Team:METUHS-Ankara/project.html

From 2014hs.igem.org

(Difference between revisions)
 
(7 intermediate revisions not shown)
Line 3: Line 3:
  <!-- BC_OBNW -->
  <!-- BC_OBNW -->
<head>
<head>
-
 
+
<style>
 +
#p-logo {display:none}
 +
#content {top: -120px}
 +
#menubar {top: -30px; font-size: 65%}
 +
#search-controls {display : none}
 +
body
 +
{
 +
background-color:#22B573;
 +
}
 +
</style>
<meta http-equiv="Content-type" content="text/html;charset=UTF-8" />
<meta http-equiv="Content-type" content="text/html;charset=UTF-8" />
<title>Project</title>
<title>Project</title>
Line 10: Line 19:
<link rel="shortcut icon" href="http://fisofowi.netii.net/images/favicon.ico?319024345" />
<link rel="shortcut icon" href="http://fisofowi.netii.net/images/favicon.ico?319024345" />
<link rel="stylesheet" type="text/css" href="http://fisofowi.netii.net/css/site_global.css?417434784" />
<link rel="stylesheet" type="text/css" href="http://fisofowi.netii.net/css/site_global.css?417434784" />
-
<link rel="stylesheet" type="text/css" href="http://fisofowi.netii.net/css/master_frame.css?173419010" />
+
<link rel="stylesheet" type="text/css" href="http://fisofowi.netii.net/css/master_frame.css?56859511" />
-
<link rel="stylesheet" type="text/css" href="http://fisofowi.netii.net/css/project.css?173314404" id="pagesheet" />
+
<link rel="stylesheet" type="text/css" href="http://fisofowi.netii.net/css/project.css?4095008318" id="pagesheet" />
<script type="text/javascript">
<script type="text/javascript">
   document.documentElement.className += ' js';
   document.documentElement.className += ' js';
Line 17: Line 26:
</script>
</script>
<script type="text/javascript">
<script type="text/javascript">
-
   document.write('\x3Cscript src="' + (document.location.protocol == 'https:' ? 'https:' : 'http:') + '//webfonts.creativecloud.com/abel:n4:all;droid-sans:n4,n7:all.js" type="text/javascript">\x3C/script>');
+
   document.write('\x3Cscript src="' + (document.location.protocol == 'https:' ? 'https:' : 'http:') + '//webfonts.creativecloud.com/droid-sans:n4,n7:all;abel:n4:all;paytone-one:n4:all.js" type="text/javascript">\x3C/script>');
</script>
</script>
<meta name="generator" content="7.0.314.244" />
<meta name="generator" content="7.0.314.244" />
Line 24: Line 33:
<!-- CSS -->
<!-- CSS -->
<!--[if lt IE 9]>
<!--[if lt IE 9]>
-
   <link rel="stylesheet" type="text/css" href="http://fisofowi.netii.net/css/iefonts_project.css?4117494052"/>
+
   <link rel="stylesheet" type="text/css" href="http://fisofowi.netii.net/css/iefonts_project.css?15414100"/>
   <![endif]-->
   <![endif]-->
<!-- Other scripts -->
<!-- Other scripts -->
Line 42: Line 51:
     <ul class="MenuBar clearfix grpelem" id="menuu2018"><!-- horizontal box -->
     <ul class="MenuBar clearfix grpelem" id="menuu2018"><!-- horizontal box -->
       <li class="MenuItemContainer clearfix grpelem" id="u2037"><!-- vertical box -->
       <li class="MenuItemContainer clearfix grpelem" id="u2037"><!-- vertical box -->
-
       <a class="nonblock nontext MenuItem MenuItemWithSubMenu clearfix colelem" id="u2040" href="index.html"><!-- horizontal box --><div class="MenuItemLabel NoWrap clearfix grpelem" id="u2041-4"><!-- content --><p>Home</p></div></a>
+
       <a class="nonblock nontext MenuItem MenuItemWithSubMenu rounded-corners clearfix colelem" id="u2040" href="/Team:METUHS-Ankara/index.html"><!-- horizontal box --><div class="MenuItemLabel NoWrap clearfix grpelem" id="u2041-4"><!-- content --><p>Home</p></div></a>
       </li>
       </li>
       <li class="MenuItemContainer clearfix grpelem" id="u2030"><!-- vertical box -->
       <li class="MenuItemContainer clearfix grpelem" id="u2030"><!-- vertical box -->
-
       <a class="nonblock nontext MenuItem MenuItemWithSubMenu MuseMenuActive clearfix colelem" id="u2033" href="project.html"><!-- horizontal box --><div class="MenuItemLabel NoWrap clearfix grpelem" id="u2035-4"><!-- content --><p>Project</p></div></a>
+
       <a class="nonblock nontext MenuItem MenuItemWithSubMenu MuseMenuActive rounded-corners clearfix colelem" id="u2033" href="/Team:METUHS-Ankara/project.html"><!-- horizontal box --><div class="MenuItemLabel NoWrap clearfix grpelem" id="u2035-4"><!-- content --><p>Project</p></div></a>
       </li>
       </li>
       <li class="MenuItemContainer clearfix grpelem" id="u2044"><!-- vertical box -->
       <li class="MenuItemContainer clearfix grpelem" id="u2044"><!-- vertical box -->
-
       <a class="nonblock nontext MenuItem MenuItemWithSubMenu clearfix colelem" id="u2047" href="team.html"><!-- horizontal box --><div class="MenuItemLabel NoWrap clearfix grpelem" id="u2048-4"><!-- content --><p>Team</p></div></a>
+
       <a class="nonblock nontext MenuItem MenuItemWithSubMenu rounded-corners clearfix colelem" id="u2047" href="/Team:METUHS-Ankara/team.html"><!-- horizontal box --><div class="MenuItemLabel NoWrap clearfix grpelem" id="u2048-4"><!-- content --><p>Team</p></div></a>
       </li>
       </li>
       <li class="MenuItemContainer clearfix grpelem" id="u2058"><!-- vertical box -->
       <li class="MenuItemContainer clearfix grpelem" id="u2058"><!-- vertical box -->
-
       <a class="nonblock nontext MenuItem MenuItemWithSubMenu clearfix colelem" id="u2059" href="extras.html"><!-- horizontal box --><div class="MenuItemLabel NoWrap clearfix grpelem" id="u2060-4"><!-- content --><p>Extras</p></div><div class="clip_frame grpelem" id="u2062"><!-- image --></div></a>
+
       <a class="nonblock nontext MenuItem MenuItemWithSubMenu rounded-corners clearfix colelem" id="u2059" href="/Team:METUHS-Ankara/extras.html"><!-- horizontal box --><div class="MenuItemLabel NoWrap clearfix grpelem" id="u2060-4"><!-- content --><p>Extras</p></div><div class="clip_frame grpelem" id="u2062"><!-- image --></div></a>
       <div class="SubMenu  MenuLevel1 clearfix" id="u2063"><!-- vertical box -->
       <div class="SubMenu  MenuLevel1 clearfix" id="u2063"><!-- vertical box -->
         <ul class="SubMenuView clearfix colelem" id="u2064"><!-- vertical box -->
         <ul class="SubMenuView clearfix colelem" id="u2064"><!-- vertical box -->
         <li class="MenuItemContainer clearfix colelem" id="u2065"><!-- horizontal box -->
         <li class="MenuItemContainer clearfix colelem" id="u2065"><!-- horizontal box -->
-
           <a class="nonblock nontext MenuItem MenuItemWithSubMenu clearfix grpelem" id="u2066" href="attributions.html"><!-- horizontal box --><div class="MenuItemLabel NoWrap clearfix grpelem" id="u2068-4"><!-- content --><p>Attributions</p></div></a>
+
           <a class="nonblock nontext MenuItem MenuItemWithSubMenu clearfix grpelem" id="u2066" href="/Team:METUHS-Ankara/attributions.html"><!-- horizontal box --><div class="MenuItemLabel NoWrap clearfix grpelem" id="u2068-4"><!-- content --><p>Attributions</p></div></a>
         </li>
         </li>
         <li class="MenuItemContainer clearfix colelem" id="u2072"><!-- horizontal box -->
         <li class="MenuItemContainer clearfix colelem" id="u2072"><!-- horizontal box -->
-
           <a class="nonblock nontext MenuItem MenuItemWithSubMenu clearfix grpelem" id="u2075" href="fun.html"><!-- horizontal box --><div class="MenuItemLabel NoWrap clearfix grpelem" id="u2077-4"><!-- content --><p>Fun</p></div></a>
+
           <a class="nonblock nontext MenuItem MenuItemWithSubMenu clearfix grpelem" id="u2075" href="/Team:METUHS-Ankara/fun.html"><!-- horizontal box --><div class="MenuItemLabel NoWrap clearfix grpelem" id="u2077-4"><!-- content --><p>Fun</p></div></a>
         </li>
         </li>
         <li class="MenuItemContainer clearfix colelem" id="u2079"><!-- horizontal box -->
         <li class="MenuItemContainer clearfix colelem" id="u2079"><!-- horizontal box -->
-
           <a class="nonblock nontext MenuItem MenuItemWithSubMenu clearfix grpelem" id="u2082" href="referance.html"><!-- horizontal box --><div class="MenuItemLabel NoWrap clearfix grpelem" id="u2085-4"><!-- content --><p>Referance</p></div></a>
+
           <a class="nonblock nontext MenuItem MenuItemWithSubMenu clearfix grpelem" id="u2082" href="/Team:METUHS-Ankara/reference.html"><!-- horizontal box --><div class="MenuItemLabel NoWrap clearfix grpelem" id="u2085-4"><!-- content --><p>Reference</p></div></a>
         </li>
         </li>
         </ul>
         </ul>
Line 67: Line 76:
       </li>
       </li>
       <li class="MenuItemContainer clearfix grpelem" id="u2051"><!-- vertical box -->
       <li class="MenuItemContainer clearfix grpelem" id="u2051"><!-- vertical box -->
-
       <a class="nonblock nontext MenuItem MenuItemWithSubMenu clearfix colelem" id="u2052" href="contact-us.html"><!-- horizontal box --><div class="MenuItemLabel NoWrap clearfix grpelem" id="u2055-4"><!-- content --><p>Contact Us</p></div></a>
+
       <a class="nonblock nontext MenuItem MenuItemWithSubMenu rounded-corners clearfix colelem" id="u2052" href="/Team:METUHS-Ankara/contact-us.html"><!-- horizontal box --><div class="MenuItemLabel NoWrap clearfix grpelem" id="u2055-4"><!-- content --><p>Contact Us</p></div></a>
       </li>
       </li>
     </ul>
     </ul>
 +
    <div class="clearfix grpelem" id="u3158-4"><!-- content -->
 +
      <div>
 +
<p>CO&#45;sense</p>
 +
</div>
 +
    </div>
 +
    <div class="clip_frame clearfix grpelem" id="u3225"><!-- image -->
 +
      <div id="u3225_clip">
 +
      <img class="position_content" id="u3225_img" src="http://fisofowi.netii.net/images/output_nl44aa.gif" alt="" width="178" height="136" />
 +
      </div>
 +
    </div>
     </div>
     </div>
     <div class="TabbedPanelsWidget clearfix colelem" id="tab-panelu326"><!-- vertical box -->
     <div class="TabbedPanelsWidget clearfix colelem" id="tab-panelu326"><!-- vertical box -->
Line 100: Line 119:
     </div>
     </div>
     <div class="TabbedPanelsContentGroup clearfix colelem" id="u327"><!-- stack box -->
     <div class="TabbedPanelsContentGroup clearfix colelem" id="u327"><!-- stack box -->
-
       <div class="TabbedPanelsContent invi clearfix grpelem" id="u328"><!-- group -->
+
       <div class="TabbedPanelsContent clearfix grpelem" id="u328"><!-- group -->
       <div class="clearfix grpelem" id="u404-8"><!-- content -->
       <div class="clearfix grpelem" id="u404-8"><!-- content -->
         <p id="u404-2">Carbon monoxide (CO) is a colorless and odorless gas which can be very poisonous and dangerous. Since it is produced from commonly used household devices and industry, in the case of any leakage it can cause severe poisonings and deaths. Many people die in Turkey because of mishandled ascot and blast heaters. According to statistics 10,154 people, meaning 14 out of every 100,000 people, were intoxicated.</p>
