 
     <p>
−
      <b>To learn more: </b> see our <a href="https://2017.igem.org/Team:INSA-UPS_France/Design">design</a>, and our<a href="https://2017.igem.org/Team:INSA-UPS_France/Results"> results</a>
+
       <b>To learn more: </b> </html><partinfo>BBa_K1800001</partinfo><html>,<a href="https://2017.igem.org/Team:INSA-UPS_France/Results">results</a>
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<h2><i>Pichia pastoris</i> D-NY15 AMP generator:
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</h2>
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    <figure>
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−
      <img src="https://static.igem.org/mediawiki/parts/7/77/T--INSA-UPS_France--pGAP-D-NY15.png
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" alt="">
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      <figcaption>
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      fig <i>Pichia pastoris</i> D-NY15 AMP generator:
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      </figcaption>
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    </figure>
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    <p>
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      This system allowed us to constitutively express BBa_K2278021 with a yeast promoter. This system was used to produce AMP at the highest concentration
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The pGAP promoter ensures genome recombination in <i>Pichia pastoris</i> genome. The α-factor sequence contains a RBS and a signal sequence to secrete the produced peptides
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The restriction enzyme sites allow to extract individually each components of the plasmid. This construction was cloned in pPICZalpha yeast vector.
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    </p>
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−
    <p>
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       <b>To learn more: </b> </html><partinfo>BBa_K2278021</partinfo><html>,<a href="https://2017.igem.org/Team:INSA-UPS_France/Results">results</a>
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<h2><i>Pichia pastoris</i> Leucrocin I AMP generator </h2>
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    <figure>
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      <img src="https://static.igem.org/mediawiki/parts/4/4f/T--INSA-UPS_France--pAOXI-leucrocinI.png" alt="">
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      <figcaption>
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      fig: <i>Pichia pastoris</i> Leucrocin I AMP generator
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      </figcaption>
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    </figure>
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    <p>
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      This system allowed us to constitutively express the BBa_K2278022 with a yeast promoter. This system was used to produce AMP at the highest concentration
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The pAOXI promoter makes genome recombination easier in <i>Pichia pastoris</i> genome. The α-factor sequence contains a RBS and a signal sequence to secrete the produced peptides
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The restriction enzyme sites allow to extract individually each components of the plasmid.
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This construction was cloned in pPICZalpha yeast vector.
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−
+
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    </p>
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−
 
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−
    <p>
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      <b>To learn more: </b> </html><partinfo>BBa_K2278022</partinfo><html>,<a href="https://2017.igem.org/Team:INSA-UPS_France/Results">results</a>
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<h2><i>Pichia pastoris</i> cOT2 AMP generator:
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</h2>
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    <figure>
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      <img src="https://static.igem.org/mediawiki/parts/9/97/T--INSA-UPS_France--pAOXI-cOT2.png
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" alt="">
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      <figcaption>
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      fig : <i>Pichia pastoris</i> cOT2 I AMP generator
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      </figcaption>
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    </figure>
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    <p>
+
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      This system allowed us to constitutively express BBa_K2278023 with a yeast promoter. This system was used to produce AMP at the highest concentration
+
−
The pAOXI promoter makes genome recombination easier in <i>Pichia pastoris</i> genome. The α-factor sequence contains a RBS and a signal sequence to secrete the produced peptides
+
−
The restriction enzyme sites allow to extract individually each components of the plasmid.
+
−
This construction was cloned in pPICZalpha yeast vector.
+
−
+
−
    </p>
+
−
 
+
−
    <p>
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−
      <b>To learn more: </b> </html><partinfo>BBa_K2278023</partinfo><html>,<a href="https://2017.igem.org/Team:INSA-UPS_France/Results">results</a>
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*************************************************
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<h2><i>Pichia pastoris</i> <i>Pichia pastoris</i> complete module :  <i>Pichia pastoris</i> complete module : 
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</h2>
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−
    <figure>
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      <img src="https://static.igem.org/mediawiki/parts/9/97/T--INSA-UPS_France--pGAP-cOT2.png
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−
" alt="">
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      <figcaption>
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fig: <i>Pichia pastoris</i> complete module
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https://static.igem.org/mediawiki/parts/5/59/T--INSA-UPS_France--ODR-10-cOT2.png
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      </figcaption>
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    </figure>
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    <p>
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      This system allowed us to constitutively express Odr10 receptor to detect diacetyl at any time. Odr10 pathway leads to the activation of pFUS promoter in case of diacetyl detection, hence the AMP are produced.
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The α-factor signal and coT2 gene are flanked with two restriction enzyme sites to clone a reporter gene such as RFP.
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This construction was cloned in pPICZalpha yeast vector.
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−
 
