Difference between revisions of "Team:Stuttgart/Design"

 
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<html>
 
<html>
  
−
</div>
+
<script>
−
<!-- start of content -->
+
−
<div class="igem_2017_content_wrapper">
+
−
    <h1>LIGHT UP THE PIPE</h1>
+
  
 +
    // This is the jquery part of your template. 
 +
    // Try not modify any of this code too much since it makes your menu work.
  
−
<h2>Keratinases</h2>
+
    $(document).ready(function() {
  
−
    <p>Blablabla</p>
+
        $("#HQ_page").attr('id','');
  
 +
        // call the functions that control the menu
 +
        menu_functionality();
 +
        hide_show_menu();
  
  
−
<h2>Esterases</h2>
 
  
−
    <p>Blablabla</p>
+
        //this function controls the expand and collapse buttons of the menu and changes the +/- symbols
 +
        function menu_functionality() {
  
 +
            //when clicking on a "menu_button", it will change the "+/-" accordingly and it will show/hide the corresponding submenu
 +
            $(".menu_button").click(function(){
  
 +
                // add or remove the class "open" , this class holds the "-"
 +
                $(this).children().toggleClass("open");
 +
                // show or hide the submenu
 +
                $(this).next('.submenu_wrapper').fadeToggle(400);
 +
            });
  
−
<h2>Lipases</h2>
+
            // when the screen size is smaller than 800px, the display_menu_control button appears and will show/hide the whole menu
 +
            $("#display_menu_control").click(function(){
 +
                $('#menu_content').fadeToggle(400);
 +
            });
  
−
    <p>Blablabla</p>
+
            // call the current page highlight function
 +
            highlight_current_page();
 +
        }
  
  
 +
        // call the highlight current page function to show it on the menu with a different background color
 +
        function highlight_current_page() {
  
−
<h2>Insertion of fragrances</h2>
+
            // select a page from the menu based on the id assigned to it and the current page name and add the class "current page" to make it change background color
 +
            $("#"+  wgPageName.substring(wgPageName.lastIndexOf("/")+1, wgPageName.length ) + "_page").addClass("current_page");
  
−
    <p>The microbial synthesis of natural flavor compounds has become a very attractive alternative to the chemical production.1 In recent years microorganisms such as E.coli and Yeast have been metabolically engineered to produce different flavors like limonene, geraniol or rose.1,2,3 For our project we discussed different approaches and choose two different scents: limonene and rose. </p>
+
            // now that the current_page class has been added to a menu item, make the submenu fade in
−
    <br>
+
            $(".current_page").parents(".submenu_wrapper").fadeIn(400);
 +
            // change the +/- symbol of the corresponding menu button
 +
            $(".current_page").parents(".submenu_wrapper").prev().children().toggleClass("open");
  
 +
        }
  
−
<h3>The limonene scent</h3>
 
  
  
−
<p>Limonene is a well-known cyclic monoterpene which can occur in two optical forms.2 (D)-Limonene is one of the most important and widespread terpenes in the flavor and fragrance industry, for example in citrus-flavored products such as soft drinks and candy.2 The (L)-Limonene form has a more harsh turpentine-like odor with a lemon-note.2 For our project we choose an enzyme-cascade, beginning with acetyl-coA and leading to the product (L)-limonene. This biosynthetic pathway was designed and inserted in E.coli.</p>
+
        // allow button on the black menu bar to show/hide the side menu
−
<br>
+
        function hide_show_menu() {
  
 +
            // in case you preview mode is selected, the menu is hidden for better visibility
 +
            if (window.location.href.indexOf("submit") >= 0) {
 +
                $(".igem_2017_menu_wrapper").hide();
 +
            }
  
−
<h3>The rose scent</h3>
+
            // if the black menu bar has been loaded
 +
            if (document.getElementById('bars_item')) {
  
