Team:Uppsala/Demonstrate
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Demonstrate
In our project we have sucessfully produced valuable pigments from the crocin pathway in E. coli. Our zeaxanthin strain succesfully produced the pathway pigments in overnight LB cultures with volumes between 5 ml and 2 L and we see great potential for scale-up of this process. In proof-of-concept trial experiments we were able to extract 13 mg of highly concentrated pigments containing zeaxanthin from 10 ml culture of our zeaxanthin strain. This extract contains multiple pigments from the pathway as demonstrated by our TLC experiment, and the exact purity of zeaxanthin is thus not known. However, due to the orange color of the extract and the clear and dominant presence of the zeaxanthin peaks in the measured absorbance spectrum, 10 % purity of zeaxanthin in the extract is a low estimate of the yield.
.With the market price of 10 % zeaxanthin being around
References
.With the market price of 10 % zeaxanthin being around
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.
