Difference between revisions of "Team:Uppsala/Results"
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| − | <img src="https://static.igem.org/mediawiki/2017/ | + | <img src="https://static.igem.org/mediawiki/2017/d/d8/Uppsala-Results_Final_Header.svg" style="width:100%; height: auto"> |
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<div class="header"> Summary </div> | <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%;"> | <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 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, CsADH2946 and UGTCs2. 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> | + | <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> |
<figure class="figure"> | <figure class="figure"> | ||
| − | <img src="https://static.igem.org/mediawiki/2017/1/18/Uppsala-ZeaPlate.png" style="display: block; margin: auto; width: | + | <img src="https://static.igem.org/mediawiki/2017/1/18/Uppsala-ZeaPlate.png" style="display: block; margin: auto; width:50%; height: auto;"> |
| − | <figcaption class="figure-caption figtext" style="padding- | + | <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> |
</figure> | </figure> | ||
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<div class="col-xs-10 textbox"> | <div class="col-xs-10 textbox"> | ||
| − | <div class="header"> Step 1: CaCCD2 </div> | + | <div class="header"> Step 1: <a href="https://2017.igem.org/Team:Uppsala/CrocinPathway">CaCCD2</a> </div> |
<div class="header"> Zeaxanthin → Crocetin dialdehyde </div> | <div class="header"> Zeaxanthin → Crocetin dialdehyde </div> | ||
<img src="https://static.igem.org/mediawiki/2017/3/30/Results_line_under_title.svg" style="margin: auto;"> | <img src="https://static.igem.org/mediawiki/2017/3/30/Results_line_under_title.svg" style="margin: auto;"> | ||
<div class="text"> | <div class="text"> | ||
<ul> | <ul> | ||
| − | <li><a href="https://2017.igem.org/Team:Uppsala/Parts">Biobrick</a> | + | <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> |
<li>Homology <a href="https://2017.igem.org/Team:Uppsala/Model">model</a></li> | <li>Homology <a href="https://2017.igem.org/Team:Uppsala/Model">model</a></li> | ||
| − | <li><a href="https://2017.igem.org/Team:Uppsala/Model">Molecular dynamics</a> | + | <li><a href="https://2017.igem.org/Team:Uppsala/Model">Molecular dynamics</a> – the model was stable!</li> |
<li>Successfully combined with pathway and transformed into <a href="https://2017.igem.org/Team:Uppsala/Zea-Strain">zeaxanthin strain</a>!</li> | <li>Successfully combined with pathway and transformed into <a href="https://2017.igem.org/Team:Uppsala/Zea-Strain">zeaxanthin strain</a>!</li> | ||
</ul> | </ul> | ||
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<div class="col-xs-10 textbox"> | <div class="col-xs-10 textbox"> | ||
| − | <div class="header"> Step 2: CsADH2946 </div> | + | <div class="header"> Step 2: <a href="https://2017.igem.org/Team:Uppsala/CrocinPathway">CsADH2946</a> </div> |
<div class="header"> Crocetin dialdehyde → Crocetin </div> | <div class="header"> Crocetin dialdehyde → Crocetin </div> | ||
<img src="https://static.igem.org/mediawiki/2017/3/30/Results_line_under_title.svg" style="margin: auto;"> | <img src="https://static.igem.org/mediawiki/2017/3/30/Results_line_under_title.svg" style="margin: auto;"> | ||
<div class="text"> | <div class="text"> | ||
<ul> | <ul> | ||
| − | <li><a href="https://2017.igem.org/Team:Uppsala/Parts">Biobrick</a> | + | <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> |
<li>Homology <a href="https://2017.igem.org/Team:Uppsala/Model">model</a></li> | <li>Homology <a href="https://2017.igem.org/Team:Uppsala/Model">model</a></li> | ||
| − | <li><a href="https://2017.igem.org/Team:Uppsala/Model">Molecular dynamics</a> | + | <li><a href="https://2017.igem.org/Team:Uppsala/Model">Molecular dynamics</a> – the model was stable!</li> |
| − | <li><a href="https://2017.igem.org/Team:Uppsala/Model">Steered Molecular Dynamics (pulling)</a> | + | <li><a href="https://2017.igem.org/Team:Uppsala/Model">Steered Molecular Dynamics (pulling)</a> – estimated K<sub>d</sub>(=4.9321 µM)</li> |
| − | <li><a href="https://2017.igem.org/Team:Uppsala/Model"> | + | <li><a href="https://2017.igem.org/Team:Uppsala/Model#KM">Measurement of K<sub>m</sub></a> (=20.7842 µM)</li> |
| − | <li><a href="https://2017.igem.org/Team:Uppsala/ | + | <li><a href="https://2017.igem.org/Team:Uppsala/CrocinPathway">Chromatogram and SDS-PAGE gel</a> showing our protein successfully expressed and purified</li> |
| − | <li><a href="https://2017.igem.org/Team:Uppsala/ | + | <li><a href="https://2017.igem.org/Team:Uppsala/CrocinPathway">Activity</a> against the conversion of crocetin dialdehyde to crocetin</li> |
<li>Successfully combined with pathway and transformed into <a href="https://2017.igem.org/Team:Uppsala/Zea-Strain">zeaxanthin strain</a>!</li> | <li>Successfully combined with pathway and transformed into <a href="https://2017.igem.org/Team:Uppsala/Zea-Strain">zeaxanthin strain</a>!</li> | ||
</ul> | </ul> | ||
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| − | <div class="header"> Step 3: UGTCs2 </div> | + | <div class="header"> Step 3: <a href="https://2017.igem.org/Team:Uppsala/CrocinPathway">UGTCs2</a></div> |
<div class="header"> Crocetin → Crocin </div> | <div class="header"> Crocetin → Crocin </div> | ||
<img src="https://static.igem.org/mediawiki/2017/3/30/Results_line_under_title.svg" style="margin: auto;"> | <img src="https://static.igem.org/mediawiki/2017/3/30/Results_line_under_title.svg" style="margin: auto;"> | ||
<div class="text"> | <div class="text"> | ||
<ul> | <ul> | ||
| − | <li><a href="https://2017.igem.org/Team:Uppsala/Parts">Biobrick</a> | + | <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> |
<li>Homology <a href="https://2017.igem.org/Team:Uppsala/Model">model</a></li> | <li>Homology <a href="https://2017.igem.org/Team:Uppsala/Model">model</a></li> | ||
| − | <li><a href="https://2017.igem.org/Team:Uppsala/Model">Molecular dynamics</a> | + | <li><a href="https://2017.igem.org/Team:Uppsala/Model">Molecular dynamics</a> – the model was stable!</li> |
<li>Successfully combined with pathway and transformed into <a href="https://2017.igem.org/Team:Uppsala/Zea-Strain">zeaxanthin strain</a>!</li> | <li>Successfully combined with pathway and transformed into <a href="https://2017.igem.org/Team:Uppsala/Zea-Strain">zeaxanthin strain</a>!</li> | ||
</ul> | </ul> | ||
Latest revision as of 20:22, 1 November 2017
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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.
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
- Successfully integrated five genes from FPP to zeaxanthin into the chromosome of E. coli.
- Successfully transformed the crocin pathway into the zeaxanthin producing strain
- Extracted zeaxanthin from the zeaxanthin producing strain
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
- 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!
- Steered Molecular Dynamics (pulling) – estimated Kd(=4.9321 µM)
- Measurement of Km (=20.7842 µM)
- 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!
Step 3: UGTCs2
Crocetin → Crocin
- 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!