         <p id="u404-2">Carbon monoxide (CO) is a colorless and odorless gas which can be very poisonous and dangerous. Since it is produced from commonly used household devices and industry, in the case of any leakage it can cause severe poisonings and deaths. Many people die in Turkey because of mishandled ascot and blast heaters. According to statistics 10,154 people, meaning 14 out of every 100,000 people, were intoxicated.</p>
Line 111: Line 130:
       </div>
       </div>
       </div>
       </div>
-
       <div class="TabbedPanelsContent clearfix grpelem" id="u336"><!-- group -->
+
       <div class="TabbedPanelsContent invi clearfix grpelem" id="u336"><!-- column -->
-
       <div class="clearfix grpelem" id="u2687-7"><!-- content -->
+
       <div class="clearfix colelem" id="pu3932-7"><!-- group -->
-
        <p id="u2687-2">CO to CO2 Converter &amp; CO Monitoring System</p>
+
        <div class="clearfix grpelem" id="u3932-7"><!-- content -->
-
        <p id="u2687-3">&nbsp;</p>
+
        <p id="u3932-2">CO to CO2 Converter &amp; CO Monitoring System</p>
-
        <p id="u2687-5">Carbon monoxide is a highly toxic gas which is undetectable by humans and it is fatal when inhaled. We’ve developed a biological device that comprises of both a qualitative detector for this dangerous gas and a conversion system to transform it into carbon dioxide. As for detection, CO sensitive promoters pCooM and pCooF from Rhodosprillum rubrum will initiate the production of fluorescent proteins in the presence of CO. Optic sensors will be used to track the production of these proteins and if the sensors pick up data indicating that CO is present; an alarm will be triggered. Meanwhile, the conversion system of our device will utilize a Cyanobacteria enzyme called Carbon Monoxide Dehydrogenase (CODH), which converts CO into CO2. We also have a kill&#45;switch design based on the lac&#45;operon. The kill&#45;switch mechanism will be activated to avoid any contamination of the environment, in case the altered bacteria escape the device.</p>
+
        <p id="u3932-3">&nbsp;</p>
 +
        <p id="u3932-5">Carbon monoxide is a highly toxic gas which is undetectable by humans and it is fatal when inhaled. We’ve developed a biological device that comprises of both a qualitative detector for this dangerous gas and a conversion system to transform it into carbon dioxide. As for detection, CO sensitive promoters pCooM and pCooF from Rhodosprillum rubrum will initiate the production of fluorescent proteins in the presence of CO. Optic sensors will be used to track the production of these proteins and if the sensors pick up data indicating that CO is present; an alarm will be triggered. Meanwhile, the conversion system of our device will utilize a Cyanobacteria enzyme called Carbon Monoxide Dehydrogenase (CODH), which converts CO into CO2. We also have a kill&#45;switch design based on the lac&#45;operon. The kill&#45;switch mechanism will be activated to avoid any contamination of the environment, in case the altered bacteria escape the device.</p>
 +
        </div>
 +
        <div class="clearfix grpelem" id="u3945-4"><!-- content -->
 +
        <p>constitutive promoter</p>
 +
        </div>
 +
      </div>
 +
      <div class="clearfix colelem" id="pu3218"><!-- group -->
 +
        <div class="clip_frame clearfix grpelem" id="u3218"><!-- image -->
 +
        <div id="u3218_clip">
 +
          <img class="position_content" id="u3218_img" src="http://fisofowi.netii.net/images/cir.jpg" alt="" width="486" height="150" />
 +
        </div>
 +
        </div>
 +
        <div class="clip_frame grpelem" id="u3221"><!-- image -->
 +
        <img class="block" id="u3221_img" src="http://fisofowi.netii.net/images/cir%202.jpg" alt="" width="342" height="140" />
 +
        </div>
 +
        <div class="clip_frame grpelem" id="u3935"><!-- image -->
 +
        <img class="block" id="u3935_img" src="http://fisofowi.netii.net/images/promoter.jpg" alt="" width="72" height="71" />
 +
        </div>
 +
        <div class="clip_frame grpelem" id="u3940"><!-- image -->
 +
        <img class="block" id="u3940_img" src="http://fisofowi.netii.net/images/section.jpg" alt="" width="247" height="108" />
 +
        </div>
 +
      </div>
 +
      <div class="clearfix colelem" id="u3933-8"><!-- content -->
 +
        <p id="u3933-2">Kill switch</p>
 +
        <p id="u3933-3">&nbsp;</p>
 +
        <p id="u3933-6">we use an mRNA interferase, MazF, which cleaves mRNA’s at specific sequences. In our kill switch, we used anti&#45;sense RNA principle as a template. According to this principle, MazF, which is constantly produced via a constitutive promoter, is got inactivated by Anti&#45;MazF construct. In order to trigger this mechanism, we used IPTG, a harmless molecule for the bee which at the same time does not appear in the honey too. When IPTG is present in the environment, LacI is inhibited by IPTG and therefore promoter gets activated. With the activated promoter of it, Anti&#45;MazF is produced and inactive MazF. As long as IPTG exists, MazF gets inactivated continuously; therefore, the bacteria maintain their lives. On the other hand, if bacteria exists in a IPTG&#45;free environment, Anti&#45;MazF producing stops, which leads to MazF producing and bacteria get killed by MazF. <span id="u3933-5">(Circuit image courtesy of METU iGEM Team 2013)</span></p>
 +
      </div>
 +
      <div class="clip_frame colelem" id="u3911"><!-- image -->
 +
        <img class="block" id="u3911_img" src="http://fisofowi.netii.net/images/kill_switch.png" alt="" width="585" height="439" />
 +
      </div>
 +
      </div>
 +
      <div class="TabbedPanelsContent invi clearfix grpelem" id="u332"><!-- column -->
 +
      <div class="clearfix colelem" id="u3934-17"><!-- content -->
 +
        <p id="u3934-2">In this page, you can find out the computational modelling for our design and finished system.</p>
 +
        <p id="u3934-3">&nbsp;</p>
 +
        <p id="u3934-5">Carbon monoxide</p>
 +
        <p id="u3934-6">&nbsp;</p>
 +
        <p id="u3934-8">With our research we were able to find out that 1500 ppm of CO gas means death in an hour. To make calculations we need to convert this value into something we can work with easily, such as moles.</p>
 +
        <p id="u3934-9">&nbsp;</p>
 +
        <p id="u3934-11">1500 ppm = 0.15%</p>
 +
        <p id="u3934-12">&nbsp;</p>
 +
        <p id="u3934-14">If we take a 30 meter squared room with 3 meter ceiling height, the overall volume of the room is 90m^3 which equates to 90000 liters.</p>
 +
        <p>&nbsp;</p>
 +
      </div>
 +
      <div class="clip_frame colelem" id="u3196"><!-- image -->
 +
        <img class="block" id="u3196_img" src="http://fisofowi.netii.net/images/calc%201.jpg" alt="" width="275" height="88" />
 +
      </div>
 +
      <div class="clearfix colelem" id="pu3195-121"><!-- group -->
 +
        <div class="clearfix grpelem" id="u3195-121"><!-- content -->
 +
        <p id="u3195-2"><span id="u3195">By using Ideal Gas Law, we can say that the amount of moles of CO present in 135 Liters is around about 4 moles. So what we have learned? In a big size room, there should be at least 4 moles of CO molecules for it to be considered as deadly.</span></p>
 +
        <p id="u3195-3">&nbsp;</p>
 +
        <p id="u3195-5">CODH Activity</p>
 +
        <p id="u3195-6">&nbsp;</p>
 +
        <p id="u3195-8"><span id="u3195-7">Even though we were not able to find detailed parameters for this particular enzyme, we were able to work out a very simple formula to calculate the amount of CO we will be able to convert each second.</span></p>
 +
        <p id="u3195-9">&nbsp;</p>
 +
        <p id="u3195-11"><span id="u3195-10">r = CODH activity rate</span></p>
 +
        <p id="u3195-13"><span id="u3195-12">p = plasmid copy number</span></p>
 +
        <p id="u3195-15"><span id="u3195-14">b = amount of bacteria</span></p>
 +
        <p id="u3195-17"><span id="u3195-16">s = synthesis rate</span></p>
 +
        <p id="u3195-19"><span id="u3195-18">[CO]=amount of CO (moles)</span></p>
 +
        <p id="u3195-21">A = fraction of CO converted</p>
 +
        <p id="u3195-22">&nbsp;</p>
 +
        <p id="u3195-24"><span id="u3195-23">*Lets call r.p.b.s: &quot;c&quot; since we can assume that it will stay constant.</span></p>
 +
        <p id="u3195-26"><span id="u3195-25">*Our calculations show that &quot;c = 31777209688000&quot; or &quot;3.2 x 10^14&quot;</span></p>
 +
        <p id="u3195-27">&nbsp;</p>
 +
        <p id="u3195-29">When we divide c by the Avagadro number we can find out what fraction of moles of CO we will be able to convert each time frame. Even though &quot;c&quot; seems like a huge number, the Avagadro number is much bigger.</p>
 +
        <p id="u3195-30">&nbsp;</p>
 +
        <p id="u3195-32">3.2 x 10^14 / 6.02 x 10^23&nbsp; =&nbsp; 0.5 x 10^&#45;9</p>
 +
        <p id="u3195-33">&nbsp;</p>
 +
        <p id="u3195-35"><span id="u3195-34">This mans that we are able to convert roughly 0.000000001 of the CO available each second.</span></p>
 +
        <p id="u3195-36">&nbsp;</p>
 +
        <p id="u3195-38"><span id="u3195-37">Here is a little script we wrote with Python programming language with modules &quot;matplotlib&quot; and &quot;numPy&quot;:</span></p>
 +
        <p id="u3195-39">&nbsp;</p>
 +
        <p id="u3195-44"><span id="u3195-40">from</span><span id="u3195-41">&nbsp;pylab</span><span id="u3195-42">&nbsp;import</span><span id="u3195-43">&nbsp;*</span></p>
 +
        <p id="u3195-45">&nbsp;</p>
 +
        <p id="u3195-47">timer = 0</p>
 +
        <p id="u3195-49">f = 0.000000001</p>
 +
        <p id="u3195-52">s = 7200 <span id="u3195-51">#seconds</span></p>
 +
        <p id="u3195-55">co = 4 <span id="u3195-54"># moles</span></p>
 +
        <p id="u3195-58">co2 =0 <span id="u3195-57"># moles</span></p>
 +
        <p id="u3195-60">values = [ ]</p>
 +
        <p id="u3195-61">&nbsp;</p>
 +
        <p id="u3195-68"><span id="u3195-62">for</span>&nbsp;i<span id="u3195-64">&nbsp;in</span>&nbsp;<span id="u3195-66">range</span>(s):</p>
 +
        <p id="u3195-70">&nbsp;&nbsp;&nbsp; co2 = co2 + co*f</p>
 +
        <p id="u3195-72">&nbsp;&nbsp;&nbsp; co = co &#45; co2</p>
 +
        <p id="u3195-76">&nbsp;&nbsp;&nbsp; <span id="u3195-74">if </span>co &lt;= 0:</p>
 +
