Difference between revisions of "Team:Uppsala/Results"

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<h1>Results</h1>
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<p>Here you can describe the results of your project and your future plans. </p>
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<li> Clearly and objectively describe the results of your work.</li>
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<li> Future plans for the project. </li>
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<li> Considerations for replicating the experiments. </li>
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<li> Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for. </li>
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<li> Show data, but remember all measurement and characterization data must be on part pages in the Registry. </li>
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<li> Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project. </li>
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<h5> Project Achievements </h5>
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<p>You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.</p>
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      <div class="header"> Summary </div>
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      <div class="text">We successfully integrated the first five steps of the crocin pathway from FPP to zeaxanthin into the <i>E. coli</i> 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 (<a href="https://2017.igem.org/Team:Uppsala/Parts">BBa_K2423005</a>), CsADH2946 (<a href="https://2017.igem.org/Team:Uppsala/Parts">BBa_K2423007</a>) and UGTCs2 (<a href="https://2017.igem.org/Team:Uppsala/Parts">BBa_K2423008</a>). We have also characterized these enzymes with experiments and simulations. <bWe are the first</b> to purify and confirm activity of CsADH2946 as well as measuring the kinetic parameters of the enzyme.</div>
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        <figcaption class="figure-caption figtext" style="padding-left: 25%; padding-right: 25%; text-align:center;"> Figure 1. Top: Wild-type <i>E. coli</i>. Bottom: Zeaxanthin producing <i>E. coli</i> strain with 5 genes inserted into the chromosome.</figcaption>
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      <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>.
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      <div class="header"> Chromosomal integration: </div>
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      <div class="header"> Farnesyl Pyrophosphate (FPP) → Zeaxanthin </div>
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<li>A list of linked bullet points of the successful results during your project</li>
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  <li>Successfully integrated five genes from FPP to zeaxanthin into the <a href="https://2017.igem.org/Team:Uppsala/Zea-Strain">chromosome</a> of <i>E. coli</i>.</li>
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<li>A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.</li>
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  <li>Successfully transformed the crocin pathway into the <a href="https://2017.igem.org/Team:Uppsala/Zea-Strain">zeaxanthin producing strain</a></li>
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  <li>Extracted zeaxanthin from the <a href="https://2017.igem.org/Team:Uppsala/Zea-Strain">zeaxanthin producing strain</a></li>
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      <div class="header"> Step 1: <a href="https://2017.igem.org/Team:Uppsala/CrocinPathway">CaCCD2</a> </div>
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      <div class="header"> Zeaxanthin → Crocetin dialdehyde </div>
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  <li><a href="https://2017.igem.org/Team:Uppsala/Parts">Biobrick</a> – Coding for the enzyme CaCCD2 with his-tag and lac-inducible promoter, characterised with correct sequencing!</li>
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  <li>Homology <a href="https://2017.igem.org/Team:Uppsala/Model">model</a></li>
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  <li><a href="https://2017.igem.org/Team:Uppsala/Model">Molecular dynamics</a> – the model was stable!</li>
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  <li>Successfully combined with pathway and transformed into <a href="https://2017.igem.org/Team:Uppsala/Zea-Strain">zeaxanthin strain</a>!</li>
 
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<h5>Inspiration</h5>
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      <div class="header"> Step 2: <a href="https://2017.igem.org/Team:Uppsala/CrocinPathway">CsADH2946</a> </div>
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<p>See how other teams presented their results.</p>
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      <div class="header"> Crocetin dialdehyde → Crocetin </div>
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<li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li>
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  <li><a href="https://2017.igem.org/Team:Uppsala/Parts">Biobrick</a> – Coding for the enzyme CsADH2946 with his-tag and lac-inducible promoter, characterised with correct sequencing and activity!</li>
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<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li>
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  <li>Homology <a href="https://2017.igem.org/Team:Uppsala/Model">model</a></li>
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<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Results">2014 Paris Bettencourt </a></li>
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  <li><a href="https://2017.igem.org/Team:Uppsala/Model">Molecular dynamics</a> – the model was stable!</li>
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  <li><a href="https://2017.igem.org/Team:Uppsala/Model">Steered Molecular Dynamics (pulling)</a> – estimated K<sub>d</sub>(=4.9321 µM)</li>
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  <li><a href="https://2017.igem.org/Team:Uppsala/Model#KM">Measurement of K<sub>m</sub></a> (=20.7842 µM)</li>
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  <li><a href="https://2017.igem.org/Team:Uppsala/CrocinPathway">Chromatogram and SDS-PAGE gel</a> showing our protein successfully expressed and purified</li>
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  <li><a href="https://2017.igem.org/Team:Uppsala/CrocinPathway">Activity</a> against the conversion of crocetin dialdehyde to crocetin</li>
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  <li>Successfully combined with pathway and transformed into <a href="https://2017.igem.org/Team:Uppsala/Zea-Strain">zeaxanthin strain</a>!</li>
 
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      <div class="header"> Step 3:  <a href="https://2017.igem.org/Team:Uppsala/CrocinPathway">UGTCs2</a></div>
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      <div class="header"> Crocetin → Crocin </div>
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  <li><a href="https://2017.igem.org/Team:Uppsala/Parts">Biobrick</a> – Coding for the enzyme <a href="https://2017.igem.org/Team:Uppsala/Improve">UGTCs2</a> with his-tag and lac-inducible promoter, characterised with correct sequencing!</li>
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  <li>Homology <a href="https://2017.igem.org/Team:Uppsala/Model">model</a></li>
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  <li><a href="https://2017.igem.org/Team:Uppsala/Model">Molecular dynamics</a> – the model was stable!</li>
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  <li>Successfully combined with pathway and transformed into <a href="https://2017.igem.org/Team:Uppsala/Zea-Strain">zeaxanthin strain</a>!</li>
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Latest revision as of 20:22, 1 November 2017

<!DOCTYPE html> Results

Summary
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 (BBa_K2423005), CsADH2946 (BBa_K2423007) and UGTCs2 (BBa_K2423008). We have also characterized these enzymes with experiments and simulations. to purify and confirm activity of CsADH2946 as well as measuring the kinetic parameters of the enzyme.
Figure 1. Top: Wild-type E. coli. Bottom: Zeaxanthin producing E. coli strain with 5 genes inserted into the chromosome.
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.
Chromosomal integration:
Farnesyl Pyrophosphate (FPP) → Zeaxanthin
Step 1: CaCCD2
Zeaxanthin → Crocetin dialdehyde
  • 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!
Step 2: CsADH2946
Crocetin dialdehyde → Crocetin
Step 3: UGTCs2
Crocetin → Crocin