Team:Uppsala/Demonstrate
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Demonstrate
We got our zeaxanthin producing strain with the whole pathway from FPP to zeaxanthin integrated into the chromosome! All of the steps were confirmed with PCR, gel electrophoresis and sequencing. We were able to extract and purify the expensive yellow zeaxanthin compound from our strain (figure 3).
In our project we have focused on extending the zeaxanthine pathway to produce compounds along the way to crocin. We have stabilized the expression of zeaxanthin by lambda red recombineering and produced zeaxanthin. Next goal was to introduce further steps in the pathway. In the first step we included the enzyme CaCDD2 with a lac promotor. The enzyme was tested by successful sequencing. Then we introduced another step: CsADH2946, the enzyme that leads to Crocetin. Crocetin is a biologically interesting and research compound. We have successfully expressed and purified this enzyme and tested its activity. Both CaCDD2 and CsADH2956 were transformed into the zeaxanthin strain and production of crocetin was observed by color change. Our third step was to produce crocin. This is done with enzyme called UGTCs2. Again after creating the biobrick and transforming we have sequenced the plasmid succesfully. We have also transformed our product into the zeaxanthine strain.
You can see all of our meaningful results in our Results page
Plate with Zeaxanthin expressing E. coli strain transformed with plasmid containing all three crocin pathway enzymes CaCCD2, CsADH2946 and UGTCs2.