        <p id="u3195-79">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; <span id="u3195-78">&nbsp;break</span></p>
 +
        <p id="u3195-81">&nbsp;&nbsp;&nbsp; values.append(co2)</p>
 +
        <p id="u3195-83">&nbsp;&nbsp;&nbsp; timer += 1</p>
 +
        <p id="u3195-84">&nbsp;</p>
 +
        <p id="u3195-86">x = arange(0,timer,1)</p>
 +
        <p id="u3195-88">plot(x,values)</p>
 +
        <p id="u3195-89">&nbsp;</p>
 +
        <p id="u3195-93">xlabel(<span id="u3195-91">'time (s)'</span>)</p>
 +
        <p id="u3195-97">ylabel(<span id="u3195-95">'CO2 (moles)'</span>)</p>
 +
        <p id="u3195-101">title(<span id="u3195-99">'CO2 &#45; Time'</span>)</p>
 +
        <p id="u3195-105">grid(<span id="u3195-103">True</span>)</p>
 +
        <p id="u3195-107">show()</p>
 +
        <p id="u3195-108">&nbsp;</p>
 +
        <p id="u3195-109">&nbsp;</p>
 +
        <p id="u3195-111"><span id="u3195-110">Please note that, even though we are subtracting the amount of CO converted from the initial CO value, the graph still does not have a visible curve to it. It is obvious that even though we are able to convert a huge number of CO molecules each given second, there are way to much CO then our system can convert.</span></p>
 +
        <p id="u3195-112">&nbsp;</p>
 +
        <p id="u3195-114"><span id="u3195-113">By increasing the surface are of the single&#45;layer e&#45;coli colonies, for example take a 10 x 10 grid of the same exact system, we can achieve 100 times more efficient converters which can shave of two 0's from the results.</span></p>
 +
        <p id="u3195-115">&nbsp;</p>
 +
        <p id="u3195-117"><span id="u3195-116">Down below you can see the photos of the colonies we were able to get and the electronic circuit design.</span></p>
 +
        <p id="u3195-118">&nbsp;</p>
 +
        <p id="u3195-119">&nbsp;</p>
 +
        </div>
 +
        <div class="rounded-corners clip_frame clearfix grpelem" id="u3201"><!-- image -->
 +
        <div id="u3201_clip">
 +
          <img class="position_content" id="u3201_img" src="http://fisofowi.netii.net/images/figure_1.jpg" alt="" width="670" height="506" />
 +
        </div>
 +
        </div>
 +
        <div class="clip_frame grpelem" id="u3878"><!-- image -->
 +
        <img class="block" id="u3878_img" src="http://fisofowi.netii.net/images/gel.jpg" alt="" width="428" height="321" />
 +
        </div>
 +
        <div class="clip_frame grpelem" id="u3883"><!-- image -->
 +
        <img class="block" id="u3883_img" src="http://fisofowi.netii.net/images/elec2.jpg" alt="" width="428" height="257" />
 +
        </div>
 +
        <div class="clip_frame grpelem" id="u3888"><!-- image -->
 +
        <img class="block" id="u3888_img" src="http://fisofowi.netii.net/images/elec1.jpg" alt="" width="421" height="253" />
 +
        </div>
 +
        <div class="clearfix grpelem" id="u3893-4"><!-- content -->
 +
        <p>Trying out the color sensor. Red pen on the sensor light up red. Blue ruler on top, blue light.</p>
 +
        </div>
 +
        <div class="clearfix grpelem" id="u3921-4"><!-- content -->
 +
        <p>Final images of the design.</p>
 +
        </div>
 +
        <div class="clip_frame grpelem" id="u3922"><!-- image -->
 +
        <img class="block" id="u3922_img" src="http://fisofowi.netii.net/images/igem%20hs%20final2.jpg" alt="" width="422" height="496" />
 +
        </div>
 +
      </div>
 +
      </div>
 +
      <div class="TabbedPanelsContent invi clearfix grpelem" id="u1382"><!-- group -->
 +
      <div class="clearfix grpelem" id="u3206-42"><!-- content -->
 +
        <p id="u3206-2">Today, improving technology and ideas lead students to be the constructors of better future. That’s why as METU DF High School iGEM 2014 team we made researches and experiments to widen the perspective of what synthetic biology can achieve. We know that synthetic biology is the way of constructing core components which can be modeled by merging them with the fundamentals of biology. We assume that by the efficient use of synthetic biology we can lighten the way through future and save many lives.</p>
 +
        <p id="u3206-3">&nbsp;</p>
 +
        <p id="u3206-5">Therefore, this year we worked hard to put forth an experiment that can lead the way to our better future. As METU HS iGEM team, we believe that synthetic biology is a tool that can turn the world to a better place. And iGEM is an opportunity for us to show people that synthetic biology is helpful for the humankind. That's why we did many events to introduce people synthetic biology. We have given a bake sale which included bacteria &amp; DNA muffins and x&#45;ray crystallography cake.</p>
 +
        <p id="u3206-6">&nbsp;</p>
 +
        <p id="u3206-7">&nbsp;</p>
 +
        <p id="u3206-8">&nbsp;</p>
 +
        <p id="u3206-9">&nbsp;</p>
 +
        <p id="u3206-10">&nbsp;</p>
 +
        <p id="u3206-11">&nbsp;</p>
 +
        <p id="u3206-12">&nbsp;</p>
 +
        <p id="u3206-13">&nbsp;</p>
 +
        <p id="u3206-14">&nbsp;</p>
 +
        <p id="u3206-15">&nbsp;</p>
 +
        <p id="u3206-16">&nbsp;</p>
 +
        <p id="u3206-17">&nbsp;</p>
 +
        <p id="u3206-18">&nbsp;</p>
 +
        <p id="u3206-19">&nbsp;</p>
 +
        <p id="u3206-20">&nbsp;</p>
 +
        <p id="u3206-21">&nbsp;</p>
 +
        <p id="u3206-22">&nbsp;</p>
 +
        <p id="u3206-23">&nbsp;</p>
 +
        <p id="u3206-25">The muffins got retweeted by the iGEM HQ!</p>
 +
        <p id="u3206-26">&nbsp;</p>
 +
        <p id="u3206-27">&nbsp;</p>
 +
        <p id="u3206-28">&nbsp;</p>
 +
        <p id="u3206-30">Furthermore we interviewed some students in our school to see how much they know about synthetic biology world. We decorated our school with DNA's and posters of our team on DNA day. Also as METU HS iGEM team we have attended to a Synthetic Biology Day to expand our knowledge. It is important for the next generations to be familiar with synthetic biology, in order to achieve this we gave a presentation to 7 and 8th graders. We have informed them about iGEM and biology. And our aim is to present iGEM to everyone to convince people that synthetic biology is useful. So we have told people about our team and iGEM at the opening of an ODTU DF school in Denizli. In order to maintain our goal we believed that a sale of work will be a nice introduction of iGEM and synthetic biology. We cooked cupcakes in order to sale and we designed them as bacteria and everything about synthetic biology. Secondly, we also designed an enormous DNA shaped gumdrop. The most critical part was we recommended iGEM and talk with them about the project we were doing. Our main purpose was to make people know what synthetic biology is and create awareness about these topics. To conclude we have tried and we are still trying to spread synthetic biology in order to acknowledge human. As METU DF High School iGEM 2014 team we work hard to settle the importance of synthetic biology by referring various projects.</p>
 +
        <p id="u3206-31">&nbsp;</p>
 +
        <p id="u3206-33">We believe that awareness and education is the key of action, acceptance and understanding. Our project contains lots of valuable experiments that can prove the useful remedies of the efficient use of synthetic biology.</p>
 +
        <p id="u3206-34">&nbsp;</p>
 +
        <p id="u3206-39">You can find every photo and video we referred to at the fun page by following this<a class="nonblock" href="https://2014hs.igem.org/Team:METUHS-Ankara/extras/fun.html">&nbsp;link</a>&nbsp;or find it at the extras page!</p>
 +
        <p id="u3206-40">&nbsp;</p>
 +
      </div>
 +
      <div class="clip_frame grpelem" id="u3207"><!-- image -->
 +
        <img class="block" id="u3207_img" src="http://fisofowi.netii.net/images/tweet.jpg" alt="" width="589" height="355" />
 +
      </div>
 +
      </div>
 +
      <div class="TabbedPanelsContent invi clearfix grpelem" id="u2488"><!-- group -->
 +
      <div class="PamphletWidget clearfix grpelem" id="pamphletu2946"><!-- none box -->
 +
        <div class="popup_anchor" id="u2952popup">
 +
        <div class="ContainerGroup clearfix" id="u2952"><!-- stack box -->
 +
          <div class="Container clearfix grpelem" id="u2958"><!-- group -->
 +
          <div class="clearfix grpelem" id="u2999-59"><!-- content -->
 +
            <p id="u2999-2">Competent Cell</p>
 +
            <p id="u2999-4">• Take 1 µL cell (3 µL cell for us )</p>
 +
            <p id="u2999-6">100 ml LB 37ºC 2h (OD 0.600 200 rpm)</p>
 +
            <p id="u2999-8">• Divide 100 ml into 2 falcons &amp; centrifuge for 10 minutes 5000 rpm +4ºC</p>
 +
            <p id="u2999-10">• Discard supernatant</p>
 +
            <p id="u2999-12">• Add 10 µL 0.1 M (molarity) CaCl2</p>
 +
            <p id="u2999-14">• Dissolve pellet by gently shaking</p>
 +
            <p id="u2999-16">• Incubate on ice 10 min.</p>
 +
            <p id="u2999-18">• Centrifuge 5000 rpm for 5 min. +4ºC</p>
 +
            <p id="u2999-20">• Discard supernatant</p>
 +
            <p id="u2999-22">• Put 2 ml CaCl2 and dissolve pellet</p>
 +
            <p id="u2999-24">• Put in ice for 5 min. &amp; keep it &#45;80ºC 80% glycerol</p>
 +
            <p id="u2999-25">&nbsp;</p>
 +
            <p id="u2999-27">Transformation</p>
 +
            <p id="u2999-29">• Thaw competent cells on ice</p>
 +
            <p id="u2999-31">• Mix ligation mix (100 µL cells 5 µL plasmids)</p>
 +
            <p id="u2999-33">• Incubate cells on ice 30 min.</p>
 +
            <p id="u2999-35">• Heat shock at 42ºC 90 sec.</p>
 +
            <p id="u2999-37">• Incubate cells on ice 5 min.</p>
 +
            <p id="u2999-39">• Add 900 µL LB</p>
 +
            <p id="u2999-41">• Incubate at 37ºC for 80 min.</p>
 +
            <p id="u2999-43">• Centrifuge at 3000 rpm for 10 min.</p>
 +
            <p id="u2999-45">• Discard supernatant</p>
 +
            <p id="u2999-47">• Resuspend the pellet in 100 µL LB</p>
 +
            <p id="u2999-49">• Spread cells on LB Agar with antibiotic</p>
 +
            <p id="u2999-51">K34F &#45; DT Chl</p>
 +
            <p id="u2999-53">K34B &#45; RFP Chl</p>
 +
            <p id="u2999-55">K25J &#45; GFP Chl</p>
 +
            <p id="u2999-56">&nbsp;</p>
 +
            <p>&nbsp;</p>
 +
          </div>
 +
          </div>
 +
          <div class="Container invi clearfix grpelem" id="u2963"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3001-25"><!-- content -->
 +
            <p id="u3001-2">Transformation again!</p>
 +
            <p id="u3001-4">Selin Hoca Transformation</p>
 +
