Difference between revisions of "Team:Uppsala/Results"

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   <li><a href="https://2017.igem.org/Team:Uppsala/Model">Steered Molecular Dynamics (pulling)</a> - estimated K<sub>d</sub></li>
 
   <li><a href="https://2017.igem.org/Team:Uppsala/Model">Steered Molecular Dynamics (pulling)</a> - estimated K<sub>d</sub></li>
 
   <li><a href="https://2017.igem.org/Team:Uppsala/Model">Estimation of K<sub>m</sub></a></li>
 
   <li><a href="https://2017.igem.org/Team:Uppsala/Model">Estimation of K<sub>m</sub></a></li>
   <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/CrocinPathway/#step2">Chromatogram and SDS-PAGE gel</a> showing our protein successfully expressed and purified</li>
   <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><a href="https://2017.igem.org/Team:Uppsala/CrocinPathway/#step2">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>
 
   <li>Successfully combined with pathway and transformed into <a href="https://2017.igem.org/Team:Uppsala/Zea-Strain">zeaxanthin strain</a>!</li>
 
</ul>
 
</ul>

Revision as of 16:49, 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, CsADH2946 and UGTCs2. 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