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| + | {{Template:Kingsborough_NY}} |
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| − | <body class="mediawiki ltr sitedir-ltr ns-10 ns-subject page-Template_TeamTemplateB skin-igem action-view">
| + | |
| | | | |
| − | <script type='text/javascript' src ='/common/tablesorter/jquery.tablesorter.min.js'></script>
| + | == Designing a Kill-Switch == |
| − | <link rel='stylesheet' type='text/css' href='/common/tablesorter/themes/groupparts/style.css' />
| + | |
| − | <link rel='stylesheet' type='text/css' href='/common/table_styles.css' />
| + | |
| | | | |
| − | <div id='globalWrapper'>
| + | <p>We initially attempted to create engineered bacteria that break down nitrogenous waste effectively in wastewater treatment plant. Since our <i>E. coli</i> can survive in saltwater, creating a kill switch for our engineered bacteria will, in theory, prevent our bacteria from thriving outside wastewater treatment plants.</p> |
| − | <div id='top_menu_under' class='noprint'></div>
| + | <p> |
| − | <div id='top_menu_14' class='noprint'>Loading menubar.....</div> <!-- Will be replaced with the jQuery.load -->
| + | We decided to vary from previous iGEM team efforts in three important ways: |
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| + | <br><br> |
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| + | 1) We appended the <i>ssrA</i> degradation tag onto our toxin of choice, MazF. This should lead any MazF expressed due to leaky control of the promoter to be destroyed, and not damage the cell. |
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| + | <br><br> |
| − |
| + | 2) We introduced, in some of our constructs, a supplmentary copy of the cI repressor gene, in cause the light-controlled copy on pDawn was insufficient to control MazF. |
| − | </script>
| + | <br><br> |
| − | <div id="content" class="mw-body" role="main">
| + | 3) For both MazF constructs we created, we had counterparts that contained the alpha fragment of LacZ instead of the toxin. This way, we can monitor expression levels without worrying about the cell death that high levels of MazF might cause. |
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| + | </p> |
| − | <h1 id="firstHeading" class="firstHeading">
| + | |
| − | <span dir="auto">Template:TeamTemplateB</span>
| + | |
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| + | |
| − | <div id="mw-content-text" lang="en" dir="ltr" class="mw-content-ltr"><p> | + | |
| | | | |
| − | <!-- Start of CSS-->
| + | == Control of Gene Expression by pDawn alone == |
| − | <style type="text/css">
| + | |
| | | | |
| − | /* WIKI SETTINGS*/
| + | 3.1 (K2268003): When light is present, c1 WILL NOT be produced. In theory, the expression of LacZ should be maximal. This is intended to be the non-lethal version of our kill-switch. |
| − |
| + | |
| − | /* Hide first heading */
| + | |
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| + | |
| − |
| + | |
| − | /*-- Remove borders, float content left and remove padding/margin */
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| + | |
| | | | |
| − | /*left align Black Menu Bar */
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| − | }
| + | |
| − | </style>
| + | |
| − | </p><p>
| + | |
| | | | |
| − | <!-- Start of CSS-->
| + | 4.1 (K2268004): When light is present, c1 WILL NOT be produced. In theory, the expression of MazF should be maximal. |
| − | <style type="text/css">
| + | [[File:KbccMidnightSlide2.jpg|800px|center]] |
| | + | <br clear=all> |
| | | | |
| − | /* PAGE LAYOUT */
| + | == Control of Gene Expression by pDawn and supplementary cI Repressor == |
| − |
| + | |
| − | /* page background color*/
| + | |
| − | body {
| + | |
| − | background-color: #000000;
| + | |
| − | }
| + | |
| | | | |
| | + | 3.2(K2268005): When light is present with no ITPG, c1 WILL NOT be produced. In theory, maximal expression of LacZ should be produced. This is a non-lethal version of our kill switch. |
| | | | |
| − | /* Creates a container that will wrap all of the content inside your wiki pages. */
| + | [[File:KbccMidnightSlide3.jpg|800px|center]] |
| − | #mainContainer {
| + | 4.2(K2268006): When light is present with no ITPG, c1 WILL NOT be produced. In theory, maximal expression of MazF should be produced. |
| − | width: 980px;
| + | |
| − | overflow: hidden;
| + | |
| − | float:left;
| + | |
| − | margin-left: 20px;
| + | |
| − | background-color: #EEEDEA;
| + | |
| − | border-bottom: 6px solid #EE3e53;
| + | |
| − | }
| + | |
| | | | |
| − | /* Creates the container for the menu */
| + | [[File:KbccMidnightSlide4.jpg|800px|center]] |
| − | #menuContainer {
| + | <br clear=all> |
| − | float:right;
| + | |
| − | width: 150px;
| + | |
| − | background-color: #0e232f;
| + | |
| − | height: 100%;
| + | |
| − | border-top: 6px solid #EE3e53;
| + | |
| − | font-family: "Arial", Helvetica, sans-serif;
| + | |