            <p id="u3001-5">&nbsp;</p>
 +
            <p id="u3001-7">Pet28a</p>
 +
            <p id="u3001-9">Pet28a+gene</p>
 +
            <p id="u3001-11">RFP+ DT</p>
 +
            <p id="u3001-12">&nbsp;</p>
 +
            <p id="u3001-14">We cannot insert the plasmid!! Something’s wrong with the competent cells!!</p>
 +
            <p id="u3001-15">&nbsp;</p>
 +
            <p id="u3001-17">Comp 1 &#45;</p>
 +
            <p id="u3001-19">Comp 2&nbsp; &#45;&nbsp;&nbsp;&nbsp;&nbsp; RFP + DT&nbsp;&nbsp;&nbsp; V&nbsp;&nbsp; Pet28a</p>
 +
            <p id="u3001-21">Comp 3&nbsp; &#45;&nbsp;&nbsp;&nbsp; Amp, Kan&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Kan</p>
 +
            <p id="u3001-22">&nbsp;</p>
 +
            <p>&nbsp;</p>
 +
          </div>
 +
          <div class="clip_frame grpelem" id="u3007"><!-- image -->
 +
            <img class="block" id="u3007_img" src="http://fisofowi.netii.net/images/tablo.jpg" alt="" width="568" height="337" />
 +
          </div>
 +
          </div>
 +
          <div class="Container invi clearfix grpelem" id="u2953"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3017-38"><!-- content -->
 +
            <p id="u3017-2">The second transformation.</p>
 +
            <p id="u3017-4">Competent cell, why didn’t they work?</p>
 +
            <p id="u3017-6">1) Lack of CaCl2</p>
 +
            <p id="u3017-8">2) CaCl2 wasn’t cold enough</p>
 +
            <p id="u3017-10">3) Non homogeneous cells (The closest)</p>
 +
            <p id="u3017-12">4) OD was not enough</p>
 +
            <p id="u3017-13">&nbsp;</p>
 +
            <p id="u3017-15">Transformation, why didn’t it work?</p>
 +
            <p id="u3017-17">1) Heat shock</p>
 +
            <p id="u3017-19">2) Cell died in centrifuge</p>
 +
            <p id="u3017-21">3) Cell died whilst spreading</p>
 +
            <p id="u3017-23">• Heat</p>
 +
            <p id="u3017-25">• Mechanic force</p>
 +
            <p id="u3017-26">&nbsp;</p>
 +
            <p id="u3017-28">Plasmid Isolation</p>
 +
            <p id="u3017-29">&nbsp;</p>
 +
            <p id="u3017-32"><span id="u3017-30">.</span>Gel Electrophoresis 10 µL&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; .Stock 15 µL</p>
 +
            <p id="u3017-34">&nbsp;Gel Extraction</p>
 +
            <p id="u3017-35">&nbsp;</p>
 +
            <p>&nbsp;</p>
 +
          </div>
 +
          </div>
 +
          <div class="Container invi clearfix grpelem" id="u3019"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3046-10"><!-- content -->
 +
            <p id="u3046-2">A1&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; K1&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; RFP</p>
 +
            <p id="u3046-4">Needed the plasmids!</p>
 +
            <p id="u3046-6">Plasmid Isolation &#45; 2 µL cell pellet, lyse down the cells take plamids!</p>
 +
            <p id="u3046-7">&nbsp;</p>
 +
            <p>&nbsp;</p>
 +
          </div>
 +
          </div>
 +
          <div class="Container invi clearfix grpelem" id="u3025"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3050-16"><!-- content -->
 +
            <p id="u3050-2">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 16.05.2014</p>
 +
            <p id="u3050-4">Transformation</p>
 +
            <p id="u3050-5">&nbsp;</p>
 +
            <p id="u3050-7">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; pCooM&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; with changes of 50 µL cell</p>
 +
            <p id="u3050-9">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; pCooF&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; + 10 µL&nbsp; plasmid</p>
 +
            <p id="u3050-10">&nbsp;</p>
 +
            <p id="u3050-11">&nbsp;</p>
 +
            <p id="u3050-12">&nbsp;</p>
 +
            <p id="u3050-13">&nbsp;</p>
 +
            <p>&nbsp;</p>
 +
          </div>
 +
          </div>
 +
          <div class="Container invi clearfix grpelem" id="u3028"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3052-46"><!-- content -->
 +
            <p id="u3052-2">Digestion</p>
 +
            <p id="u3052-4">NEB Buffer&nbsp;&nbsp; 5 µL</p>
 +
            <p id="u3052-6">BSA&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 0.5 µL</p>
 +
            <p id="u3052-8">Enzyme 1&nbsp;&nbsp;&nbsp;&nbsp; 0.5 µL</p>
 +
            <p id="u3052-10">Enzyme 2&nbsp;&nbsp;&nbsp;&nbsp; 0.5 µL µL</p>
 +
            <p id="u3052-12">Plasmid&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 1000 ng/µL</p>
 +
            <p id="u3052-13">&nbsp;</p>
 +
            <p id="u3052-15">To complete to 50 µL, add ddH2O/dH2O</p>
 +
            <p id="u3052-17">Keep it in water bath for 2.5 hours at 37ºC</p>
 +
            <p id="u3052-18">&nbsp;</p>
 +
            <p id="u3052-20">LIGATION</p>
 +
            <p id="u3052-22">10 x T4 ligase buffer&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 2 µL</p>
 +
            <p id="u3052-24">6:1 M ratio to invert to vector</p>
 +
            <p id="u3052-26">Add dH2O</p>
 +
            <p id="u3052-28">T4 ligase&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 1 µL</p>
 +
            <p id="u3052-29">&nbsp;</p>
 +
            <p id="u3052-30">&nbsp;</p>
 +
            <p id="u3052-32">Incubate at room temtperature&nbsp; 1 hours and 15 min. at 65ºC for enzyme activation</p>
 +
            <p id="u3052-34">Alternative: +4ºC over night</p>
 +
            <p id="u3052-35">&nbsp;</p>
 +
            <p id="u3052-37">Calculatory Insert Amount</p>
 +
            <p id="u3052-38">&nbsp;</p>
 +
            <p id="u3052-40">Insert mass in ng = 6 x&nbsp; insert length in 6 µL / Vector length 6 g...</p>
 +
            <p id="u3052-42">&nbsp; ...x vector mass in mg</p>
 +
            <p id="u3052-43">&nbsp;</p>
 +
            <p>&nbsp;</p>
 +
          </div>
 +
          </div>
 +
          <div class="Container invi clearfix grpelem" id="u3030"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3054-15"><!-- content -->
 +
            <p id="u3054-2">&#45;Gel</p>
 +
            <p id="u3054-4">&#45;Gel Extraction</p>
 +
            <p id="u3054-5">&nbsp;</p>
 +
            <p id="u3054-7">Vector &#45; 2500 bp</p>
 +
            <p id="u3054-9">Insert&nbsp; &#45; 500 bp</p>
 +
            <p id="u3054-11">1300 &#45; to prepare the agarose gel 1.95 g of agarose (nearly 2%)</p>
 +
            <p id="u3054-12">&nbsp;</p>
 +
            <p>&nbsp;</p>
 +
          </div>
 +
          </div>
 +
          <div class="Container invi clearfix grpelem" id="u3032"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3056-74"><!-- content -->
 +
            <p id="u3056-2">&#45;Gel Results</p>
 +
            <p id="u3056-4">&#45;PCR</p>
 +
            <p id="u3056-6">&#45;Gel</p>
 +
            <p id="u3056-7">&nbsp;</p>
 +
            <p id="u3056-9">Why didn’t it result correct?</p>
 +
            <p id="u3056-11">There was no DNA</p>
 +
            <p id="u3056-13">Problem with gel</p>
 +
            <p id="u3056-15">Part we don’t want in the gel</p>
 +
            <p id="u3056-17">Lack of the amount</p>
 +
            <p id="u3056-19">TAE?</p>
 +
            <p id="u3056-21">Agarose</p>
 +
            <p id="u3056-23">6x loading dye</p>
 +
            <p id="u3056-25">Plasmid&nbsp; &#45;&nbsp; Digestion, Ligation</p>
 +
            <p id="u3056-27">EtBr</p>
 +
            <p id="u3056-29">DNA degredation</p>
 +
            <p id="u3056-30">&nbsp;</p>
 +
            <p id="u3056-31">&nbsp;</p>
 +
            <p id="u3056-32">&nbsp;</p>
 +
            <p id="u3056-33">&nbsp;</p>
 +
            <p id="u3056-34">&nbsp;</p>
 +
            <p id="u3056-35">&nbsp;</p>
 +
            <p id="u3056-36">&nbsp;</p>
 +
            <p id="u3056-37">&nbsp;</p>
 +
            <p id="u3056-38">&nbsp;</p>
 +
            <p id="u3056-39">&nbsp;</p>
 +
            <p id="u3056-40">&nbsp;</p>
 +
            <p id="u3056-41">&nbsp;</p>
 +
            <p id="u3056-42">&nbsp;</p>
 +
            <p id="u3056-43">&nbsp;</p>
 +
            <p id="u3056-44">&nbsp;</p>
 +
            <p id="u3056-45">&nbsp;</p>
 +
            <p id="u3056-47">Conditions</p>
 +
            <p id="u3056-49">Initial denaturation&nbsp;&nbsp; &#45;&nbsp;&nbsp; 94ºC&nbsp;&nbsp;&nbsp;&nbsp; &#45;30 sec</p>
 +
            <p id="u3056-51">Cycles&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 94ºC&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 10&#45;30 sec</p>
 +
            <p id="u3056-53">(30&#45;35)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 45&#45;65ºC (Tm= 58 ºC) 15&#45;60 sec&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 2000+1800 bp</p>
 +
            <p id="u3056-55">(32&#45;33)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 65ºC&nbsp;&nbsp; 50 sec/kb (⁓2 min)</p>
 +
            <p id="u3056-57">Final extension&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 65ºC&nbsp; 10 min</p>
 +
            <p id="u3056-59">Hold&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; +4ºC</p>
 +
            <p id="u3056-60">&nbsp;</p>
 +
            <p id="u3056-62">8F 9R&nbsp;&nbsp;&nbsp; /&nbsp;&nbsp; 10F 11R &#45; primers used!</p>
 +
            <p id="u3056-64">Nutrient agar plate inoculation</p>
 +
            <p id="u3056-66">RFP&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; GFP</p>
 +
            <p id="u3056-68">IPTG +</p>
 +
            <p id="u3056-70">IPTG &#45;</p>
 +
            <p id="u3056-71">&nbsp;</p>
 +
            <p>&nbsp;</p>
 +
          </div>
 +
          <div class="clip_frame grpelem" id="u3111"><!-- image -->
 +
            <img class="block" id="u3111_img" src="http://fisofowi.netii.net/images/tablo%202.jpg" alt="" width="546" height="340" />
 +
          </div>
 +
          </div>
 +
          <div class="Container invi clearfix grpelem" id="u3034"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3069-14"><!-- content -->
 +
            <p id="u3069-2">Nutrient agar plate inoculation</p>
 +
            <p id="u3069-4">IPTG + and –</p>
 +
            <p id="u3069-6">Inoculation on broth media</p>
 +
            <p id="u3069-8">IPTG x2</p>
 +
            <p id="u3069-10">MP x4</p>
 +
            <p id="u3069-11">&nbsp;</p>
 +
            <p>&nbsp;</p>
 +
          </div>
 +
          </div>
 +
          <div class="Container invi clearfix grpelem" id="u3036"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3071-6"><!-- content -->
 +