−
<p>As second fragrance we want to install a lovely scent of rose in our microbial system. Hair are commonly made of Keratin (90%) and small amounts of amino acids, such as L-phenylalanine. This amino acid can be used as substrate for the production of 2-Phenylethylacetate (2-PEAc), which has a rose-like odor.1 Therefor this odor can act as an indicator for keratin degradation.
+
                // when the "bars_item" has been clicked
−
In recent studies from Guo et al the 2-PEAc biosynthetic pathway was successfully designed and expressed in E.coli.1 This pathway comprised four steps (Fig.1): Aminotransferase (ARO 8) for transamination of L-phenylalanine to phenylpyruvate, 2-keto acid decarboxylase KDC for the decarboxylation of the phenylpyruvate to phenylacetaldehyde, aldehyde reductase YjgB for the reduction of phenylacetaldehyde to 2-Phenylethanol, alcohol acetyltransferase ATF1 for the esterification of 2-PE to 2-PEAc.</p>
+
                $("#bars_item").click(function() {
−
<br>
+
                    $("#sideMenu").hide();
  
−
     <img  />
+
                    // show/hide the menu wrapper
 +
                    $(".igem_2017_menu_wrapper").fadeToggle("100");
 +
                });
 +
            }
 +
 
 +
            // because the black menu bars loads at a different time than the rest of the page, this function is set on a time out so it can run again in case it has not been loaded yet
 +
            else {
 +
                setTimeout(hide_show_menu, 15);
 +
            }
 +
        }
 +
 
 +
 
 +
     });
 +
 
 +
 
 +
</script>
 +
 
 +
<style>
 +
 
 +
 
 +
 
 +
    /***************************************************** DEFAULT WIKI SETTINGS  ****************************************************/
 +
 
 +
    /* Clear the default wiki settings */
 +
 
 +
    #home_logo, #sideMenu { display:none; }
 +
    #sideMenu, #top_title, .patrollink  {display:none;}
 +
    #content { width:100%; padding:0px;  margin-top:-7px; margin-left:0px;}
 +
    body {background-color:white; }
 +
    #bodyContent h1, #bodyContent h2, #bodyContent h3, #bodyContent h4, #bodyContent h5 { margin-bottom: 0px; }
 +
 
 +
 
 +
 
 +
 
 +
    /**************************************************************** MENU ***************************************************************/
 +
    /* Wrapper for the menu */
 +
    .igem_2017_menu_wrapper {
 +
        width: 15%;
 +
        height:100vh;
 +
        position:fixed;
 +
        right:0;
 +
        padding:0;
 +
        float:right;
 +
        border-left: 1px solid #F8CE63;
 +
        background-color:#190707;
 +
        text-align:left;
 +
        font-family:Tahoma, Geneva, sans-serif;
 +
        overflow-y: auto;
 +
        overflow-x: hidden;
 +
    }
 +
 
 +
    /* this hides the scrollbar to keep view consistency */
 +
    .igem_2017_menu_wrappe::-webkit-scrollbar {
 +
        display: none;
 +
    }
 +
 
 +
 
 +
    /* styling for links in the menu, removes the line under text */
 +
    .igem_2017_menu_wrapper a {
 +
        text-decoration: none;
 +
    }
 +
 
 +
 
 +
    /* styling for the images in the menu */
 +
    .igem_2017_menu_wrapper img {
 +
        width: 100%;
 +
    }
 +
 
 +
    /* styling for the menu buttons */
 +
    .igem_2017_menu_wrapper .menu_button {
 +
        width: 100%;
 +
        padding: 10px 0px 10px 15px;
 +
        float:left;
 +
        border-bottom: 1px solid #F8CE63;
 +
        font-size: 20px;
 +
        font-weight: bold;
 +
        color: #F8CE63;
 +
        cursor: pointer;
 +
    }
 +
 
 +
 
 +
    .igem_2017_menu_wrapper .menu_bottom_padding {
 +
        width: 100%;
 +
        height: 30px;
 +
        float:left;
 +
    }
 +
 
 +
    .menu_button.direct_to_page {
 +
        padding-left: 36px;
 +
    }
 +
 
 +
 
 +
    .igem_2017_menu_wrapper .menu_button .expand_collapse_icon {
 +
        width:10%;
 +
        float:left;
 +
    }
 +
 
 +
    .igem_2017_menu_wrapper .menu_button .expand_collapse_icon::before {
 +
        content: "+";
 +
    }
 +
 
