Difference between revisions of "Team:Exeter/Proof"

 
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{{Exeter}}
 
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<html>
 
<html>
 +
</div></div></div></div></div>
 +
  <meta charset="utf-8">
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  <meta name="viewport" content="width=device-width, initial-scale=1">
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  <link rel="stylesheet" href="http://maxcdn.bootstrapcdn.com/bootstrap/3.3.6/css/bootstrap.min.css">
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  <script src="https://ajax.googleapis.com/ajax/libs/jquery/1.12.4/jquery.min.js"></script>
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  <script src="http://maxcdn.bootstrapcdn.com/bootstrap/3.3.6/js/bootstrap.min.js"></script>
 +
 
 +
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</head>
 +
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<body>
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<style type="text/css">
 +
  @media (max-width: 920px) {
 +
  .navbar-header {
 +
      float: none;
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  }
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  .navbar-left,.navbar-right {
 +
      float: none !important;
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  .navbar-toggle {
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      display: block;
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  }
 +
  .navbar-collapse {
 +
      box-shadow: inset 0 0 0 rgba(255,255,255,0.1);
 +
  }
 +
  .navbar-fixed-top {
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      top: 0;
 +
      border-width: 0 0 1px;
 +
  }
 +
  .navbar-collapse.collapse {
 +
      display: none!important;
 +
  }
 +
 
 +
  .navbar-nav {
 +
      float: none!important;
 +
      margin-top: 7.5px;
 +
  }
 +
  .navbar-nav>li {
 +
      float: none;
 +
  }
 +
  .navbar-nav>li>a {
 +
      padding-top: 10px;
 +
      padding-bottom: 10px;
 +
  }
 +
  .collapse.in{
 +
      display:block !important;
 +
  }
 +
  #soc_1, #soc_2, #soc_3{
 +
display:none;
 +
  }
 +
}
 +
#links{
 +
color:#47BCC2;
 +
/*font-size:20px;*/
 +
font-size:1.2vw;
 +
margin:0 0.9vw 0.5vh 0.9vw;
 +
padding:0;
 +
line-height:0px;
 +
padding-top:28px;
 +
}
 +
/*Fixes font size when nav-bar is collapsed*/
 +
@media (max-width: 920px) {
 +
#links{
 +
font-size:20px;
 +
}
 +
#links_drop{
 +
margin:0.9vw -0.4vw 0 -0.2vw;
 +
}
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}
 +
#links:hover{
 +
color:#339499;
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}
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#links:active{
 +
color:#339499;
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}
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.navbar{
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background-color:#e8e8e8;
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border-bottom:none;
 +
min-height:54px;
 +
    margin:4px 0 0 0;
 +
}
 +
.navbar-inverse .navbar-toggle .icon-bar {/*teal toggle */
 +
    background-color:#47BCC2;
 +
}
 +
.navbar-inverse .navbar-toggle { /*Removes toggle bobrder*/
 +
border:none;
 +
background-color:#e8e8e8;
 +
}
 +
.navbar-inverse .navbar-toggle{ /*Removes toggle bobrder*/
 +
border:none;
 +
background-color:#e8e8e8;
 +
}
 +
.navbar-inverse .navbar-toggle collapse{ /*Removes toggle bobrder*/
 +
border:none;
 +
background-color:#e8e8e8;
 +
}
 +
.navbar-inverse .navbar-toggle:hover {
 +
background-color:#47BCC2;
 +
}
 +
.navbar-inverse .navbar-toggle:focus, .navbar-inverse .navbar-toggle:hover {
 +
    background-color:#47BCC2;
 +
}
 +
.navbar-inverse .navbar-toggle:hover .icon-bar, .navbar-inverse .navbar-toggle:focus .icon-bar{
 +
background-color:#eeeeee;
 +
}
 +
/*soc classes controll css for media icons*/
 +
#soc{
 +
margin-top:-12px;
 +
 +
}
 +
/****Social Media navbar icons****/
 +
/*youtube*/
 +
#soc_1{
 +
margin-right:3px;
 +
height:50px;
 +
width:48px;
 +
}
 +
/*facebook*/
 +
#soc_2{
 +
margin-right:3px;
 +
height:50px;
 +
width:48px;
 +
}
 +
/*Twitter*/
 +
#soc_3{
 +
margin-right:3px;
 +
margin-right:18px;
 +
height:50px;
 +
width:48px;
 +
}
  
