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| − | {{Uppsala/CSS}}
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| − | {{Uppsala/Navbar}}
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| − | <!DOCTYPE html>
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| − | <html lang="en">
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| − | <head>
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| − | <title>Alpha Crocin</title>
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| − | <meta charset="utf-8">
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| − | <meta name="viewport" content="width=device-width, initial-scale=1">
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| − | <link href="https://fonts.googleapis.com/css?family=Raleway" rel="stylesheet">
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| − | <link href="https://fonts.googleapis.com/css?family=Roboto+Slab" rel="stylesheet">
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| − | <style type="text/css">
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| − | .mainheader {
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| − | font-family: "Raleway";
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| − | font-size: 60pt;
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| − | text-align: center;
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| − | color: #D92627;
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| − | .text {
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| − | font-family: "Roboto Slab";
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| − | font-size: 17px;
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| − | .miniheader {
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| − | font-family: "Roboto Slab";
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| − | font-size: 17px;
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| − | font-weight: bold;
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| − | .title {
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| − | font-family: "Raleway";
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| − | font-size: 5vh;
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| − | font-weight: bold;
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| − | padding-top: 3%;
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| − | padding-bottom: 2%;
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| − | </style>
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| − | </head>
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| − | <body>
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| − | <div class="container-fluid">
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| − | <div class="row">
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| − | <div class="col-xs-12" style="height:60px;"></div>
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| − | </div>
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| − | <div class="row">
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| − | <div class="mainheader"> PROJECT DESCRIPTION </div>
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| − | <div class="col-xs-4 textbox">
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| − | <img src="https://static.igem.org/mediawiki/2017/c/c2/Description_Red_Flask.png" style="margin: auto; display: block; width: 65%; height: auto; padding-top: 50px; padding-bottom: 50px;">
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| − | </div>
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| − | <div class="col-xs-4"></div>
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| − | </div>
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| − | <div class="row">
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| − | <div class="mainheader"> CROCIN PATHWAY </div>
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| − | </div>
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| − | <div class="row">
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| − | <div class="col-xs-12" style="height:60px;"></div>
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| − | </div>
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| − | <div class="row text">
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| − | <div class= "col-xs-1"></div>
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| − | <div class= "col-xs-10">
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| − | <div> We at Uppsala this year, are planning to make Alpha crocin in E.coli.
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| − | Alpha-crocin, an apocarotenoid found in Crocus and Gardenia, is responsible for the red color of Saffron. Recent studies suggest that crocin may have several medicinal properties.Due to its colour, it could also be potentially used as a dye. It being a powerful antioxidant with interesting and not yet fully studied medicinal capabilities, large scale mass production of crocin would be of interest to further study its effects on the human body. Our team from 2013 already did the groundwork for us by developing zeaxanthin accumulating strain of E.coli. This year's project is building up on that. We identified three enzymatic steps leading from zeaxanthin to crocin. We at Uppsala this year, are planning to make Alpha crocin in E.coli.