            <p><span id="u3071">​</span><span><span class="actAsInlineDiv normal_text" id="u3073"><!-- content --><span class="actAsDiv clearfix excludeFromNormalFlow" id="u3072-8"><!-- content --><span class="actAsPara" id="u3072-4"><span id="u3072">​</span><span><span class="actAsInlineDiv normal_text" id="u3075"><!-- content --><span class="actAsDiv clearfix excludeFromNormalFlow" id="u3074-21"><!-- content --><span class="actAsPara" id="u3074-2">• IPTG induction in Broth Media (1)</span><span class="actAsPara" id="u3074-4">• Miniprep pCooM, pCooF (1)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Holin&#45; Antiholin</span><span class="actAsPara" id="u3074-6">• CODH from BCG PCR (2)</span><span class="actAsPara" id="u3074-8">• Kill Switch Transformation (3)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; MazF&#45; AntimazF</span><span class="actAsPara" id="u3074-10">• Digestion &amp; Ligation (?)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; BCG &#45;primer(Rhodospirillum rubrum&nbsp; primer)</span><span class="actAsPara" id="u3074-11">&nbsp;</span><span class="actAsPara" id="u3074-12">&nbsp;</span><span class="actAsPara" id="u3074-13">&nbsp;</span><span class="actAsPara">&nbsp;</span><span class="actAsPara">&nbsp;</span><span class="actAsPara">&nbsp;</span><span class="actAsPara">&nbsp;</span><span class="actAsPara">&nbsp;</span><span class="actAsPara">&nbsp;</span></span></span></span><span></span></span><span class="actAsPara" id="u3072-5">&nbsp;</span><span class="actAsPara">&nbsp;</span></span></span></span><span></span></p>
 +
          </div>
 +
          <div class="clip_frame grpelem" id="u3076"><!-- image -->
 +
            <img class="block" id="u3076_img" src="http://fisofowi.netii.net/images/cir.jpg" alt="" width="486" height="150" />
 +
          </div>
 +
          <div class="clip_frame grpelem" id="u3081"><!-- image -->
 +
            <img class="block" id="u3081_img" src="http://fisofowi.netii.net/images/cir%202.jpg" alt="" width="342" height="140" />
 +
          </div>
 +
          </div>
 +
          <div class="Container invi clearfix grpelem" id="u3038"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3087-4"><!-- content -->
 +
            <p>Transformation of kill switch (with changes of LB &#45; 150 µL at last step)</p>
 +
          </div>
 +
          <div class="clip_frame clearfix grpelem" id="u3088"><!-- image -->
 +
            <div id="u3088_clip">
 +
            <img class="position_content" id="u3088_img" src="http://fisofowi.netii.net/images/tablo%203.jpg" alt="" width="311" height="189" />
 +
            </div>
 +
          </div>
 +
          </div>
 +
          <div class="Container invi clearfix grpelem" id="u3040"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3094-44"><!-- content -->
 +
            <p id="u3094-2">Gel Extraction</p>
 +
            <p id="u3094-4">Genomic DNA Isolation</p>
 +
            <p id="u3094-6">Chl &amp; Tet Inoculation</p>
 +
            <p id="u3094-8">&nbsp;&nbsp;&nbsp; Empty: MazF, AntimazF (streak plates) (4 inoculations)</p>
 +
            <p id="u3094-10">Genomic DNA Isolation</p>
 +
            <p id="u3094-12">• Suspend colonies in 120 µL TEN Buffer</p>
 +
            <p id="u3094-14">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 40 µM Tris, 1 µM EDTA, 150 µM NaCl</p>
 +
            <p id="u3094-16">• Incubate at 100ºC for 10 min</p>
 +
            <p id="u3094-18">• Centrifuge at 13.000 rpm</p>
 +
            <p id="u3094-20">• Take supernatant store at +4ºC</p>
 +
            <p id="u3094-22">Gel Extraction</p>
 +
            <p id="u3094-24">• Slice the gel</p>
 +
            <p id="u3094-26">• Add 1:1 volume binding buffer</p>
 +
            <p id="u3094-28">• Incubate 50&#45;60ºC for 10 min.</p>
 +
            <p id="u3094-30">• Transfer up to 800 µL of the solubilized gel solution to the purification column</p>
 +
            <p id="u3094-32">• Cent. 1 min, discard flow&#45;through</p>
 +
            <p id="u3094-34">• 100 µL binding buffer cent. 1 min, discard flow&#45;through</p>
 +
            <p id="u3094-36">• 700 µL wash buffer, cent. 2 min discard flow&#45;through</p>
 +
            <p id="u3094-38">• Column to eppendorf 50 µL Elution buffer dH2O, cent. 1 min.</p>
 +
            <p id="u3094-40">• Discard column store it at &#45;20ºC</p>
 +
            <p id="u3094-41">&nbsp;</p>
 +
            <p>&nbsp;</p>
 +
          </div>
 +
          </div>
 +
          <div class="Container invi clearfix grpelem" id="u3042"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3096-55"><!-- content -->
 +
            <p id="u3096-2">1) PCR</p>
 +
            <p id="u3096-4">2) Gel Electrophoresis &#45; Genomic DNA + Gel Extraction</p>
 +
            <p id="u3096-5">&nbsp;</p>
 +
            <p id="u3096-7">&#45;PCR</p>
 +
            <p id="u3096-8">&nbsp;</p>
 +
            <p id="u3096-9">&nbsp;</p>
 +
            <p id="u3096-10">&nbsp;</p>
 +
            <p id="u3096-11">&nbsp;</p>
 +
            <p id="u3096-12">&nbsp;</p>
 +
            <p id="u3096-13">&nbsp;</p>
 +
            <p id="u3096-14">&nbsp;</p>
 +
            <p id="u3096-15">&nbsp;</p>
 +
            <p id="u3096-16">&nbsp;</p>
 +
            <p id="u3096-17">&nbsp;</p>
 +
            <p id="u3096-18">&nbsp;</p>
 +
            <p id="u3096-19">&nbsp;</p>
 +
            <p id="u3096-20">&nbsp;</p>
 +
            <p id="u3096-21">&nbsp;</p>
 +
            <p id="u3096-22">&nbsp;</p>
 +
            <p id="u3096-23">&nbsp;</p>
 +
            <p id="u3096-24">&nbsp;</p>
 +
            <p id="u3096-25">&nbsp;</p>
 +
            <p id="u3096-26">&nbsp;</p>
 +
            <p id="u3096-27">&nbsp;</p>
 +
            <p id="u3096-28">&nbsp;</p>
 +
            <p id="u3096-29">&nbsp;</p>
 +
            <p id="u3096-30">&nbsp;</p>
 +
            <p id="u3096-31">&nbsp;</p>
 +
            <p id="u3096-32">&nbsp;</p>
 +
            <p id="u3096-33">&nbsp;</p>
 +
            <p id="u3096-34">&nbsp;</p>
 +
            <p id="u3096-35">&nbsp;</p>
 +
            <p id="u3096-36">&nbsp;</p>
 +
            <p id="u3096-37">&nbsp;</p>
 +
            <p id="u3096-38">&nbsp;</p>
 +
            <p id="u3096-39">&nbsp;</p>
 +
            <p id="u3096-40">&nbsp;</p>
 +
            <p id="u3096-41">&nbsp;</p>
 +
            <p id="u3096-42">&nbsp;</p>
 +
            <p id="u3096-43">&nbsp;</p>
 +
            <p id="u3096-44">&nbsp;</p>
 +
            <p id="u3096-45">&nbsp;</p>
 +
            <p>&nbsp;</p>
 +
            <p>&nbsp;</p>
 +
            <p>&nbsp;</p>
 +
            <p>&nbsp;</p>
 +
            <p>&nbsp;</p>
 +
            <p>&nbsp;</p>
 +
            <p>&nbsp;</p>
 +
            <p>&nbsp;</p>
 +
          </div>
 +
          <div class="clip_frame grpelem" id="u3097"><!-- image -->
 +
            <img class="block" id="u3097_img" src="http://fisofowi.netii.net/images/tablo%204.jpg" alt="" width="350" height="427" />
 +
          </div>
 +
          <div class="clip_frame grpelem" id="u3105"><!-- image -->
 +
            <img class="block" id="u3105_img" src="http://fisofowi.netii.net/images/tablo%205.jpg" alt="" width="477" height="363" />
 +
          </div>
 +
          </div>
 +
          <div class="Container invi clearfix grpelem" id="u3044"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3110-22"><!-- content -->
 +
            <p id="u3110-2">L&nbsp;&nbsp;&nbsp; Uncut 2&nbsp;&nbsp;&nbsp; 2.2&nbsp;&nbsp; 2.1&nbsp;&nbsp; Uncut 4&nbsp;&nbsp; 4&nbsp;&nbsp; BA&nbsp;&nbsp; BK&nbsp;&nbsp;&nbsp; L&nbsp;&nbsp; 2T&nbsp;&nbsp; 4T&nbsp;&nbsp;&nbsp; 4L</p>
 +
            <p id="u3110-3">&nbsp;</p>
 +
            <p id="u3110-5">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; = Gel Electrophoresis</p>
 +
            <p id="u3110-6">&nbsp;</p>
 +
            <p id="u3110-8">Gel Extraction</p>
 +
            <p id="u3110-10">Nutrient Agar plate inoculation</p>
 +
            <p id="u3110-12">LB at the last step of transformation is 150 µL</p>
 +
            <p id="u3110-14">Ligation:</p>
 +
            <p id="u3110-16">Same with 13.06.2014 but overnight +4ºC incubation  prepared at 19.45</p>
 +
            <p id="u3110-18">17.06.2014 &#45; at 11:45, will be transformed.</p>
 +
            <p id="u3110-19">&nbsp;</p>
 +
            <p>&nbsp;</p>
 +
          </div>
 +
          </div>
 +
          <div class="Container invi clearfix grpelem" id="u3805"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3811-18"><!-- content -->
 +
            <p id="u3811-2">17.06.14 &#45; Transformation</p>
 +
            <p id="u3811-4">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 100 &#45; 10</p>
 +
            <p id="u3811-6">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 50&nbsp;&nbsp; &#45; 5</p>
 +
            <p id="u3811-8">Transformation &#45; heat shock 75’’</p>
 +
            <p id="u3811-10">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 100&nbsp;&nbsp; LB at the last step</p>
 +
            <p id="u3811-12">Selin inoculation &#45; amount of LB was more than needed.</p>
 +
            <p id="u3811-14">0.5 eppendorf &#45; it was opened before centrifuge (contamination)</p>
 +
            <p id="u3811-15">&nbsp;</p>
 +
            <p>&nbsp;</p>
 +
          </div>
 +
          </div>
 +
          <div class="Container invi clearfix grpelem" id="u3812"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3817-84"><!-- content -->
 +
            <p id="u3817-2">4F&nbsp;&nbsp; 9R &#45; primers&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 1&#45; 2 GE&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 3&#45; 2.2</p>
 +
            <p id="u3817-4">PCR &#45; 2. Sample&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 2&#45; 2.1</p>
 +
            <p id="u3817-5">&nbsp;</p>
 +
            <p id="u3817-7">LIGATION</p>
 +
            <p id="u3817-9">T4 ligase&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 1 µL</p>
 +
            <p id="u3817-11">T4 ligase buffer&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 1 µL</p>
 +
            <p id="u3817-13">Vector&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 4&nbsp;&nbsp;&nbsp;&nbsp; 6&nbsp;&nbsp;&nbsp;&nbsp; 2&nbsp;&nbsp;&nbsp;&nbsp; 5&nbsp;&nbsp;&nbsp;&nbsp; 3</p>
 +
            <p id="u3817-15">Insert&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 4&nbsp;&nbsp;&nbsp;&nbsp; 2&nbsp;&nbsp;&nbsp;&nbsp; 6&nbsp;&nbsp;&nbsp;&nbsp; 3&nbsp;&nbsp;&nbsp;&nbsp; 5</p>
 +
            <p id="u3817-17">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 1:1&nbsp;&nbsp; 3:1&nbsp; 1:3&nbsp; 5:3&nbsp;&nbsp; 3:5</p>
 +
            <p id="u3817-19">16ºC&nbsp;&nbsp;&nbsp;&nbsp; 30’</p>
 +
            <p id="u3817-21">RT&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 30’</p>