 +
/*Navbar exgem logo styling*/
 +
#logo, #logo_inverse{
 +
margin-top:-12px;
 +
vertical-align:middle;
 +
width:48px;
 +
height:48px;
 +
/*Alligns logo with toggled list on thin screens*/
 +
margin-left:13px;
 +
margin-right:-12px;
 +
}
 +
/*Logo is visable on page load by default*/
 +
#logo{
 +
display:block;
 +
}
 +
/*The inverse logo is not visable by default, but becomes visable*/
 +
/*when #logo_button is hovered over (found in jquery)*/
 +
#logo_inverse{
 +
display:none;
 +
}
  
 +
/*Displays brand inline with ExGem*/
 +
.navbar-brand > img {
 +
    display: inline;
 +
}
 +
.navbar-right a{
 +
height:50px;
 +
width:48px;
 +
}
 +
#Top_link.navbar-brand{/*Top link styling*/
 +
color:#47BCC2;
 +
font-size:25px;
 +
margin-bottom:5px;
 +
opacity:0.6
 +
}
 +
#topnav{
 +
padding-bottom:0px;
 +
position:fixed;
 +
}
 +
#topnav (max-width: 767px) {
 +
display:none;
 +
}
 +
#text{/*Top link text*/
 +
color:#47BCC2;
 +
}
 +
#myNavbar{
 +
border: none;
 +
}
 +
/*sticky footer*/
 +
.wrap {
 +
  min-height: 100%;
 +
  margin-bottom: -65px;  /* equal to footer height */
 +
}
 +
.wrap:after {
 +
  content: "";
 +
  display: block;
 +
}
 +
.footer, .wrap:after {
 +
  height: 60px;
 +
  padding-top:5px;
 +
}
 +
.flare .wrap:after{
 +
height:5px;
 +
}
 +
.flare{
 +
background-color:#8cd5d9; 
 +
padding-top:3px;
 +
position:relative;
 +
bottom:0px;
 +
width:100%;
 +
}
 +
.footer{
 +
background-color:#e8e8e8;
 +
}
 +
/*Hides igm logo and team name*/
 +
#sideMenu, #top_title {display:none;}
 +
#bodyContent h1, #bodyContent h2, #bodyContent h3, #bodyContent h4, #bodyContent h5 { margin-bottom: 0px;}
 +
#top_title{visibility: hidden; background:#eeeeee;}
 +
#bodyContent{background:#eeeeee;}
 +
#mw-content-text{background:#eeeeee;}
 +
#HQ_page{background:none; height:0px; width:0px;}
 +
#content{background:none;}
 +
#globalWrapper{height:0px;background:#eeeeee;}
 +
/*end of hacky css :) */
 +
/*There is a <p></p> at bottom of iGEM page. In order to keep footer*/
 +
/*On bottom of page we have styled <p> tags to have no margin or border*/
 +
/*Therefore on all of our paragraphs need to id "pp" to work properly*/
 +
p{
 +
        margin:0;
 +
        padding:0;
 +
        border-style:none;
 +
}
 +
#pp{
 +
padding-top:20px;
 +
padding-right:40px;
 +
padding-left:40px;
 +
font-size:150%;
 +
}
 +
.container ul, .container ol{
 +
font-size:140%;
 +
padding-right:40px;
 +
padding-left:60px;
 +
padding-top:10px;
 +
}
 +
/*Heading styles*/
 +
h1, h2, h3, h4, h5{
 +
text-align:center;
 +
color:#339499;
 +
font-weight:500;
 +
    padding-left: 40px;
 +
    padding-right: 40px;
 +
padding-top: 40px;
 +
padding-bottom:0;
 +
}
 +
h1{font-size:340%;}
 +
h2{font-size:320%;}
 +
h3{font-size:290%;}
 +
h4{font-size:260%;}
 +
h5{font-size:220%;}
 +
h6{
 +
color:#339499;
 +
font-weight:500;
 +
font-size:200%;
 +
padding-left:40px;
 +
padding-top: 20px;
 +
padding-bottom:-10px;
 +
}
 +
/*Italics for Quotes using i tag*/
 +
q{
 +
text-align:center;
 +
color:#339499;
 +
padding-left:10%;
 +
padding-right:10%;
 +
font-size:120%;
 +
font-style: italic;
 +
}
 +
/*Styling for links*/
 +
a{
 +
color:#339499;
 +
font-weight:550;
 +
}
 +
/**page content styling starts**/
 +
body{
 +
background-color:#eeeeee;
 +
}
 +
h1{
 +
text-align:center;
 +
        border-style:none;
 +
}
 +
#Section_Link, #Section_Link:visited{
 +
display:block;
 +
margin:0 auto 10px auto;
 +
width:14px;
 +
}
 +
#Section_Link:hover{
  