 +
    .open::before {
 +
        content: "-" !important;
 +
    }
 +
 
 +
 
 +
 
 +
    /* styling for the menu buttons on hover */
 +
    .igem_2017_menu_wrapper .menu_button:hover, .igem_2017_menu_wrapper .submenu_button:hover , .submenu_button.current_page:hover {
 +
        background-color: #F8CE63;
 +
        text-decoration: none;
 +
        color:#190707;
 +
    }
 +
 
 +
    /* styling for the menu button when it is the current page */
 +
    .current_page {
 +
        background-color:#190707  !important;
 +
        color:#F8CE63 !important;
 +
    }
 +
 
 +
 
 +
    /* styling for the submenu buttons */
 +
    .igem_2017_menu_wrapper .submenu_button {
 +
        width: 100%;
 +
        padding: 10px 0px 10px 34px;
 +
        float:left;
 +
        background-color:#190707;
 +
        border-bottom: 1px solid #F8CE63;
 +
        font-size: 15px;
 +
        color: #F8CE63;
 +
        cursor: pointer;
 +
    }
 +
 
 +
    /* wrapper for the submenu items, they are hidden by default*/
 +
    .igem_2017_menu_wrapper .submenu_wrapper {
 +
        display:none;
 +
    }
 +
 
 +
    /* when the page size is bigger than 800px, this show/hide control is hidden by default */
 +
    .igem_2017_menu_wrapper #display_menu_control {
 +
        display:none;
 +
        text-align:center;
 +
    }
 +
 
 +
 
 +
    /***************************************************** CONTENT OF THE PAGE ****************************************************/
 +
 
 +
    /* Wrapper for the content */
 +
    .igem_2017_content_wrapper {
 +
        width: 81%;
 +
        margin: 2%;
 +
        display:block;
 +
        float:left;
 +
        background-color: #190707;
 +
        font-family:Tahoma, Geneva, sans-serif;
 +
    }
 +
 
 +
 
 +
    /********************************* HTML STYLING  *********************************/
 +
 
 +
    /* styling for the titles h1 h2 */
 +
    .igem_2017_content_wrapper h1, .igem_2017_content_wrapper h2 {
 +
        color:#F8CE63;
 +
        padding:15px 0 25px 0;
 +
        border-bottom: 0;
 +
    }
 +
 
 +
    /* styling for the titles  h3 h4 h5 h6*/
 +
    .igem_2017_content_wrapper h3, .igem_2017_content_wrapper h4, .igem_2017_content_wrapper h5, .igem_2017_content_wrapper h6 {
 +
        padding:5px 15px;
 +
        border-bottom:0;
 +
    }
 +
 
 +
    /* font and text */
 +
    .igem_2017_content_wrapper p {
 +
        padding: 15px 0;
 +
        font-size: 15px;
 +
    }
 +
    .igem_2017_content_wrapper img {
 +
        padding: 15px 0;
 +
    }
 +
 
 +
    /* Links */
 +
    .igem_2017_content_wrapper a {
 +
        font-weight: bold;
 +
        text-decoration: underline;
 +
        text-decoration-color: #190707;
 +
        color:  #3399ff;
 +
        -webkit-transition: all 0.4s ease;
 +
        -moz-transition: all 0.4s ease;
 +
        -ms-transition: all 0.4s ease;
 +
        -o-transition: all 0.4s ease;
 +
        transition: all 0.4s ease;
 +
    }
 +
 
 +
    /* hover for the links */
 +
    .igem_2017_content_wrapper a:hover {
 +
        text-decoration:none;
 +
        color:#000000;
 +
    }
 +
 
 +
    /* non numbered lists */
 +
    .igem_2017_content_wrapper ul {
 +
        padding:0px 20px;
 +
        font-size: 15px;
 +
        font-family:Tahoma, Geneva, sans-serif;
 +
    }
 +
 
 +
    /* numbered lists */
 +
    .igem_2017_content_wrapper ol {
 +
        padding:0px;
 +
        font-size: 15px;
 +
        font-family:Tahoma, Geneva, sans-serif;
 +
    }
 +
 
 +
    /* Table */
 +
    .igem_2017_content_wrapper table {
 +
        width: 97%;
 +
        margin:15px 10px;
 +
        border: 2px solid #190707;
 +
        border-collapse: collapse;
 +
    }
 +
 
