Difference between revisions of "Team:Uppsala/Demonstrate"

 
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      <div style="padding-right:3%; padding-left:3%; text-align:justify;"><br>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>
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  <div style="padding-bottom:3%"> <b>We got our zeaxanthin producing strain with the whole pathway from FPP to zeaxanthin integrated into the chromosome!</b> 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).</div>
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        <figcaption class="figure-caption figtext" style="padding-bottom: 3%; padding-left:0%"> Figure 3. Left: Large scale expression of zeaxanthin from the zeaxanthin producing <i>E. coli</i> strain. Right: Extracted and purified zeaxanthin.</figcaption>
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      <div style="padding-right:3%; padding-left:3%; text-align:justify;">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.</div>
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<div style="padding-right:3%; padding-left:3%; text-align:justify;">You can see all of our meaningful results in our <a href= "https://2017.igem.org/Team:Uppsala/Results"> Results page </a></di>
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<img src="https://static.igem.org/mediawiki/2017/3/3f/T--Uppsala--demonstrate_zeastrain.png" alt="Plate with colonies" style="width:60%;margin:auto;"></img>
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<div class="textbox" style="font-size: 2vh;">Plate with Zeaxanthin expressing E. coli strain transformed with plasmid containing all three crocin pathway enzymes CaCCD2, CsADH2946 and UGTCs2.
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<i>E. coli </i> culture producing colorful pigments from the crocin pathway.
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Latest revision as of 02:30, 2 November 2017

<!DOCTYPE html> Results

Demonstrate

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.