−
<h3>★  ALERT! </h3>
+
}
−
<p>This page is used by the judges to evaluate your team for the <a href="https://2016.igem.org/Judging/Medals">gold medal criterion for proof of concept</a>. </p>
+
#sectionGap, #sectionGap:focus, #contentTitle{
 +
min-width:100%;
 +
min-height:10vh;
 +
background:#e8e8e8;
 +
display:block;
 +
font-size:400%;
 +
text-align:center;
 +
color:#47BCC2;
 +
text-decoration:none;
 +
border-style:none none solid none;
 +
border-width:3px;
 +
border-color:#8cd5d9;
 +
}
 +
#sectionGap:hover, #sectionGap:active{
 +
color:#339499;
 +
}
 +
.container{
 +
min-width:100%;
 +
padding:0;
 +
}
 +
.div_vl{
 +
padding-top:2%;
 +
height:35vw;
 +
width:95vw;
 +
display:block;
 +
margin-left:auto;
 +
margin-right:auto;
 +
}
 +
.div_vl.backgroundimage{
 +
background-image: url('https://static.igem.org/mediawiki/2016/9/93/T--Exeter--Home_Background1.png');
 +
background-repeat:no-repeat;
 +
background-size: 100% auto;
 +
width:100%;
 +
height:88vh;
 +
}
 +
#title{
 +
style="font-family: 'Arial';
 +
color:#4e4e4e;
 +
text-align:center;
 +
margin-bottom:20px;
 +
font-size:400%;
 +
}
 +
.div_content{
 +
padding:0;
 +
min-height:100vh;
 +
width:100%;
 +
background:#eeeeee;
 +
}
  
 +
/*div_banner contains to links at the top of each page to link to*/
 +
/*each section of the page*/
 +
.div_banner{
 +
max-width:90vw;
 +
margin:12vh auto 5vh auto;
 +
display:block;
 +
height:8vh;
 +
}
  