 +
            <p id="u3817-23">RT&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 30’</p>
 +
            <p id="u3817-25">+4ºC&nbsp;&nbsp;&nbsp; Overnight</p>
 +
            <p id="u3817-26">&nbsp;</p>
 +
            <p id="u3817-27">&nbsp;</p>
 +
            <p id="u3817-28">&nbsp;</p>
 +
            <p id="u3817-29">&nbsp;</p>
 +
            <p id="u3817-30">&nbsp;</p>
 +
            <p id="u3817-31">&nbsp;</p>
 +
            <p id="u3817-32">&nbsp;</p>
 +
            <p id="u3817-33">&nbsp;</p>
 +
            <p id="u3817-34">&nbsp;</p>
 +
            <p id="u3817-35">&nbsp;</p>
 +
            <p id="u3817-36">&nbsp;</p>
 +
            <p id="u3817-37">&nbsp;</p>
 +
            <p id="u3817-38">&nbsp;</p>
 +
            <p id="u3817-39">&nbsp;</p>
 +
            <p id="u3817-40">&nbsp;</p>
 +
            <p id="u3817-41">&nbsp;</p>
 +
            <p id="u3817-42">&nbsp;</p>
 +
            <p id="u3817-43">&nbsp;</p>
 +
            <p id="u3817-44">&nbsp;</p>
 +
            <p id="u3817-45">&nbsp;</p>
 +
            <p id="u3817-46">&nbsp;</p>
 +
            <p id="u3817-47">&nbsp;</p>
 +
            <p id="u3817-48">&nbsp;</p>
 +
            <p id="u3817-49">&nbsp;</p>
 +
            <p id="u3817-50">&nbsp;</p>
 +
            <p id="u3817-51">&nbsp;</p>
 +
            <p id="u3817-52">&nbsp;</p>
 +
            <p id="u3817-53">&nbsp;</p>
 +
            <p id="u3817-54">&nbsp;</p>
 +
            <p id="u3817-55">&nbsp;</p>
 +
            <p id="u3817-56">&nbsp;</p>
 +
            <p id="u3817-57">&nbsp;</p>
 +
            <p id="u3817-58">&nbsp;</p>
 +
            <p id="u3817-59">&nbsp;</p>
 +
            <p id="u3817-60">&nbsp;</p>
 +
            <p id="u3817-61">&nbsp;</p>
 +
            <p id="u3817-62">&nbsp;</p>
 +
            <p id="u3817-63">&nbsp;</p>
 +
            <p id="u3817-64">&nbsp;</p>
 +
            <p id="u3817-65">&nbsp;</p>
 +
            <p id="u3817-66">&nbsp;</p>
 +
            <p id="u3817-67">&nbsp;</p>
 +
            <p id="u3817-68">&nbsp;</p>
 +
            <p id="u3817-70">Transformation:</p>
 +
            <p id="u3817-72">Öykü: 1st: (3) 2 GE 2h A (3:1)&nbsp; &#45;&nbsp; 2nd: 2 GE A 301</p>
 +
            <p id="u3817-74">Can (Öykü): 1st: (4) 2 GE A 2h (1:3) &#45; 2nd: 2 GE A 301</p>
 +
            <p id="u3817-76">Almira: 1st: (2) 2GE A 2h 1:1 (3:5) &#45; 2nd: 2 GE A 301 – (5) 2GE A 301</p>
 +
            <p id="u3817-78">Martin: 1st: (1) 2GE A 2h 5:3 (1:1) &#45; 2nd: 2 GE A 301</p>
 +
            <p id="u3817-79">&nbsp;</p>
 +
            <p id="u3817-80">&nbsp;</p>
 +
            <p id="u3817-81">&nbsp;</p>
 +
            <p>&nbsp;</p>
 +
          </div>
 +
          <div class="clip_frame grpelem" id="u3818"><!-- image -->
 +
            <img class="block" id="u3818_img" src="http://fisofowi.netii.net/images/plasmid.jpg" alt="" width="569" height="261" />
 +
          </div>
 +
          <div class="clip_frame grpelem" id="u3823"><!-- image -->
 +
            <img class="block" id="u3823_img" src="http://fisofowi.netii.net/images/foto1.jpg" alt="" width="410" height="288" />
 +
          </div>
 +
          <div class="clip_frame grpelem" id="u3828"><!-- image -->
 +
            <img class="block" id="u3828_img" src="http://fisofowi.netii.net/images/foto2.jpg" alt="" width="410" height="329" />
 +
          </div>
 +
          </div>
 +
          <div class="Container invi clearfix grpelem" id="u3833"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3847-29"><!-- content -->
 +
            <p id="u3847-2">Transformation &#45; +4 ONC</p>
 +
            <p id="u3847-4">PCR &#45; 2 &#45; 2’</p>
 +
            <p id="u3847-6">PCR Purification</p>
 +
            <p id="u3847-8">Digestion</p>
 +
            <p id="u3847-10">Gel</p>
 +
            <p id="u3847-12">Gel Extraction</p>
 +
            <p id="u3847-14">Ligation 16ºC&nbsp;&nbsp; 1h</p>
 +
            <p id="u3847-16">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; RT&nbsp;&nbsp;&nbsp;&nbsp; 2h</p>
 +
            <p id="u3847-18">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; +4&nbsp;&nbsp;&nbsp;&nbsp; ONC</p>
 +
            <p id="u3847-20">Gel</p>
 +
            <p id="u3847-22">Gel Extraction</p>
 +
            <p id="u3847-24">Transformation</p>
 +
            <p id="u3847-25">&nbsp;</p>
 +
            <p id="u3847-26">&nbsp;</p>
 +
            <p>&nbsp;</p>
 +
          </div>
 +
          <div class="clip_frame grpelem" id="u3848"><!-- image -->
 +
            <img class="block" id="u3848_img" src="http://fisofowi.netii.net/images/foto3.jpg" alt="" width="619" height="338" />
 +
          </div>
 +
          </div>
 +
          <div class="Container invi clearfix grpelem" id="u3853"><!-- group -->
 +
          <div class="clip_frame grpelem" id="u3861"><!-- image -->
 +
            <img class="block" id="u3861_img" src="http://fisofowi.netii.net/images/foto4.jpg" alt="" width="645" height="258" />
 +
          </div>
 +
          </div>
 +
        </div>
 +
        </div>
 +
        <div class="ThumbGroup clearfix grpelem" id="u2974"><!-- none box -->
 +
        <div class="popup_anchor">
 +
          <div class="Thumb popup_element clearfix" id="u2975"><!-- group -->
 +
          <div class="clearfix grpelem" id="u2976-4"><!-- content -->
 +
            <p>23.04.2014</p>
 +
          </div>
 +
          </div>
 +
        </div>
 +
        <div class="popup_anchor">
 +
          <div class="Thumb popup_element clearfix" id="u2979"><!-- group -->
 +
          <div class="clearfix grpelem" id="u2980-4"><!-- content -->
 +
            <p>28.04.2014</p>
 +
          </div>
 +
          </div>
 +
        </div>
 +
        <div class="popup_anchor">
 +
          <div class="Thumb popup_element clearfix" id="u2977"><!-- group -->
 +
          <div class="clearfix grpelem" id="u2978-4"><!-- content -->
 +
            <p><span id="u2978">29.04.2014</span></p>
 +
          </div>
 +
          </div>
 +
        </div>
 +
        <div class="popup_anchor">
 +
          <div class="Thumb popup_element clearfix" id="u3021"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3024-4"><!-- content -->
 +
            <p><span id="u3024">01.05.2014</span></p>
 +
          </div>
 +
          </div>
 +
        </div>
 +
        <div class="popup_anchor">
 +
          <div class="Thumb popup_element clearfix" id="u3026"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3047-4"><!-- content -->
 +
            <p><span id="u3047">16.05.2014</span></p>
 +
          </div>
 +
          </div>
 +
        </div>
 +
        <div class="popup_anchor">
 +
          <div class="Thumb popup_element clearfix" id="u3029"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3051-4"><!-- content -->
 +
            <p><span id="u3051">23.05.2014</span></p>
 +
          </div>
 +
          </div>
 +
        </div>
 +
        <div class="popup_anchor">
 +
          <div class="Thumb popup_element clearfix" id="u3031"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3053-4"><!-- content -->
 +
            <p><span id="u3053">25.05.2014</span></p>
 +
          </div>
 +
          </div>
 +
        </div>
 +
        <div class="popup_anchor">
 +
          <div class="Thumb popup_element clearfix" id="u3033"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3055-4"><!-- content -->
 +
            <p><span id="u3055">30.05.2014</span></p>
 +
          </div>
 +
          </div>
 +
        </div>
 +
        <div class="popup_anchor">
 +
          <div class="Thumb popup_element clearfix" id="u3035"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3068-4"><!-- content -->
 +
            <p><span id="u3068">09.06.2014</span></p>
 +
          </div>
 +
          </div>
 +
        </div>
 +
        <div class="popup_anchor">
 +
          <div class="Thumb popup_element clearfix" id="u3037"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3070-4"><!-- content -->
 +
            <p><span id="u3070">10.06.2014</span></p>
 +
          </div>
 +
          </div>
 +
        </div>
 +
        <div class="popup_anchor">
 +
          <div class="Thumb popup_element clearfix" id="u3039"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3086-4"><!-- content -->
 +
            <p><span id="u3086">11.06.2014</span></p>
 +
          </div>
 +
          </div>
 +
        </div>
 +
        <div class="popup_anchor">
 +
          <div class="Thumb popup_element clearfix" id="u3041"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3093-4"><!-- content -->
 +
            <p><span id="u3093">12.06.2014</span></p>
 +
          </div>
 +
          </div>
 +
        </div>
 +
        <div class="popup_anchor">
 +
          <div class="Thumb popup_element clearfix" id="u3043"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3095-4"><!-- content -->
 +
            <p><span id="u3095">13.06.2014</span></p>
 +
          </div>
 +
          </div>
 +
        </div>
 +
        <div class="popup_anchor">
 +
          <div class="Thumb popup_element clearfix" id="u3045"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3102-4"><!-- content -->
 +
            <p><span id="u3102">16.06.2014</span></p>
 +
          </div>
 +
          </div>
 +
        </div>
 +
        <div class="popup_anchor">
 +
          <div class="Thumb popup_element clearfix" id="u3807"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3809-4"><!-- content -->
 +
            <p><span id="u3809">17.06.2014</span></p>
 +
          </div>
 +
          </div>
 +
        </div>
 +
        <div class="popup_anchor">
 +
          <div class="Thumb popup_element clearfix" id="u3814"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3816-4"><!-- content -->
 +
            <p><span id="u3816">18.06.2014</span></p>
 +
          </div>
 +
          </div>
 +
        </div>
 +
        <div class="popup_anchor">
 +
          <div class="Thumb popup_element clearfix" id="u3844"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3846-4"><!-- content -->
 +
            <p><span id="u3846">19.06.2014</span></p>
 +
          </div>
 +
          </div>
 +
        </div>
 +
        <div class="popup_anchor">
 +
          <div class="Thumb popup_element clearfix" id="u3858"><!-- group -->
 +
          <div class="clearfix grpelem" id="u3860-4"><!-- content -->
 +
            <p><span id="u3860">20.06.2014</span></p>
 +
          </div>
 +
          </div>
 +
        </div>
 +
        </div>
       </div>
       </div>
       </div>
       </div>
-
      <div class="TabbedPanelsContent invi grpelem" id="u332"><!-- simple frame --></div>
 