 +
    /* table cells */
 +
    .igem_2017_content_wrapper  td {
 +
        padding: 10px;
 +
        vertical-align: text-top;
 +
        border: 1px solid #190707;
 +
        border-collapse: collapse;
 +
    }
 +
 
 +
    /* table headers */
 +
    .igem_2017_content_wrapper th {
 +
        padding: 10px;
 +
        vertical-align: text-top;
 +
        border: 1px solid #190707;
 +
        border-collapse: collapse;
 +
        background-color:##F8CE63;
 +
    }
 +
 
 +
 
 +
    /**********************************LAYOUT CLASSES **********************************/
 +
 
 +
    /* general class for column divs */
 +
    .igem_2017_content_wrapper .column  {
 +
        padding: 10px 0px;
 +
        float:left;
 +
    }
 +
 
 +
    /* class for a full width column */
 +
    .column .full_size {
 +
        width:100%;
 +
    }
 +
 
 +
    /* styling for images in a full width column*/
 +
    .column.full_size img {
 +
        width:97%;
 +
        padding: 10px 15px;
 +
    }
 +
 
 +
    /* class for a half width column */
 +
    .column.half_size {
 +
        width: 50%;
 +
    }
 +
    /* styling for images in a half width column*/
 +
    .column.half_size img {
 +
        width: 94.5%;
 +
        padding: 10px 15px;
 +
    }
 +
 
 +
 
 +
 
 +
 
 +
    /********************************* SUPPORT CLASSES ********************************/
 +
 
 +
    /* class that clears content below*/
 +
    .igem_2017_content_wrapper .clear {
 +
        clear:both;
 +
    }
 +
 
 +
 
 +
    /* adds extra spacing when clearing content */
 +
    .igem_2017_content_wrapper  .clear.extra_space {
 +
        height: 30px;
 +
    }
 +
 
 +
 
 +
    /* highlight class, makes content slightly smaller */
 +
    .igem_2017_content_wrapper .highlight {
 +
        margin: 0px 15px;
 +
        padding: 15px 0px;
 +
    }
 +
 
 +
 
 +
    /* highlight class, adds a gray background */
 +
    .igem_2017_content_wrapper .highlight.gray {
 +
        background-color: #190707;
 +
    }
 +
 
 +
    /* highlight with decoration blue line on top */
 +
    .igem_2017_content_wrapper .highlight.blue_top {
 +
        border-top: 4px solid #190707;
 +
    }
 +
 
 +
    /* highlight with a full blue border decoration */
 +
    .igem_2017_content_wrapper .highlight.blue_border {
 +
        border: 4px solid  #190707;
 +
    }
 +
 
 +
 
 +
    /* button class */
 +
    .igem_2017_content_wrapper .button{
 +
        max-width: 35%;
 +
        margin: 30px auto;
 +
        padding: 12px 10px;
 +
        background-color: #190707;
 +
        text-align: center;
 +
        color: #F8CE63;
 +
        -webkit-transition: all 0.4s ease;
 +
        -moz-transition: all 0.4s ease;
 +
        -ms-transition: all 0.4s ease;
 +
        -o-transition: all 0.4s ease; transition: all 0.4s ease;
 +
    }
 +
 
 +
    /* styling for button on hover */
 +
    .igem_2017_content_wrapper .button:hover{
 +
        background-color: #190707;
 +
        color: #F8CE63;
 +
    }
 +
 
 +
 
 +
 
 +
 
 +
    /***************************************************** RESPONSIVE STYLING ****************************************************/
 +
 
 +
    /* IF THE SCREEN IS LESS THAN 1200PX */
 +
    @media only screen and (max-width: 1200px) {
 +
 
 +
        #content {width:100%; }
 +
        .igem_2017_menu_wrapper {width:15%; right:0;}
 +
        .highlight {padding:10px 0px;}
 +
        .igem_2017_menu_wrapper #display_menu_control { display:none; }
 +
        #menu_content { display:block;}
 +
        .menu_button.direct_to_page {padding-left: 17px;}
 +
 
 +
    }
 +
 
 +
    /* IF THE SCREEN IS LESS THAN 800PX */
 +
    @media only screen and (max-width: 800px) {
 +
 