−
<p> Delete this box in order to be evaluated for this medal. See more information at <a href="https://2016.igem.org/Judging/Pages_for_Awards/Instructions"> Instructions for Pages for awards</a>.</p>
+
/*Contains styling for the left and right pictures of the banner*/
 +
.subdiv_banner{
 +
height:8vh;
 +
padding:0;
 +
margin:auto;
 +
position:relative;
 +
bottom:0;
 +
right:0;
 +
}
 +
.subdiv_banner.left{
 +
float:left;
 +
display:block;
 +
}
 +
.subdiv_banner.right{
 +
float:right;
 +
display:block;
 +
}
 +
.subdiv_banner.middle{
 +
height:100%;
 +
}
 +
/*Positions the images in correct place in the divs*/
 +
.subdiv_banner.left img, .subdiv_banner.right img{
 +
width:100%;
 +
position: relative;
 +
top: 38%;
 +
-ms-transform: translateY(-50%) /* IE 9 */
 +
    -webkit-transform: translateY(-50%) /* Chrome, Safari, Opera */
 +
    transform: translateY(-50%);
 +
}
 +
.banner_link,.banner_link:visited{
 +
position: relative;
 +
height:100%;
 +
padding:0, 2px!important;
 +
color:#47BCC2;
 +
text-align:center;
 +
font-size:1.5vw;
 +
/*Max font size where "section" and "1" don't appear on*/
 +
}
 +
.banner_link span.oneline{
 +
position: relative;
 +
top: 34.5%;
 +
-ms-transform: translateY(-50%) /* IE 9 */
 +
    -webkit-transform: translateY(-50%) /* Chrome, Safari, Opera */
 +
    transform: translateY(-50%);
 +
}
 +
.banner_link span.twoline{
 +
position: relative;
 +
top: 7.5%;
 +
-ms-transform: translateY(-50%) /* IE 9 */
 +
    -webkit-transform: translateY(-50%) /* Chrome, Safari, Opera */
 +
    transform: translateY(-50%);
 +
}
 +
.banner_link:hover{
 +
text-decoration:none;
 +
color:#339499;
 +
}
 +
.link_fix{
 +
display:block;
 +
position:relative;
 +
height:1px;
 +
top:-15px;
 +
}
 +
/*Mobile and small screen css*/
 +
@media (max-width: 767px){
 +
.link_fix{
 +
display:none;
 +
}
 +
.div_vl.backgroundimage{
 +
background-image: url('#');
 +
background:#ddd;
 +
}
 +
#title{
 +
font-size:150%;
 +
}
 +
/*Makes side pictures on banner invisible on small screens*/
 +
/*as it clutters screen*/
 +
.subdiv_banner.left, .subdiv_banner.right{
 +
display:none;
 +
}
 +
}
 +
#dropdownMenu1{
 +
margin-top:-0.15vw;
 +
background:#e8e8e8;
 +
border-style:none;
 +
}
 +
@media (min-width: 767px){
 +
.dropdown-menu{
 +
margin-top:24px;
 +
left:-19px;
 +
border-radius:0px;
 +
padding:4px;
 +
}
 +
}
 +
/*Mobile and small screen css*/
 +
@media (max-width: 767px){
 +
.div_vl.backgroundimage{
 +
height:37vh;
 +
background-size: auto 200%;
 +
}
 +
.div_l{
 +
display:none;
 +
}
 +
#logo_Banner_Desktop{
 +
display:none;
 +
}
 +
#logo_Banner_Mobile{
 +
display:block;
 +
width:100%;
 +
max-width:200px;
 +
margin:auto;
 +
}
 +
#dropdownMenu1{
 +
padding:2.5% 0 2% 0;
 +
margin:-1.6% auto -1.4% 0.9%;
 +
}
 +
#links{
 +
padding:10px 0;
 +
margin-left:0.85vw;
 +
}
 +
}
 +
</style>
 +
<div id="top_page" class="link_fix"></div>
 +
<nav id="Top" class="navbar navbar-inverse navbar-static-top">
 +
  <div class="container-fluid">
 +
    <div class="navbar-header">
 +
        <button type="button" class="navbar-toggle" data-toggle="collapse" data-target="#myNavbar">
 +
          <span class="icon-bar"></span>
 +
          <span class="icon-bar"></span>
 +
          <span class="icon-bar"></span>                       
 +
</button>
 +
      <a id="logo_button" class="navbar-brand" href="https://2016.igem.org/Team:Exeter">
 +
<img id="logo" src="https://static.igem.org/mediawiki/2016/7/75/T--Exeter--Template--Logo2.png"/>
 +
<img id="logo_inverse" src="https://static.igem.org/mediawiki/2016/2/23/T--Exeter--Template_Logo2_inverse.png"/>
 +
  </a>
 +
 