| − | }
| + | |
| | | | |
| − | /* Creates the container for the content */
| + | == Light control by pDawn == |
| − | #contentContainer {
| + | |
| − | background-color: #EEEDEA;
| + | |
| − | width: 810px;
| + | |
| − | float: left;
| + | |
| − | padding: 10px;
| + | |
| − | border-top: 6px solid #EE3e53;
| + | |
| − | }
| + | |
| | | | |
| | + | In order for our design to work, the light induction module present on pDawn must be operational. We tested pDawn alone, in the dark and in light, to verify that expression of the RFP reporter only occurs in the light (see petri dish below). We also observed this red fluorescence when pDawn was used in conjunction with our constructs (data not shown). |
| | | | |
| − | /*Set up height place holder for the banner*/
| + | [[File:T--Kingsborough_NY--pDawn1.png]] |
| − | #bannerContainer {
| + | |
| − | height:200px;
| + | |
| − | margin:auto;
| + | |
| − | text-align:center;
| + | |
| − | color: #24B694;
| + | |
| − | }
| + | |
| − | | + | |
| − | /*VISUAL STYLE (TABLES, FONTS, LINKS, IMAGES, UL) */
| + | |
| − | | + | |
| − | | + | |
| − | /*Change the styling of text for everything inside main container*/
| + | |
| − | #mainContainer p {
| + | |
| − | font-family: "Arial", Helvetica, sans-serif;
| + | |
| − | font-size: 12px;
| + | |
| − | color: #0e232f;
| + | |
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| + | |
| − |
| + | |
| − | /*This changes the color and font family */
| + | |
| − | #contentContainer h1, h2, h3, h4, h5, h6 {
| + | |
| − | font-family: "Arial", Helvetica, sans-serif;
| + | |
| − | color: #0e232f;
| + | |
| − | border-bottom: none;
| + | |
| − | font-weight: bold;
| + | |
| − | text-transform: uppercase;
| + | |
| − | }
| + | |
| − | | + | |
| − | /*Style of the links - links are different inside the menu */
| + | |
| − | #contentContainer a {
| + | |
| − | font-weight: bold;
| + | |
| − | color: #ee3e53;
| + | |
| − | }
| + | |
| − | | + | |
| − | /* Styling links on hover- links are different inside the menu */
| + | |
| − | #contentContainer a:hover{
| + | |
| − | color: #f4b248;
| + | |
| − | }
| + | |
| − | | + | |
| − | /*Change the styling of tables */
| + | |
| − | #contentContainer table {
| + | |
| − | border: 1px solid #0e232f;
| + | |
| − | border-collapse: collapse;
| + | |
| − | width: 90%
| + | |
| − | margin: auto;
| + | |
| − | margin-bottom: 15px;
| + | |
| − | margin-top: 15px;
| + | |
| − | margin-right: 10px;
| + | |
| − | margin-left: 10px;
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| + | |
| − | | + | |
| − | /*Change the styling of table cells*/
| + | |
| − | #contentContainer td {
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| − | border: 1px solid #0e232f;
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| + | |
| − | background-color: #EEEDEA;
| + | |
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| + | |
| − | | + | |
| − | /*Change the styling of table headers */
| + | |
| − | #contentContainer th {
| + | |
| − | padding: 10px;
| + | |
| − | border: 1px solid #0e232f;
| + | |
| − | border-collapse: collapse;
| + | |
| − | vertical-align: text-top;
| + | |
| − | background-color: #e8d5b7;
| + | |
| − | }
| + | |
| − | | + | |
| − | | + | |
| − | /*MENU STYLING */
| + | |
| − | | + | |
| − | /*Styling for the links in the menu */
| + | |
| − | #menuContainer a {
| + | |
| − | color: #EEEDEA;
| + | |
| − | text-decoration:none;
| + | |
| − | font-weight: bold;
| + | |
| − | }
| + | |
| − | | + | |
| − | /* Sets the style for lists inside menuContainer */
| + | |
| − | #menuContainer ul {
| + | |
| − | width:100%;
| + | |
| − | list-style: none;
| + | |
| − | margin: auto;
| + | |
| − | padding-top: 10px;
| + | |
| − | padding-bottom: 10px;
| + | |
| − | | + | |
| − | }
| + | |
| − | | + | |
| − | /*Styles the list items to become menu buttons */
| + | |
| − | #menuContainer ul li {
| + | |
| − | display: block;
| + | |
| − | position: relative;
| + | |
| − | padding: 10px;
| + | |
| − | margin-bottom:5px;
| + | |
| − | padding-left: -20px;
| + | |
| − | }
| + | |
| − | | + | |
| − | #menuContainer ul li:hover {
| + | |
| − | background: #F4B248;
| + | |
| − | color: #0e232f;
| + | |
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| + | |
| − | | + | |
| − | | + | |
| − | /*HIde the submenus and removes the padding */
| + | |
| − | #menuContainer li ul {
| + | |
| − | display: none;
| + | |
| − | padding-top:15px;
| + | |
| − | margin-left: -9px;
| + | |
| − | }
| + | |
| − | | + | |
| − | /*Shows the submenus once you hover*/
| + | |
| − | #menuContainer li:hover ul {
| + | |
| − | display: block;
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| + | |
| − | | + | |
| − | /*style the submenu buttons*/
| + | |
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| − | | + | |
| − | | + | |
| − | | + | |
| − | /*CLASSES */
| + | |
| − | /*Clear class for all the pages, adds spacing too*/
| + | |
| − | .clear{