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| − | Alpha-crocin, an apocarotenoid found in Crocus and Gardenia, is responsible for the red color of Saffron. Recent studies suggest that crocin may have several medicinal properties.Due to its colour, it could also be potentially used as a dye. It being a powerful antioxidant with interesting and not yet fully studied medicinal capabilities, large scale mass production of crocin would be of interest to further study its effects on the human body. Our team from 2013 already did the groundwork for us by developing zeaxanthin accumulating strain of E.coli. This year's project is building up on that. We identified three enzymatic steps leading from zeaxanthin to crocin. </div>
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| − | </div>
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| − | <div class= "col-xs-1"></div>
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| − | </div>
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| − | <div class="row">
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| − | <div class="col-xs-12" style="height:40px;"></div>
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| − | </div>
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| − | <div class="row">
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| − | <img src="https://static.igem.org/mediawiki/2017/2/29/Description_Grey_Bar_Step_1.png" style="width:100%; height: auto">
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| − | </div>
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| − | <div class="row title">
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| − | <div class="col-xs-1"></div>
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| − | <div class="col-xs-10">
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| − | <div> Step 1: Zeaxanthin → Crocetin dialdehyde</div>
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| − | </div>
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| − | <div class= "col-xs-1"></div>
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| − | </div>
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| − | <div class="row text">
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| − | <div class= "col-xs-1"></div>
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| − | <div class= "col-xs-10">
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| − | <div> We successfully made a sequence verified BioBrick of CaCCD2 with His-tag. The BioBrick was also combined with the other steps in the pathway and inserted into the zeaxanthin producing E. coli strain for a complete pathway from FPP to crocin. See the result here! </div>
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| − | <figure class="figure">
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| − | <img src="https://static.igem.org/mediawiki/2017/6/61/CraftingCrocinStep1.png" class="figure-img img-fluid" style="display: block; margin: auto; width: 55%; height: auto; padding-top: 5%; padding-bottom: 2%;">
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| − | <figcaption class="figure-caption" style="padding-bottom: 3%; padding-left: 25%;"> Figure 1. blabla </figcaption>
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| − | </figure>
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| − | <div class="miniheader"> Modelling of CaCCD2 </div>
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| − | <div> Since the enzyme is poorly characterized, we created a homology model and performed stability simulations to verify that our model was reasonable. The resulting structure of the homology modelling and its corresponding RMSD plot from simulations can be seen in figure X. The RMSD plot indicated that the model conforms to a stable structure. Read more about the homology modelling and dynamics modelling in the Modelling section. </div>
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| − | <figure class="figure">
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| − | <img src="https://static.igem.org/mediawiki/2017/8/82/CraftingCrocinModeling.png" class="figure-img img-fluid" style="display: block; margin: auto; width: 65%; height: auto; padding-top: 5%; padding-bottom: 2%;">
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| − | <figcaption class="figure-caption" style="padding-bottom: 3%; padding-left: 20%; padding-right: 20%;"> Figure 2. Homology model of CaCCD2 and RMSD plot for the same model. The simulation was run for 100 ns and displayed a stable homology model.</figcaption>
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| − | </figure>
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| − | </div>
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| − | <div class= "col-xs-1"></div>
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| − | </div>
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| − | <div class="row">
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| − | <div class="col-xs-12" style="height:40px;"></div>
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| − | </div>
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| − | <div class="row">
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| − | <img src="https://static.igem.org/mediawiki/2017/8/85/Description_Grey_Bar_Step_2.png" style="width:100%; height: auto">
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| − | </div>
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| − | <div class="row title">
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| − | <div class="col-xs-1"></div>
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| − | <div> Step 2: Crocetin dialdehyde → Crocetin </div>
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| − | </div>
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| − | <div class= "col-xs-1"></div>
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| − | </div>
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| − | <div class="row text">
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| − | <div class= "col-xs-1"></div>
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| − | <div class= "col-xs-10">