 +
    </div>
 +
    <div>
 +
        <div class="collapse navbar-collapse" id="myNavbar">
 +
<ul class="nav navbar-nav">
 +
<li><div id="links" style="margin:0;" class="dropdown">
 +
  <button class="dropdown-toggle" type="button" id="dropdownMenu1" data-toggle="dropdown" aria-haspopup="true" aria-expanded="true">
 +
    <span style="margin-bottom:4px;">Lab</span>
 +
    <span class="caret"></span>
 +
  </button>
 +
  <ul class="dropdown-menu" style="background:#e8e8e8;margin-left:25px;" aria-labelledby="dropdownMenu1">
 +
<li><a id="links" style="margin:10px 0 30px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/Project">Lab Project</a></li>
 +
    <li><a id="links" style="margin:30px 0 10px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/Labbook">Lab Book</a></li>
  
 +
  </ul>
 +
</div></li>
 +
<li ><a id="links"href="https://2016.igem.org/Team:Exeter/Parts">Parts</a></li>
 +
<li ><a id="links"href="https://2016.igem.org/Team:Exeter/Team">Team</a></li>
 +
<li ><a id="links" href="https://2016.igem.org/Team:Exeter/Interlab">InterLab</a></li>
 +
<li><div id="links" style="margin:0;" class="dropdown">
 +
  <button class="dropdown-toggle" type="button" id="dropdownMenu1" data-toggle="dropdown" aria-haspopup="true" aria-expanded="true">
 +
    <span style="margin-bottom:4px;">Human Practices</span>
 +
    <span class="caret"></span>
 +
  </button>
 +
  <ul class="dropdown-menu" style="background:#e8e8e8;margin-left:25px;" aria-labelledby="dropdownMenu1"> 
 +
    <li><a id="links" style="margin:10px 0 10px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/Integrated_Practices">Integrated</a></li>
 +
<li><a id="links" style="background:none;line-height:0.7vh;margin:30px 0 10px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/Engagement">Public Engagement<br /><br /><br /> & Education</a></li>
 +
<li><a id="links" style="margin:30px 0 10px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/Log">Log</a></li>
  
 +
  </ul>
 +
</div></li>
  
 +
<li ><a id="links"href="https://2016.igem.org/Team:Exeter/Attributions">Attributions</a></li>
  
 +
<li><div id="links" style="margin:0;" class="dropdown">
 +
  <button class="dropdown-toggle" type="button" id="dropdownMenu1" data-toggle="dropdown" aria-haspopup="true" aria-expanded="true">
 +
    <span style="margin-bottom:4px;">Awards</span>
 +
    <span class="caret"></span>
 +
  </button>
 +
  <ul class="dropdown-menu" style="background:#e8e8e8;margin-left:25px;" aria-labelledby="dropdownMenu1">
 +
 
 +
<li><a id="links" style="margin:10px 0 30px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/Awards">Medals</a></li>
 +
<li><span style="margin:10px 0 30px 2px;padding:0;">Special pages</span></li>
 +
<li><a id="links" style="margin:30px 0 10px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/HP/Silver">HP Silver</a></li>
 +
<li><a id="links" style="margin:30px 0 10px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/HP/Gold">HP Gold</a></li>
  
 +
  </ul>
 +
</div></li>
 +
<li ><a id="links"href="https://2016.igem.org/Team:Exeter/Model">Models</a></li>
 +
<li ><a id="links"href="https://2016.igem.org/Team:Exeter/Collaborations">Collaborations</a></li>
  
−
<div class="column full_size">
 
  
  