| + | |
| − | clear:both;
| + | |
| − | height: 10px;
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| − | }
| + | |
| − | | + | |
| − | /* highlight box for special messages */
| + | |
| − | .highlightBox {
| + | |
| − | width:500px;
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| − | margin:auto;
| + | |
| − | background-color: #E8D5B7;
| + | |
| − | margin-bottom: 15px;
| + | |
| − | margin-top: 15px;
| + | |
| − | padding: 15px;
| + | |
| − | padding-top: 5px;
| + | |
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| + | |
| − | | + | |
| − | </style>
| + | |
| − | <!-- End of CSS -->
| + | |
| − | | + | |
| − | | + | |
| − | <!-- Start of the template html elements. -->
| + | |
| − | <div id ="mainContainer"><!--This div will close on the page.-->
| + | |
| − | | + | |
| − | <div id="bannerContainer">
| + | |
| − | <br><br>
| + | |
| − |
| + | |
| − | <img src="https://static.igem.org/mediawiki/2017/b/b1/Kbcc-banner.jpg" width="980" height="450" alt="Computer Hope">
| + | |
| − |
| + | |
| − |
| + | |
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| + | |
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| + | |
| − | <!-- This list is your menu, every list item is a menu button and nested listed become submenu buttons -->
| + | |
| − | <ul>
| + | |
| − | <a href="https://2017.igem.org/Team:Kingsborough_NY"><li>HOME</li></a>
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | <a href="#"><li>PROJECT
| + | |
| − | <ul>
| + | |
| − | <a href="https://2017.igem.org/Team:Kingsborough_NY/Design"><li>Description</li></a>
| + | |
| − | <a href="https://2017.igem.org/Team:Kingsborough_NY/Experiments"><li>Experiments & Protocols</li></a>
| + | |
| − | <a href="https://2017.igem.org/Team:Kingsborough_NY/Results"><li>Results</li></a>
| + | |
| − | </ul>
| + | |
| − | </li></a>
| + | |
| − | | + | |
| − | <a href="#"><li>PARTS
| + | |
| − | <ul>
| + | |
| − | <a href=""><li>Team Parts</li></a>
| + | |
| − | | + | |
| − |
| + | |
| − | <a href="https://2017.igem.org/Team:Kingsborough_NY/Attributions"><li>ATTRIBUTIONS</li></a>
| + | |
| − | | + | |
| − | <a href="https://2017.igem.org/Team:Kingsborough_NY/Collaborations"><li>COLLABORATIONS</li></a>
| + | |
| − | | + | |
| − | <a href="https://2017.igem.org/Team:Kingsborough_NY/HP/Silver"><li>HUMAN practices</li></a>
| + | |
| − | | + | |
| − | </ul>
| + | |
| − | </div>
| + | |
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| + | |
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| + | |
| − |
| + | |
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| + | |
| − | | + | |
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| + | |
| − |
| + | |
| − | <p>.</p>
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| − | </div>
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| + | |
Designing a Kill-Switch
We initially attempted to create engineered bacteria that break down nitrogenous waste effectively in wastewater treatment plant. Since our E. coli can survive in saltwater, creating a kill switch for our engineered bacteria will, in theory, prevent our bacteria from thriving outside wastewater treatment plants.
We decided to vary from previous iGEM team efforts in three important ways:
1) We appended the ssrA degradation tag onto our toxin of choice, MazF. This should lead any MazF expressed due to leaky control of the promoter to be destroyed, and not damage the cell.
2) We introduced, in some of our constructs, a supplmentary copy of the cI repressor gene, in cause the light-controlled copy on pDawn was insufficient to control MazF.
3) For both MazF constructs we created, we had counterparts that contained the alpha fragment of LacZ instead of the toxin. This way, we can monitor expression levels without worrying about the cell death that high levels of MazF might cause.
Control of Gene Expression by pDawn alone
3.1 (K2268003): When light is present, c1 WILL NOT be produced. In theory, the expression of LacZ should be maximal. This is intended to be the non-lethal version of our kill-switch.
4.1 (K2268004): When light is present, c1 WILL NOT be produced. In theory, the expression of MazF should be maximal.
Control of Gene Expression by pDawn and supplementary cI Repressor
3.2(K2268005): When light is present with no ITPG, c1 WILL NOT be produced. In theory, maximal expression of LacZ should be produced. This is a non-lethal version of our kill switch.
4.2(K2268006): When light is present with no ITPG, c1 WILL NOT be produced. In theory, maximal expression of MazF should be produced.
Light control by pDawn
In order for our design to work, the light induction module present on pDawn must be operational. We tested pDawn alone, in the dark and in light, to verify that expression of the RFP reporter only occurs in the light (see petri dish below). We also observed this red fluorescence when pDawn was used in conjunction with our constructs (data not shown).
