Difference between revisions of "Team:Uppsala/Results"

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      <div class="header"> Chromosomal integration: </div>
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      <div class="header"> Farnesyl Pyrophosphate (FPP) → Zeaxanthin </div>
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  <li>Successfully integrated five genes from FPP to zeaxanthin into the chromosome of E. coli.</li>
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  <li>Successfully transformed the  crocin pathway into the <a href="https://2017.igem.org/Team:Uppsala/Zea-Strain">zeaxanthin strain</a></li>
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  <li>Extracted zeaxanthin from the zeaxanthin producing <a href="https://2017.igem.org/Team:Uppsala/Zea-Strain">strain</a></li>
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      <div class="header"> Step 1: CaCCD2 </div>
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      <div class="header"> Zeaxanthin → Crocetin dialdehyde </div>
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  <li><a href="https://2017.igem.org/Team:Uppsala/Parts">Biobrick</a> - Coding for the enzyme CaCCD2 with His-tag and Lac-inducible promoter, characterised with correct sequencing!</li>
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  <li>Homology <a href="https://2017.igem.org/Team:Uppsala/Model">model</a></li>
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  <li><a href="https://2017.igem.org/Team:Uppsala/Model">Molecular dynamics</a> - the model was stable!</li>
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  <li>Successfully combined with pathway and transformed into <a href="https://2017.igem.org/Team:Uppsala/Zea-Strain">zeaxanthin strain</a>!</li>
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</ul>
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      <div class="header"> Step 2: CsADH2946 </div>
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      <div class="header"> Crocetin dialdehyde → Crocetin </div>
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  <li><a href="https://2017.igem.org/Team:Uppsala/Parts">Biobrick</a> - Coding for the enzyme CsADH2946 with His-tag and Lac-inducible promoter, characterised with correct sequencing and activity!</li>
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  <li>Homology <a href="https://2017.igem.org/Team:Uppsala/Model">model</a></li>
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  <li><a href="https://2017.igem.org/Team:Uppsala/Model">Molecular dynamics</a> - the model was stable!</li>
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  <li><a href="https://2017.igem.org/Team:Uppsala/Model">Pulling simulation</a> - estimated binding energy and KD</li>
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  <li><a href="https://2017.igem.org/Team:Uppsala/Model">Estimation of Km</a></li>
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  <li><a href="https://2017.igem.org/Team:Uppsala/AlphaCrocin/#step2">Chromatogram and SDS-PAGE gel</a> showing our protein successfully expressed and purified</li>
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  <li><a href="https://2017.igem.org/Team:Uppsala/AlphaCrocin/#step2">Activity</a> against the conversion of crocetin dialdehyde to crocetin</li>
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  <li>Successfully combined with pathway and transformed into <a href="https://2017.igem.org/Team:Uppsala/Zea-Strain">zeaxanthin strain</a>!</li>
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</ul>
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</div>
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Revision as of 22:13, 30 October 2017

<!DOCTYPE html> Results

Summary
We created and combined the zeaxanthin producing strain and with a plasmid containing the extended crocin pathway which gave us an E.coli strain including the entire production pathway from FPP to crocin. In the end, we were able to identify, create and extensively characterize the pathway for crafting crocin and confirm that modern production of crocin in E.coli is red-y.
The three enzyme BioBricks in the zeaxanthin-crocin pathway was assembled to one plasmid using 3A assembly(link 3A assembly protocol) and was inserted into the zeaxanthin producing E.coli strain using electroporation(link Electroporation protocol). The color of the colonies changes at each addition of another enzyme construct (another step in the crocin pathway). This is an indication that something is indeed happening with the bacterial production when we introduce our pathway steps, see figure X.
Chromosomal integration:
Farnesyl Pyrophosphate (FPP) → Zeaxanthin
  • Successfully integrated five genes from FPP to zeaxanthin into the chromosome of E. coli.
  • Successfully transformed the crocin pathway into the zeaxanthin strain
  • Extracted zeaxanthin from the zeaxanthin producing strain
Step 1: CaCCD2
Zeaxanthin → Crocetin dialdehyde
  • Biobrick - Coding for the enzyme CaCCD2 with His-tag and Lac-inducible promoter, characterised with correct sequencing!
  • Homology model
  • Molecular dynamics - the model was stable!
  • Successfully combined with pathway and transformed into zeaxanthin strain!
Step 2: CsADH2946
Crocetin dialdehyde → Crocetin
Chromosomal integration:
Farnesyl Pyrophosphate (FPP) → Zeaxanthin
  • Successfully integrated five genes from FPP to zeaxanthin into the chromosome of E. coli.
  • Successfully transformed the crocin pathway into the zeaxanthin strain
  • Extracted zeaxanthin from the zeaxanthin producing strain