−
<p>
+
</ul>
−
iGEM teams are great at making things work! We value teams not only doing an incredible job with theoretical models and experiments, but also in taking the first steps to make their project real.  
+
<ul class="nav navbar-nav navbar-right" >
−
</p>
+
<li class="media_icon">
 +
<a  id="soc_1" href="https://www.youtube.com/channel/UC31qfG4hnm8gRHDCkrBtAiQ">
 +
<img id = "soc" src="https://static.igem.org/mediawiki/2016/2/23/You.png"/>
 +
</a>
 +
</li>
 +
    <li class="media_icon" >
 +
<a id="soc_2"href="https://www.facebook.com/ExeteriGEM2016/?ref=aymt_homepage_panel">
 +
<img id = "soc" src="https://static.igem.org/mediawiki/2016/5/51/Fb.png"/>
 +
</a>
 +
</li>
 +
    <li class="media_icon" >
 +
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<div class="container">
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<h1>Proof of Concept</h1>
 +
<h3>Continuous culture</h3>
 +
<p id="pp">The main focus of Project: Exepire in the lab was the robustness of kill switches in real world conditions. By looking at the effectiveness of the kill switches in continuous culture we have begun to show potential failure rates over time. By simulating a continuous culture that would take place on a much larger scale in industry, we have shown the potential failures that need to be addressed if kill switches are to replace traditional chemical and physical bio-containment. </p>
  
−
 
+
<p id="pp">Before starting the project we spoke to Prof. Robert Beardmore EPSRC Leadership Fellow in the Mathematical Biosciences at Exeter University. Much of his research has been into antibiotic resistance. We discussed how high selection pressure is applied by prolonged use of antibiotics and how kill switches may be analogous to this. It is clear that cells which develop a mutation that inactivates the kill switch would be strongly selected for. It was estimated that functional loss of the kill switch would occur in a short amount of time as a result, and if this was the case, could have strong implications for kill switch longevity. To test this we decided to use a ministat to perform a continuous culture. The ministat was developed in the Dunham lab at the University of Washington (Miller <i>et al</i>, 2013). Each ministat chamber is fed from its own media container via a peristaltic pump, with the culture volume set by the height of the effluent needle in the chamber. Air is bubbled through flasks of water to hydrate it and then used to agitate the culture. Chambers were inoculated with freshly transformed <i>E. coli</i> BL21 (DE3) and samples taken to test if the kill switches were still viable. By simulating in miniature how a kill switch might behave in an industrial setting, the ministat provides a proof of concept for how a kill switch might be maintained in larger chemostats during a continuous culture. A protocol for running experiments in the ministat can be found <a href="#MinistatProt">here</a>
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<h4> What should we do for our proof of concept? </h4>
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−
<p>
+
−
You can assemble a device from BioBricks and show it works. You could build some equipment if you're competing for the hardware award. You can create a working model of your software for the software award. Please note that this not an exhaustive list of activities you can do to fulfill the gold medal criterion. As always, your aim is to impress the judges!
+
 
</p>
 
</p>
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<br>
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<br>
  