 +
        .igem_2017_menu_wrapper { width:100%; height: 15%; position:relative; left:0%;}
 +
        .igem_2017_content_wrapper {width:100%; margin-left:0px;}
 +
        .column.half_size  {width:100%; }
 +
        .column.full_size img, .column.half_size img {  width: 100%; padding: 10px 0px;}
 +
        .highlight {padding:15px 5px;}
 +
        .igem_2017_menu_wrapper #display_menu_control { display:block; }
 +
        #menu_content { display:none;}
 +
        .igem_2017_menu_wrapper .menu_button .expand_collapse_icon { width: 5%; }
 +
        .menu_bottom_padding {display:none;}
 +
        .menu_button.direct_to_page { padding-left: 36px; }
 +
    }
 +
 
 +
 
 +
 
 +
 
 +
    /* special class that the system uses to make sure the team wants a page to be evaluated */
 +
    .judges-will-not-evaluate {
 +
        width: 96.6%;
 +
        margin: 5px 15px;
 +
        display: block;
 +
        border: 4px solid #3399ff;
 +
        font-weight: bold;
 +
    }
 +
 
 +
</style>
 +
 
 +
 
 +
<!--- THIS IS WHERE THE HTML BEGINS --->
 +
 
 +
<head>
 +
 
 +
    <!-- This tells the browser that your page is responsive -->
 +
    <meta name="viewport" content="width=device-width, initial-scale=1">
 +
 
 +
</head>
 +
 
 +
 
 +
<!--MENU-->
 +
<div class="igem_2017_menu_wrapper" >
 +
 
 +
 
 +
 
 +
    <a href="https://2017.igem.org/Team:Stuttgart">
 +
        <img src="https://static.igem.org/mediawiki/2017/4/45/Bildschirmfoto_2017-10-17_um_14.23.20.png">
 +
    </a>
 +
 
 +
 
 +
    <!-- this div is hidden by default and will only be displayed if the screen size is too small -->
 +
    <div class="menu_button" id="display_menu_control">
 +
        MENU
 +
    </div>
 +
 
 +
    <div id="menu_content">
 +
 
 +
 
 +
 
 +
 
 +
        <a href="https://2017.igem.org/Team:Stuttgart">
 +
            <div class="menu_button direct_to_page">
 +
                HOME
 +
            </div>
 +
        </a>
 +
 
 +
 
 +
 
 +
        <div class="menu_button">
 +
            <div class="expand_collapse_icon">  </div> TEAM
 +
        </div>
 +
 
 +
        <div class="submenu_wrapper" id="team_submenu">
 +
 
 +
            <a href="https://2017.igem.org/Team:Stuttgart/Team">
 +
                <div class="submenu_button" id="Team_page">
 +
                    Team
 +
                </div>
 +
            </a>
 +
 
 +
            <a href="https://2017.igem.org/Team:Stuttgart/Collaborations">
 +
                <div class="submenu_button"  id="Collaborations_page">
 +
                    Collaborations
 +
                </div>
 +
            </a>
 +
 
 +
 
 +
        </div>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
        <div class="menu_button">
 +
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−
(1)Metabolic engineering of Escherichia coli for production of 2-Phenylethylacetate from L-phenylalanine (2017). Daoyi Guo, Lihua Zhang, Hong Pan and Xun Li.
 
−
(2) Biotechnological production of limonene in microorganisms (2016). Esmer Jongedijk, Katarina Cankar, Markus Buchhaupt, Jens Schrader, Harro Bouwmeester and Jules Beekwilder.
 
−
(3)Utilization of alkaline phosphatase PhoA in the bioproduction of geraniol by metabolically engineered Escherichia coli (2015). Wei Liu, Rubing Zhang, Ning Tian, Xin Xu, Yujing Cao, Mo Xian and Huizhou Liu.
 
−
(4)Rose Scent: Genomics Approach to Discovering Novel Floral Fragrance–Related Genes (2002). Inna Guterman, Moshe Shalit, Naama Menda, Dan Piestun, Mery Dafny-Yelin, Gil Shalev, Einat Bar, Olga Davydov, Mariana Ovadis, Michal Emanuel, Jihong Wang, Zach Adam, Eran Pichersky, Efraim Lewinsohn, Dani Zamir, Alexander Vainstein, and David Weiss.
 