+
−
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−
    </p>
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−
 
+
−
    <p>
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      <b>To learn more: </b> </html><partinfo>BBa_K2278023</partinfo><html>,<a href="https://2017.igem.org/Team:INSA-UPS_France/Results">results</a>
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−
 
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<h2><i>Pichia pastoris</i> <i>Pichia pastoris</i> complete Respond/Effector Module :
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</h2>
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−
 
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−
    <figure>
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−
      <img src="https://static.igem.org/mediawiki/parts/9/97/T--INSA-UPS_France--pGAP-cAOXI.png
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−
" alt="">
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      <figcaption>
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https://static.igem.org/mediawiki/parts/3/37/T--INSA-UPS_France--ODR-10-rfp.png
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+
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      </figcaption>
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    </figure>
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−
 
+
−
    <p>
+
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      This system allowed us to constitutively express BBa_K2278023 with a yeast promoter. This system was used to produce AMP at the highest concentration
+
−
The pGAP promoter makes genome recombination easier in <i>Pichia pastoris</i> genome. The α-factor sequence contains a RBS and a signal sequence to secrete the produced peptides
+
−
The restriction enzyme sites allow to extract individually each components of the plasmid.
+
−
This construction was cloned in pPICZalpha yeast vector.
+
−
+
−
    </p>
+
−
 
+
−
    <p>
+
−
      <b>To learn more: </b> </html><partinfo>BBa_K2278023</partinfo><html>,<a href="https://2017.igem.org/Team:INSA-UPS_France/design">design</a>
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−
 
+
  
 +
 
 +
  </section>
  
−
</section>
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  <section>
 +
    <h1>Parts used in our project but not submitted to the registry </h1>
 +
  </section>
  
 
      
 
      

Revision as of 00:15, 24 October 2017

Croc'n Cholera
iGEM UPS-INSA Toulouse 2017

Parts

New parts submitted to the registry

Name Function Type Part State
<partinfo>BBa_K2278001</partinfo> QS molecule generator basic working
<partinfo>BBa_K2278002</partinfo> QS molecule generator basic not released
<partinfo>BBa_K2278011</partinfo> Diacetyl generator basic issues
<partinfo>BBa_K2278021</partinfo> D-NY15 AMP generator basic Working
<partinfo>BBa_K2278022</partinfo> Leucrocin I AMP generator basic unsuccessful
<partinfo>BBa_K2278023</partinfo> coT2 AMP generator basic unsuccessful

Existing Parts we have contributed to characterized

<partinfo>BBa_J04450</partinfo>: RFP coding device

BBa_J04450 biobrick conjugated in Vibrio harveyi

BBa_J04450 was tested in the Vibrio harveyi background. BBa_J04450 biobrick was cloned in a broad host range plasmid (pBBR1MCS-4) and conjugated into Vibrio harveyi to demonstrate the production of RFP in this chassis.

To learn more: <partinfo>BBa_J04450</partinfo>, results

<partinfo>BBa_K431009</partinfo>: glyceraldehyde 3-phosphate dehydrogenase promoter (pGAP)

Figure 2: construction to characterized BBa_K431009

The pGAP promoter was used as a constitutive promoter and as a homology region to make easier the genomic recombination of our synthetic construction in Pichia pastoris genome. The promoter was characterized by RT-qPCR using d-ny15 as reporter gene.

To learn more: <partinfo>BBa_K431009</partinfo> , results

<partinfo>BBa_K1800001</partinfo>: alpha factor secretion signal

fig 1 : construction to characterized BBa_K1800001

The α-factor sequence contains a kozak region and a signal sequence to secrete the produced peptides. The functionality of the signal factor was investigated by demonstrating that AMP are present in the supernatant through a toxicity assay.

To learn more: <partinfo>BBa_K1800001</partinfo>,results

Parts used in our project but not submitted to the registry