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<span class="caption">Media container used to feed a single ministat chamber.</span>
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<img style="max-width:100%;" src="https://static.igem.org/mediawiki/2016/6/6a/T--Exeter--pump.JPG"> 
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<span class="caption">Peristaltic pump</span>
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<img style="max-width:100%;" src="https://static.igem.org/mediawiki/2016/a/a3/T--Exeter--effluent.JPG"> 
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<span class="caption">Ministat chambers in heatblock and 1 litre Duran bottle used to collect effluent</span>
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<div class="col-xs-12">
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<img style="max-width:100%;padding: 5px 30% 5px 30%;" src="https://static.igem.org/mediawiki/2016/8/8a/T--Exeter--ministat.jpg">
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<div class="col-xs-3"></div>
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<div class="col-xs-6"><span class="caption" style="padding: 5px 30% 5px 30%;">Ministat running a preliminary experiment to calibrate parameters such as dilution rate and temperature of the heat block. 50 ml burettes used here to accurately measure effluent levels. 1 litre Duran bottles were used for effluent collection in the main experiment due to greater volumes of effluent. </span></div>
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<div class="col-xs-3"></div>
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<p id="pp">Our own growth curve was performed to determine the maximum specific growth rate of <i>E. coli </i> BL21 (DE3) in our lab, but could not be conducted for a sufficient length of time to be accurate. A maximum specific growth rate value of 1.730 was used (Cox, 2004). The ministat must be set to a flow rate at which dilution rate is less than maximum specific growth rate. This prevents the culture being washed out of the growth chambers. The dilution rate of the culture was calculated by measuring flow rate at a setting of 7.5 rpm on the peristaltic pump. For practical reasons the pump could not be run faster than this due to the amount of media needed. The dilution rate was set at 0.2 which produced cultures that grew at an average OD of 3.47 for KillerRed samples, 3.64 for KillerOrange samples and 3.17 for lysozyme samples. The ministat must be set to flow rate at which dilution rate is below the maximum specific growth rate. This prevents the culture being washed out of the chamber. OD was measured daily with a Bug Lab OD scanner. When the same sample was measured in a tecan infinite 200 pro plate reader the Bug Lab showed reading approximately three times higher. The difference between the samples was consistent regardless of the method used to measure OD.
 +
</p>
 +
<h6><u>Ministat experiment</u></h6>
 +
<p id="pp">All samples from the ministat were tested using the KillerRed, KillerOrange protocol found <a href="#KRKOProt">
 +
here</a>. Glycerol stocks were made from the samples taken at each time interval, testing was done using these glycerol stocks.</p>
 +
<p id="pp">The following graphs show the average number of colonies of samples taken at 0 h, 24 h, 120 h and 168 h of continuous culture and then tested in the light box. Values were
 +
averaged across three biological repeats. Colonies were not counted above 300 and so this is the maximum value given. All samples were induced to a final concentration of 0.2 nM IPTG. All samples were
 +
diluted 1000 times in a final volume of 4.5 ml liquid broth (LB). Error bars represent the standard error of the mean.</p>
  
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        <img src="https://static.igem.org/mediawiki/2016/7/78/T--Exeter--KRcont.jpg"
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style="max-width:100%;margin:auto;display:block;">
 +
            <span class="caption">Fig. 14. Comparison of CFUs formed by KillerRed exposed to light and kept in the dark for each sample taken from the ministat. The efficiency of the kill switch decreases over time as shown by the increasing number of CFUs.</span>
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<br>
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    <div class="col-xs-6" style="padding:5px 10% 5px 2%;margin:0;">
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        <img src="https://static.igem.org/mediawiki/2016/d/d0/T--Exeter--KOcont.jpg"
 +
style="max-width:100%;margin:auto;display:block;">
 +
            <span class="caption">Fig. 15. Comparison of CFUs formed by KillerOrange exposed to light and kept in the dark. The efficiency of the kill switch decreases over time as shown by the increasing number of CFUs. The effect is not as obvious in KillerOrange compared to KillerRed as the starting efficiency of KillerOrange is lower. </span>
 +
<br>
 +
<br>
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    </div>
 
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</div>
 +
 +
 +
<h6>Discussion</h6>
 +
 +
<p id="pp">The continuous culture of KillerRed showed a 15 fold increase in the percentage of viable cells after 168 hrs. A similar pattern is shown for KillerOrange but with around a two fold increase. Both KillerRed and KillerOrange show greater numbers of colonies forming over time (Fig. 14 & 15). This number approaches the amount produced in the dark condition by 168 hrs.
 +
The average fluorescence reading for 0 hr KillerRed samples was 506.3 A.U (recorded at an average OD of of 0.745). After 168
 +
hrs the average fluorescence reading  was 436 A.U (at an average OD of 0.96). It seems unlikely due to the readings being
 +
similar that a mutation has occurred in the kill switch itself. As fluorescence is proportional to the amount of ROS
 +
being produced, up regulation of native <i>E. coli</i> enzymes that mitigate the effects of ROS may be the cause of the increase
 +
in cell survival. Future transcriptome analysis could provide interesting data on the mechanism of this change, this was
 +
unfortunately beyond the scope of this project. This shows that there may be many ways for bacteria to circumvent the effects of a kill switch given the high selection pressure they pose.</p>
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Latest revision as of 14:46, 19 October 2016