-
      <div class="TabbedPanelsContent invi grpelem" id="u1382"><!-- simple frame --></div>
 
-
      <div class="TabbedPanelsContent invi grpelem" id="u2488"><!-- simple frame --></div>
 
     </div>
     </div>
     </div>
     </div>
Line 126: Line 896:
   <div class="verticalspacer"></div>
   <div class="verticalspacer"></div>
   <a class="nonblock nontext clip_frame grpelem" id="u2117" href="https://2014hs.igem.org"><!-- image --><img class="block" id="u2117_img" src="http://fisofowi.netii.net/images/wiki.jpg" alt="" width="418" height="70" /></a>
   <a class="nonblock nontext clip_frame grpelem" id="u2117" href="https://2014hs.igem.org"><!-- image --><img class="block" id="u2117_img" src="http://fisofowi.netii.net/images/wiki.jpg" alt="" width="418" height="70" /></a>
 +
  <a class="nonblock nontext clip_frame grpelem" id="u2751" href="https://2014hs.igem.org/Team:METUHS-Ankara"><!-- image --><img class="block" id="u2751_img" src="http://fisofowi.netii.net/images/metuhslogo_alpha-u2751.png" alt="" width="323" height="70" /></a>
   <a class="nonblock nontext clip_frame grpelem" id="u2123" href="http://odtugvo.k12.tr"><!-- image --><img class="block" id="u2123_img" src="http://fisofowi.netii.net/images/logo%20alpha.jpg" alt="" width="141" height="70" /></a>
   <a class="nonblock nontext clip_frame grpelem" id="u2123" href="http://odtugvo.k12.tr"><!-- image --><img class="block" id="u2123_img" src="http://fisofowi.netii.net/images/logo%20alpha.jpg" alt="" width="141" height="70" /></a>
   </div>
   </div>
Line 138: Line 909:
   <script src="http://fisofowi.netii.net/scripts/jquery.musemenu.js?3788803530" type="text/javascript"></script>
   <script src="http://fisofowi.netii.net/scripts/jquery.musemenu.js?3788803530" type="text/javascript"></script>
   <script src="http://fisofowi.netii.net/scripts/webpro.js?3903299128" type="text/javascript"></script>
   <script src="http://fisofowi.netii.net/scripts/webpro.js?3903299128" type="text/javascript"></script>
 +
  <script src="http://fisofowi.netii.net/scripts/musewpslideshow.js?138381373" type="text/javascript"></script>
 +
  <script src="http://fisofowi.netii.net/scripts/jquery.museoverlay.js?4250894771" type="text/javascript"></script>
 +
  <script src="http://fisofowi.netii.net/scripts/touchswipe.js?4156838003" type="text/javascript"></script>
   <script src="http://fisofowi.netii.net/scripts/musewpdisclosure.js?4285748861" type="text/javascript"></script>
   <script src="http://fisofowi.netii.net/scripts/musewpdisclosure.js?4285748861" type="text/javascript"></script>
   <script src="http://fisofowi.netii.net/scripts/jquery.watch.js?4068933136" type="text/javascript"></script>
   <script src="http://fisofowi.netii.net/scripts/jquery.watch.js?4068933136" type="text/javascript"></script>
Line 146: Line 920:
Muse.Utils.prepHyperlinks(true);/* body*/
Muse.Utils.prepHyperlinks(true);/* body*/
Muse.Utils.initWidget('.MenuBar', function(elem) { return $(elem).museMenu(); });/* unifiedNavBar*/
Muse.Utils.initWidget('.MenuBar', function(elem) { return $(elem).museMenu(); });/* unifiedNavBar*/
-
Muse.Utils.initWidget('#tab-panelu326', function(elem) { return new WebPro.Widget.TabbedPanels(elem, {event:'click',defaultIndex:1}); });/* #tab-panelu326*/
+
Muse.Utils.initWidget('#pamphletu2946', function(elem) { new WebPro.Widget.ContentSlideShow(elem, {contentLayout_runtime:'stack',event:'click',deactivationEvent:'none',autoPlay:false,displayInterval:3000,transitionStyle:'fading',transitionDuration:300,hideAllContentsFirst:false,shuffle:false,enableSwipe:true}); });/* #pamphletu2946*/
 +
Muse.Utils.initWidget('#tab-panelu326', function(elem) { return new WebPro.Widget.TabbedPanels(elem, {event:'click',defaultIndex:0}); });/* #tab-panelu326*/
Muse.Utils.fullPage('#page');/* 100% height page*/
Muse.Utils.fullPage('#page');/* 100% height page*/
Muse.Utils.showWidgetsWhenReady();/* body*/
Muse.Utils.showWidgetsWhenReady();/* body*/
Line 152: Line 927:
} catch(e) { Muse.Assert.fail('Error calling selector function:' + e); }});
} catch(e) { Muse.Assert.fail('Error calling selector function:' + e); }});
</script>
</script>
-
 
