Difference between revisions of "Team:Uppsala/Results"

Revision as of 17:29, 31 October 2017

<!DOCTYPE html> Results

We successfully integrated all five steps of the FPP to zeaxanthin pathway into the E. coli chromosome. The result is a E. coli strain expressing zeaxanthin. We have created six BioBricks in two versions. One version which is sequence verified, producing our enzymes CaCCD2, CsADH2946 and UGTCs2 respectively with inducible promoters. The other version include the three respective enzymes with a consecutive promoter. We have also modeled all our enzymes. In addition to this we are the first to purify and confirm activity of CsADH2946 as well as estimating the kinetic parameters.

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 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!
Pulling simulation - estimated binding energy and KD
Estimation of Km
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!

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        <div class="header"> Summary </div>
        <img src="https://static.igem.org/mediawiki/2017/3/30/Results_line_under_title.svg" style="margin: auto; padding-bottom:2%;">
-       <div class="text">We created and combined the zeaxanthin producing strain and 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 and confirm that modern production of crocin in E.coli is red-y.</div>
+      <div class="text">We successfully integrated all five steps of the FPP to zeaxanthin pathway into the E. coli chromosome. The result is a E. coli strain expressing zeaxanthin. We have created six BioBricks in two versions. One version which is sequence verified, producing our enzymes CaCCD2, CsADH2946 and UGTCs2 respectively with inducible promoters. The other version include the three respective enzymes with a consecutive promoter. We have also modeled all our enzymes. In addition to this <b>we are the first</b> to purify and confirm activity of CsADH2946 as well as estimating the kinetic parameters.</div>
-      <div class="text">The three enzyme BioBricks in the zeaxanthin-crocin pathway was assembled to one plasmid using 3A assembly(link 3A assembly protocol) and was inserted into the zeaxanthin producing E.coli strain using electroporation(link Electroporation protocol). The color of the colonies changes at each addition of another enzyme construct (another step in the crocin pathway). This is an indication that something is indeed happening with the bacterial production when we introduce our pathway steps, see figure X.</div>
+       <div class="text">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. <b>In the end, we were able to identify, create and extensively characterize the pathway for crafting crocin.</b></div>
        <img src="https://static.igem.org/mediawiki/2017/0/07/Results_title_footer.svg" style="margin: auto; width: 60%; padding-top: 2%;">
      </div>