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| − | <div> We successfully made a sequence verified BioBrick of CaADH2946 with His-tag. The BioBrick was also combined with the other steps in the pathway and inserted into the zeaxanthin producing E. coli strain for a complete pathway from FPP to crocin. See the result here! </div>
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| − | <figure class="figure">
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| − | <img src="https://static.igem.org/mediawiki/2017/7/70/CraftingCrocinStep2.png" class="figure-img img-fluid" style="display: block; margin: auto; width: 55%; height: auto; padding-top: 5%; padding-bottom: 2%;">
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| − | <figcaption class="figure-caption" style="padding-bottom: 3%; padding-left: 25%;"> Figure 1. blabla </figcaption>
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| − | </figure>
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| − | <div class="miniheader"> Purification of CsADH </div>
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| − | <div> CsADH2946 was transformed and expressed in E. coli strain BL21 (DE3*) and purified using IMAC(link protocol) on an ÄKTA protein purification system. We used a gradient of imidazole concentration from 20-500 mM, in order to get our enzyme as separated as possible from other proteins that ends up in the fractions. The peak pointed at by the arrow in the chromatogram (Figure X) indicates protein that elutes at high imidazole concentration, i.e our desired His-tagged CsADH2946. The purification was followed by SDS-PAGE to analyse the fractions, control purity and verify the protein product. In figure X the band at around 60 kDa in the crude pellet indicate an overexpression of a protein in that size range. In the SDS gel of fractions 16-26 collected between 115 - 145 mL elution volume there is a strong band at 60 kDa corresponding to the molecular weight of CsADH2946, indicating that our protein was successfully overexpressed and well-separated.</div>
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| − | <figure class="figure">
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| − | <img src="https://static.igem.org/mediawiki/2017/b/bf/CraftingCrocinElutionStep2.png" class="figure-img img-fluid" style="display: block; margin: auto; width: 40%; height: auto; padding-top: 5%; padding-bottom: 2%;">
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| − | <figcaption class="figure-caption" style="padding-bottom: 3%; padding-left: 20%; padding-right: 20%"> Figure X. Chromatogram from IMAC-purification of CsADH2946. </figcaption>
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| − | </figure>
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| − | <figure class="figure">
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| − | <img src="https://static.igem.org/mediawiki/2017/9/9e/CraftingCrocinSDS-PAGE1.png" class="figure-img img-fluid" style="display: block; margin: auto; width: 40%; height: auto; padding-top: 5%; padding-bottom: 2%;">
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| − | <figcaption class="figure-caption" style="padding-bottom: 3%; padding-left: 20%; padding-right: 20%"> Figure X. SDS-PAGE gel of from IMAC purification. 1) Crude pellet. 2) Pellet after lysis. 3) Supernatant after lysis. 4) Flow through. 5) Wash with buffer A. 6) PageRuler protein prestained ladder. </figcaption>
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| − | </figure>
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| − | <figure class="figure">
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| − | <img src="https://static.igem.org/mediawiki/2017/c/cd/CraftingCrocinSDS-PAGE2.png" class="figure-img img-fluid" style="display: block; margin: auto; width: 40%; height: auto; padding-top: 5%; padding-bottom: 2%;">
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| − | <figcaption class="figure-caption" style="padding-bottom: 3%; padding-left: 20%; padding-right: 20%"> Figure X. SDS-PAGE gel from IMAC purification. Fractions 16-26 were collected between 115 and 145 ml elution volume . A band at about 60 kDa is clearly visible. </figcaption>
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| − | </figure>
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| − | <div class="miniheader"> Activity measurements of purified CsADH2946 </div>
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| − | <div> To verify the activity of our purified enzyme CsADH2946 to convert crocetin dialdehyde to crocetin, an activity measurements(link protocol) assay was performed on a plate reader measuring absorbance of the substrate and product of the reaction. For the experiment we used a 96-well plate in which we included wells with enzyme from pooled fractions + substrate, as well as positive and negative controls, see table Z for the specifics. </div>
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| − | <img src="https://static.igem.org/mediawiki/2017/b/b2/Description_Grey_Bar_Step_3.png" style="width:100%; height: auto">
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| − | </div>
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| − | <div class="col-xs-12" style="height:40px;"></div>
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| − | </div>
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| − | <div class="row text">
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| − | <div class= "col-xs-1"></div>
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| − | <div class= "col-xs-10">
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| − | <p> We at Uppsala this year, are planning to make Alpha crocin in E.coli.
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| − | Alpha-crocin, an apocarotenoid found in Crocus and Gardenia, is responsible for the red color of Saffron. Recent studies suggest that crocin may have several medicinal properties.Due to its colour, it could also be potentially used as a dye. It being a powerful antioxidant with interesting and not yet fully studied medicinal capabilities, large scale mass production of crocin would be of interest to further study its effects on the human body. Our team from 2013 already did the groundwork for us by developing zeaxanthin accumulating strain of E.coli. This year's project is building up on that. We identified three enzymatic steps leading from zeaxanthin to crocin. </p>
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