  
  
Line 57: Line 739:
  
  
 +
<!-- start of content -->
 +
<div class="igem_2017_content_wrapper">
  
 +
<h1 align=middle > LIGHT UP THE PIPE - THREE PARTS FOR A BETTER FLOW </h1>
  
 +
<br><h3>For the cleaning and degradation process of clogged drains by e.coli we created a genetic circuit with different enzymes (model Link). Three parts are needed for a better flow: </h3>
  
  
 +
<table>
 +
<tr>
 +
<td bgcolor=#F8CE63>
 +
<img src="https://static.igem.org/mediawiki/2017/6/63/EsteraseSTUTTGART.jpeg"/ width=390px height= 360px align="left" hspace=20 vspace=0>
  
 +
<br><h3><span style="color:#2E9AFE">PART I - ESTERASES and LIPASES</span style="color:#2E9AFE"></h3><br>
 +
Hair is surrounded by a layer of grease and waxes which first need to be removed to make the hair-keratin available for keratinases. For the first degradation step we choose a combination of  esterases and lipases.
 +
We investigated two different esterases for their enzyme activity. One esterase from the registry (EstCS2 BBa_K1149002) and one esterase (LipB) supplied by Dr. Eggert from Evoxx were compared. Additionally we choose the lipase TliA to support the esterases at the fat degradation and to accelerate the entire degradation process. For the extracellular secretion of the enzymes we attached different signal sequences (pelB, OmpA and phoA) that were provided on the iGEM plates.
 +
<br>
 +
<br>
 +
<h4>EstCS2</h4>
 +
EstCS2 from the iGEM Imperial College 2013 was proved to be active. In their project the cells expressing these construct were grown and lysed by sonication and were utilized in a colourimetric assay with the substrate analog para-Nitrophenyl butyrate. In our project we didn’t purify the esterases but used the supernatant for the enzyme activity assay.
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<h4>LipB</h4>
 +
LipB showed an enzyme activity of 2,8 U/mL in the supernatant. First, we repeated the enzyme activity assay from the iGEM TU Darmstadt 2012 to determine the esterase with the highest enzyme activity.
 +
<br>
 +
<br>
 +
<h4>TliA</h4>
 +
The lipase that is used for this project is the TliA lipase from Pseudomonas fluorescens. TliA is secreted by the ABC (ATP binding cassette) export system (prtDEF gene cluster) from Dickeya dadantii (formerly known as Erwinia chrysanthemi). The LARD secretion tag for the ABC export system is added directly to the TliA gene, which hopefully leads to a good export yield and a high extracellular activity.
 +
In order to control gene expression, the TliA gene is expressed by the pBAD promoter, which can be induced by arabinose. The prtDEF gene cluster is expressed by a constitutive promoter of mediocre strength. So, the ABC export system is always expressed at a certain level and can export the LARD-tagged TliA lipase, if expressed.
 +
</td>
 +
</tr>
 +
</table>
 +
 +
 +
<table>
 +
<tr>
 +
<td bgcolor="#F8CE63"> 
 +
<img src=" https://static.igem.org/mediawiki/2017/8/8e/Keratinasen.jpeg"/ width=390px height= 360px align="right" hspace=20 vspace=0 >
 +
<br><h3><span style="color:#2E9AFE">PART II - KERATINASES</span style="color:#2E9AFE"></h3>
 +
<br>
 +
For efficient hair degradation we chose different keratinases (KerUS, KerA, KerBL and KerP). Moreover we combined this keratinases with different anderson-promotors (BBa_K206000, BBa_J23114 and BBaJ23102) and signal sequences (PelB, OmpA and PhoA) for extracellular transport of the keratinases.
 +
</td>
 +
</table>
 +
 +
 +
<table>
 +
<td height=0 bgcolor="#F8CE63">
 +
<img src="https://static.igem.org/mediawiki/2017/3/38/Rosenduft.jpeg"/ width=390px height= 360px align="left" hspace=20 vspace=0>
 +
 +
<br><h3><span style="color:#2E9AFE">PART III - A LOVELY SCENT OF ... </span style="color:#2E9AFE"></h3><br>
 +
