Team:Uppsala/Results

<!DOCTYPE html> Results

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.

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

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!

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 K_d(=4.9321 µM)
Measurement of K_m (=20.7842 µM)
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!

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!