+
</body>
-
 
+
-
 
+
-
 
+
-
 
+
-
          <div id="report-abuse"><a href="http://www.businesscatalyst.com/report-abuse?URL=metuhsigem.businesscatalyst.com%2fproject.html"><span>Report Abuse</span></a></div>
+
-
      </body>
+
</html>
</html>

Latest revision as of 03:52, 21 June 2014

Project

Project Description

Design

Results

Human Practices

Lab Notebook

Carbon monoxide (CO) is a colorless and odorless gas which can be very poisonous and dangerous. Since it is produced from commonly used household devices and industry, in the case of any leakage it can cause severe poisonings and deaths. Many people die in Turkey because of mishandled ascot and blast heaters. According to statistics 10,154 people, meaning 14 out of every 100,000 people, were intoxicated.

 

The aim of our project is to prevent these cases. In order to achieve this, we plan to develop a biological device which will include a detection and a conversion system. Firstly, our detection mechanism is based on light dependent sensors. These sensors gather data from the bacteria, in turn triggering an alarm system. Secondly, in the event of CO presence, our conversion system will be activated in order to convert carbon monoxide into carbon dioxide (CO2). To accomplish this transformation, we are using an enzyme called Carbon Monoxide Dehydrogenase (CODH). Finally, as a safety measure we will include a kill switch mechanism that aims to inactivate the system.

 

CO to CO2 Converter & CO Monitoring System

 

Carbon monoxide is a highly toxic gas which is undetectable by humans and it is fatal when inhaled. We’ve developed a biological device that comprises of both a qualitative detector for this dangerous gas and a conversion system to transform it into carbon dioxide. As for detection, CO sensitive promoters pCooM and pCooF from Rhodosprillum rubrum will initiate the production of fluorescent proteins in the presence of CO. Optic sensors will be used to track the production of these proteins and if the sensors pick up data indicating that CO is present; an alarm will be triggered. Meanwhile, the conversion system of our device will utilize a Cyanobacteria enzyme called Carbon Monoxide Dehydrogenase (CODH), which converts CO into CO2. We also have a kill-switch design based on the lac-operon. The kill-switch mechanism will be activated to avoid any contamination of the environment, in case the altered bacteria escape the device.

constitutive promoter

Kill switch

 

we use an mRNA interferase, MazF, which cleaves mRNA’s at specific sequences. In our kill switch, we used anti-sense RNA principle as a template. According to this principle, MazF, which is constantly produced via a constitutive promoter, is got inactivated by Anti-MazF construct. In order to trigger this mechanism, we used IPTG, a harmless molecule for the bee which at the same time does not appear in the honey too. When IPTG is present in the environment, LacI is inhibited by IPTG and therefore promoter gets activated. With the activated promoter of it, Anti-MazF is produced and inactive MazF. As long as IPTG exists, MazF gets inactivated continuously; therefore, the bacteria maintain their lives. On the other hand, if bacteria exists in a IPTG-free environment, Anti-MazF producing stops, which leads to MazF producing and bacteria get killed by MazF. (Circuit image courtesy of METU iGEM Team 2013)

In this page, you can find out the computational modelling for our design and finished system.

 

Carbon monoxide

 

With our research we were able to find out that 1500 ppm of CO gas means death in an hour. To make calculations we need to convert this value into something we can work with easily, such as moles.

 

1500 ppm = 0.15%

 

If we take a 30 meter squared room with 3 meter ceiling height, the overall volume of the room is 90m^3 which equates to 90000 liters.

 

By using Ideal Gas Law, we can say that the amount of moles of CO present in 135 Liters is around about 4 moles. So what we have learned? In a big size room, there should be at least 4 moles of CO molecules for it to be considered as deadly.

 

CODH Activity

 

Even though we were not able to find detailed parameters for this particular enzyme, we were able to work out a very simple formula to calculate the amount of CO we will be able to convert each second.

 

r = CODH activity rate

p = plasmid copy number

b = amount of bacteria

s = synthesis rate

[CO]=amount of CO (moles)

A = fraction of CO converted

 

*Lets call r.p.b.s: "c" since we can assume that it will stay constant.

*Our calculations show that "c = 31777209688000" or "3.2 x 10^14"

 

When we divide c by the Avagadro number we can find out what fraction of moles of CO we will be able to convert each time frame. Even though "c" seems like a huge number, the Avagadro number is much bigger.

 

3.2 x 10^14 / 6.02 x 10^23  =  0.5 x 10^-9

 

This mans that we are able to convert roughly 0.000000001 of the CO available each second.

 

Here is a little script we wrote with Python programming language with modules "matplotlib" and "numPy":

 

from pylab import *

 

timer = 0

f = 0.000000001

s = 7200 #seconds

co = 4 # moles

co2 =0 # moles

values = [ ]

 

for i in range(s):

    co2 = co2 + co*f

    co = co - co2

    if co <= 0:

        break

    values.append(co2)

    timer += 1

 

x = arange(0,timer,1)

plot(x,values)

 

xlabel('time (s)')

ylabel('CO2 (moles)')

title('CO2 - Time')

grid(True)

show()

 

 

Please note that, even though we are subtracting the amount of CO converted from the initial CO value, the graph still does not have a visible curve to it. It is obvious that even though we are able to convert a huge number of CO molecules each given second, there are way to much CO then our system can convert.

 

By increasing the surface are of the single-layer e-coli colonies, for example take a 10 x 10 grid of the same exact system, we can achieve 100 times more efficient converters which can shave of two 0's from the results.

 

Down below you can see the photos of the colonies we were able to get and the electronic circuit design.

 

 

Trying out the color sensor. Red pen on the sensor light up red. Blue ruler on top, blue light.

Final images of the design.

Today, improving technology and ideas lead students to be the constructors of better future. That’s why as METU DF High School iGEM 2014 team we made researches and experiments to widen the perspective of what synthetic biology can achieve. We know that synthetic biology is the way of constructing core components which can be modeled by merging them with the fundamentals of biology. We assume that by the efficient use of synthetic biology we can lighten the way through future and save many lives.

 

Therefore, this year we worked hard to put forth an experiment that can lead the way to our better future. As METU HS iGEM team, we believe that synthetic biology is a tool that can turn the world to a better place. And iGEM is an opportunity for us to show people that synthetic biology is helpful for the humankind. That's why we did many events to introduce people synthetic biology. We have given a bake sale which included bacteria & DNA muffins and x-ray crystallography cake.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

The muffins got retweeted by the iGEM HQ!

 

 

 

Furthermore we interviewed some students in our school to see how much they know about synthetic biology world. We decorated our school with DNA's and posters of our team on DNA day. Also as METU HS iGEM team we have attended to a Synthetic Biology Day to expand our knowledge. It is important for the next generations to be familiar with synthetic biology, in order to achieve this we gave a presentation to 7 and 8th graders. We have informed them about iGEM and biology. And our aim is to present iGEM to everyone to convince people that synthetic biology is useful. So we have told people about our team and iGEM at the opening of an ODTU DF school in Denizli. In order to maintain our goal we believed that a sale of work will be a nice introduction of iGEM and synthetic biology. We cooked cupcakes in order to sale and we designed them as bacteria and everything about synthetic biology. Secondly, we also designed an enormous DNA shaped gumdrop. The most critical part was we recommended iGEM and talk with them about the project we were doing. Our main purpose was to make people know what synthetic biology is and create awareness about these topics. To conclude we have tried and we are still trying to spread synthetic biology in order to acknowledge human. As METU DF High School iGEM 2014 team we work hard to settle the importance of synthetic biology by referring various projects.

 

We believe that awareness and education is the key of action, acceptance and understanding. Our project contains lots of valuable experiments that can prove the useful remedies of the efficient use of synthetic biology.

 

You can find every photo and video we referred to at the fun page by following this link or find it at the extras page!