Team:Uppsala/Results
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Summary
We successfully integrated all five steps of the FPP to zeaxanthin pathway into the E. coli chromosome. The result is an E. coli strain expressing zeaxanthin. We have created six BioBricks in two versions. One version which is sequence verified, producing our enzymes CaCCD2, CsADH2946 and UGTCs2 respectively with inducible promoters. The other version include the three respective enzymes with a consecutive promoter. We have also modeled all our enzymes. Above all, we are the first to purify and confirm activity of CsADH2946 as well as estimating the kinetic parameters.
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
- Successfully integrated five genes from FPP to zeaxanthin into the chromosome of E. coli.
- Successfully transformed the crocin pathway into the zeaxanthin producing 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
- Biobrick - Coding for the enzyme CsADH2946 with His-tag and Lac-inducible promoter, characterised with correct sequencing and activity!
- Homology model
- Molecular dynamics - the model was stable!
- Steered Molecular Dynamics (pulling) - estimated Kd
- Estimation of Km
- Chromatogram and SDS-PAGE gel showing our protein successfully expressed and purified
- Activity against the conversion of crocetin dialdehyde to crocetin
- Successfully combined with pathway and transformed into zeaxanthin strain!
Step 3: UGTCs2
Crocetin → Crocin
- Biobrick - Coding for the enzyme UGTCs2 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!
