Difference between revisions of "Team:Uppsala/Demonstrate"

Revision as of 02:29, 2 November 2017

<!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, CsADH2946 and UGTCs2. We have also characterized these enzymes with experiments and simulations. We are the first to purify and confirm activity of CsADH2946 as well as measuring the kinetic parameters of the enzyme. 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.

Detailed results that show that our project works can be found on our Results page

Plate with colonies — *E. coli* culture producing colorful pigments from the crocin pathway.

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-       <div style="padding-right:3%; padding-left:3%; text-align:justify;">In our project we have focused on producing the colorful and valuable molecules from the crocin pathway, naturally found in saffron. We have stabilized the expression of the saffron component zeaxanthin by lambda red recombineering and produced zeaxanthin in <i>E. coli </i>. The 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 construct was verified by successful sequencing. Then we introduced another step: CsADH2946, the enzyme that leads to Crocetin. We have successfully expressed and purified this enzyme and tested its activity. Both CaCDD2 and CsADH2946 were transformed into the zeaxanthin strain and production of crocetin was observed by a change in color. The next step was to produce crocin from crocetin, a reaction catalyzed by the enzyme UGTCs2. Again after creating the biobrick and transforming it into <i>E. coli</i> we have sequenced the plasmid succesfully. We have also transformed the combined BioBrick with CaCCD2, CsADH2946 and UGTCs2 into the zeaxanthin strain.</div>
+       <div style="padding-right:3%; padding-left:3%; text-align:justify;">We successfully integrated the first five steps of the crocin pathway from FPP to zeaxanthin into the E. coli chromosome. The result is a <i>E. coli</i> 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. We are the first to purify and confirm activity of CsADH2946 as well as measuring the kinetic parameters of the enzyme. We created and combined the zeaxanthin producing strain with a plasmid containing the extended crocin pathway which gave us an <i>E. coli</i> strain including the entire production pathway from FPP to crocin.</div>
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 <div style="padding-right:3%; padding-left:3%; text-align:justify;">Detailed results that show that our project works can be found on our <a href= "https://2017.igem.org/Team:Uppsala/Results"> Results page </a></di>