The microbial synthesis of natural flavor compounds has become a very attractive alternative to the chemical production (1). In recent years microorganisms such as E.coli and Yeast have been metabolically engineered to produce different flavors like limonene, geraniol or rose (1,2,3). For our project we discussed different approaches and choose two different scents: rose and fir.
 +
<br>
 +
<br>
 +
<br>
 +
<h4>... ROSE FRAGRANCE </h4>
 +
As first special fragrance we want to install a lovely scent of rose in our microbial system. Hair are commonly made of Keratin (90%) and small amounts of amino acids, such as L-phenylalanine. This amino acid can be used as substrate for the production of 2-Phenylethylacetate (2-PEAc), which has a rose-like odor (1).
 +
Therefor this odor can act as an indicator for keratin degradation. In recent studies from Guo et al the 2-PEAc biosynthetic pathway was successfully designed and expressed in E.coli (1). This pathway was used for our project and comprised four steps (Fig.1):
 +
<br>
 +
<img src="https://static.igem.org/mediawiki/2017/7/7a/Stuttgartpathwayrose.png"/ align="right" valign= width=560 height=180 hspace=20 vspace=0>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<li> Aminotransferase (ARO 8) for transamination of L-phenylalanine to phenylpyruvate</li>
 +
<li> 2-keto acid decarboxylase KDC for the decarboxylation of the phenylpyruvate to phenylacetaldehyde</li>
 +
<li> Aldehyde reductase YjgB for the reduction of phenylacetaldehyde to 2-Phenylethanol</li>
 +
<li> Alcohol acetyltransferase ATF1 for the esterification of 2-PE to 2-PEAc.</li>
 +
<br>             
 +
<br>
 +
<h4> ... FIR FRAGRANCE</h4>
 +
Limonene is a well-known cyclic monoterpene which can occur in two optical forms (2). (D)-Limonene is one of the most important and widespread terpenes in the flavor and fragrance industry, for example in citrus-flavored products such as soft drinks and candy (2). The (L)-Limonene form has a more harsh fir-like odor with a lemon-note (2). For our project we choose an enzyme-cascade, beginning with acetyl-coA and leading to the product (L)-limonene. This biosynthetic pathway was designed and inserted in E.coli (Fig.2)
 +
<img src="https://static.igem.org/mediawiki/2017/8/8a/Limonenepathway.png"/ align="middle" width=650 height=260 hspace=20 vspace=0>
 +
</td>
 +
</table>
 +
 +
 +
 +
 +
<foot>
 +
<h3>REFERENCES</h3>
 +
<li>(1) Metabolic engineering of Escherichia coli for production of 2-Phenylethylacetate from L-phenylalanine (2017), D. Guo and L. Zhang et. al.</li>
 +
<li>(2) Biotechnological production of limonene in microorganisms (2016), E. Jongedijk and K. Cankar et. al. </li>
 +
<li>(3) Utilization of alkaline phosphatase PhoA in the bioproduction of geraniol by metabolically engineered Escherichia coli (2015), W. Liu and R. Zhang et. al. </li>
 +
<li>(4) Rose Scent: Genomics Approach to Discovering Novel Floral Fragrance–Related Genes (2002), I. Guterman and M. Shalit et. al. </li>
 +
</foot>
 +
</div>
  
  

Latest revision as of 10:23, 21 October 2017

HOME
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JUDGING FORM

LIGHT UP THE PIPE - THREE PARTS FOR A BETTER FLOW


For the cleaning and degradation process of clogged drains by e.coli we created a genetic circuit with different enzymes (model Link). Three parts are needed for a better flow:


PART I - ESTERASES and LIPASES


Hair is surrounded by a layer of grease and waxes which first need to be removed to make the hair-keratin available for keratinases. For the first degradation step we choose a combination of esterases and lipases. We investigated two different esterases for their enzyme activity. One esterase from the registry (EstCS2 BBa_K1149002) and one esterase (LipB) supplied by Dr. Eggert from Evoxx were compared. Additionally we choose the lipase TliA to support the esterases at the fat degradation and to accelerate the entire degradation process. For the extracellular secretion of the enzymes we attached different signal sequences (pelB, OmpA and phoA) that were provided on the iGEM plates.

