Difference between revisions of "Team:Uppsala/Results"

 
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       <div class="header"> Summary </div>
 
       <div class="header"> Summary </div>
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       <div class="text">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.</div>
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      <div class="text">We successfully integrated the first five steps of the crocin pathway from FPP to zeaxanthin into the <i>E. coli</i> chromosome. The result is a <i>E. coli</i> strain expressing zeaxanthin. We have created sequence verified BioBricks of our enzymes in the extended crocin pathway: CaCCD2 (<a href="https://2017.igem.org/Team:Uppsala/Parts">BBa_K2423005</a>), CsADH2946 (<a href="https://2017.igem.org/Team:Uppsala/Parts">BBa_K2423007</a>) and UGTCs2 (<a href="https://2017.igem.org/Team:Uppsala/Parts">BBa_K2423008</a>). We have also characterized these enzymes with experiments and simulations. <bWe are the first</b> to purify and confirm activity of CsADH2946 as well as measuring the kinetic parameters of the enzyme.</div>
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      <div class="text">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.</div>
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        <figcaption class="figure-caption figtext" style="padding-left: 25%; padding-right: 25%; text-align:center;"> Figure 1. Top: Wild-type <i>E. coli</i>. Bottom: Zeaxanthin producing <i>E. coli</i> strain with 5 genes inserted into the chromosome.</figcaption>
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       <div class="text">We created and combined the zeaxanthin producing strain with a plasmid containing the extended crocin pathway which gave us an <i>E. coli</i> strain including the entire production pathway from FPP to crocin. <b>In the end, we were able to identify, create and extensively characterize the pathway for crafting crocin</b>.
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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 integrated five genes from FPP to zeaxanthin into the <a href="https://2017.igem.org/Team:Uppsala/Zea-Strain">chromosome</a> of <i>E. coli</i>.</li>
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   <li>Successfully transformed the  crocin pathway into the zeaxanthin strain</li>
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   <li>Successfully transformed the  crocin pathway into the <a href="https://2017.igem.org/Team:Uppsala/Zea-Strain">zeaxanthin producing strain</a></li>
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   <li>Extracted zeaxanthin from the zeaxanthin producing strain</li>
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   <li>Extracted zeaxanthin from the <a href="https://2017.igem.org/Team:Uppsala/Zea-Strain">zeaxanthin producing strain</a></li>
 
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      <div class="header"> Step 1: <a href="https://2017.igem.org/Team:Uppsala/CrocinPathway">CaCCD2</a> </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: <a href="https://2017.igem.org/Team:Uppsala/CrocinPathway">CsADH2946</a> </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">Steered Molecular Dynamics (pulling)</a> – estimated K<sub>d</sub>(=4.9321 µM)</li>
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  <li><a href="https://2017.igem.org/Team:Uppsala/Model#KM">Measurement of K<sub>m</sub></a> (=20.7842 µM)</li>
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  <li><a href="https://2017.igem.org/Team:Uppsala/CrocinPathway">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/CrocinPathway">Activity</a> against the conversion of crocetin dialdehyde to crocetin</li>
 +
  <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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      <div class="header"> Step 3:  <a href="https://2017.igem.org/Team:Uppsala/CrocinPathway">UGTCs2</a></div>
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      <div class="header"> Crocetin → Crocin </div>
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  <li><a href="https://2017.igem.org/Team:Uppsala/Parts">Biobrick</a> – Coding for the enzyme <a href="https://2017.igem.org/Team:Uppsala/Improve">UGTCs2</a> with his-tag and lac-inducible promoter, characterised with correct sequencing!</li>
 +
  <li>Homology <a href="https://2017.igem.org/Team:Uppsala/Model">model</a></li>
 +
  <li><a href="https://2017.igem.org/Team:Uppsala/Model">Molecular dynamics</a> – the model was stable!</li>
 +
  <li>Successfully combined with pathway and transformed into <a href="https://2017.igem.org/Team:Uppsala/Zea-Strain">zeaxanthin strain</a>!</li>
 +
</ul>
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</div>
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Latest revision as of 20:22, 1 November 2017

<!DOCTYPE html> Results

Summary
We successfully integrated the first five steps of the crocin pathway from FPP to zeaxanthin into the E. coli chromosome. The result is a E. coli strain expressing zeaxanthin. We have created sequence verified BioBricks of our enzymes in the extended crocin pathway: CaCCD2 (BBa_K2423005), CsADH2946 (BBa_K2423007) and UGTCs2 (BBa_K2423008). We have also characterized these enzymes with experiments and simulations. to purify and confirm activity of CsADH2946 as well as measuring the kinetic parameters of the enzyme.
Figure 1. Top: Wild-type E. coli. Bottom: Zeaxanthin producing E. coli strain with 5 genes inserted into the chromosome.
We created and combined the zeaxanthin producing strain 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.
Chromosomal integration:
Farnesyl Pyrophosphate (FPP) → Zeaxanthin
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
Step 3: UGTCs2
Crocetin → Crocin