EstCS2

EstCS2 from the iGEM Imperial College 2013 was proved to be active. In their project the cells expressing these construct were grown and lysed by sonication and were utilized in a colourimetric assay with the substrate analog para-Nitrophenyl butyrate. In our project we didn’t purify the esterases but used the supernatant for the enzyme activity assay.




LipB

LipB showed an enzyme activity of 2,8 U/mL in the supernatant. First, we repeated the enzyme activity assay from the iGEM TU Darmstadt 2012 to determine the esterase with the highest enzyme activity.

TliA

The lipase that is used for this project is the TliA lipase from Pseudomonas fluorescens. TliA is secreted by the ABC (ATP binding cassette) export system (prtDEF gene cluster) from Dickeya dadantii (formerly known as Erwinia chrysanthemi). The LARD secretion tag for the ABC export system is added directly to the TliA gene, which hopefully leads to a good export yield and a high extracellular activity. In order to control gene expression, the TliA gene is expressed by the pBAD promoter, which can be induced by arabinose. The prtDEF gene cluster is expressed by a constitutive promoter of mediocre strength. So, the ABC export system is always expressed at a certain level and can export the LARD-tagged TliA lipase, if expressed.

PART II - KERATINASES


For efficient hair degradation we chose different keratinases (KerUS, KerA, KerBL and KerP). Moreover we combined this keratinases with different anderson-promotors (BBa_K206000, BBa_J23114 and BBaJ23102) and signal sequences (PelB, OmpA and PhoA) for extracellular transport of the keratinases.

PART III - A LOVELY SCENT OF ...


The microbial synthesis of natural flavor compounds has become a very attractive alternative to the chemical production (1). In recent years microorganisms such as E.coli and Yeast have been metabolically engineered to produce different flavors like limonene, geraniol or rose (1,2,3). For our project we discussed different approaches and choose two different scents: rose and fir.


... ROSE FRAGRANCE

As first special fragrance we want to install a lovely scent of rose in our microbial system. Hair are commonly made of Keratin (90%) and small amounts of amino acids, such as L-phenylalanine. This amino acid can be used as substrate for the production of 2-Phenylethylacetate (2-PEAc), which has a rose-like odor (1). Therefor this odor can act as an indicator for keratin degradation. In recent studies from Guo et al the 2-PEAc biosynthetic pathway was successfully designed and expressed in E.coli (1). This pathway was used for our project and comprised four steps (Fig.1):




  • Aminotransferase (ARO 8) for transamination of L-phenylalanine to phenylpyruvate
  • 2-keto acid decarboxylase KDC for the decarboxylation of the phenylpyruvate to phenylacetaldehyde
  • Aldehyde reductase YjgB for the reduction of phenylacetaldehyde to 2-Phenylethanol
  • Alcohol acetyltransferase ATF1 for the esterification of 2-PE to 2-PEAc.


    • ... FIR FRAGRANCE

    Limonene is a well-known cyclic monoterpene which can occur in two optical forms (2). (D)-Limonene is one of the most important and widespread terpenes in the flavor and fragrance industry, for example in citrus-flavored products such as soft drinks and candy (2). The (L)-Limonene form has a more harsh fir-like odor with a lemon-note (2). For our project we choose an enzyme-cascade, beginning with acetyl-coA and leading to the product (L)-limonene. This biosynthetic pathway was designed and inserted in E.coli (Fig.2)

    REFERENCES

  • (1) Metabolic engineering of Escherichia coli for production of 2-Phenylethylacetate from L-phenylalanine (2017), D. Guo and L. Zhang et. al.
  • (2) Biotechnological production of limonene in microorganisms (2016), E. Jongedijk and K. Cankar et. al.
  • (3) Utilization of alkaline phosphatase PhoA in the bioproduction of geraniol by metabolically engineered Escherichia coli (2015), W. Liu and R. Zhang et. al.
  • (4) Rose Scent: Genomics Approach to Discovering Novel Floral Fragrance–Related Genes (2002), I. Guterman and M. Shalit et. al.