Team:METU/KillSwitchOverview

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                        <li><a href="https://2012.igem.org/Team:METU/test/KillSwitchOverview"><span>Kill-Switch</span></a>
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<h2><b> Cell Limiter </b></h2>
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<span class="toctoggle">&nbsp;[<a href="#" class="internal" id="togglelink">gizle</a>]&nbsp;</span></div>
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                                        <li><a href="https://2012.igem.org/Team:METU/test/KillSwitchOverview#Overview">Overview</a></li>
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                                        <li><a href="https://2012.igem.org/Team:METU/KillSwitch/Modelling#Modelling">Modelling</a></li>
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<li class="toclevel-1 tocsection-1"><a href="#overview"><span class="tocnumber"></span> <span class="toctext">Overview</span></a></li>
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                                        <li><a href="https://2012.igem.org/Team:METU/test/KillSwitchOverview#Results">Results</a></li>
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                        <li><a href="https://2012.igem.org/Team:METU/test/CellLimiter"><span>Cell Limiter</span></a>
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                                        <li><a href="https://2012.igem.org/Team:METU/test/CellLimiter#Overview">Overview</a></li>
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                                        <li><a href="https://2012.igem.org/Team:METU/test/CellLimiter#Modelling">Modelling</a></li>
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                                        <li><a href="https://2012.igem.org/Team:METU/test/Overview">Overview</a></li>
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                                        <li><a href="/Team:METU/Timeline">Timeline</a></li>
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                    <h1>Kill Switch</h1>
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                         <h2>Overview</h2>
                         <h2>Overview</h2>
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                             <p>
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                                 In our project we aimed to construct a biofilm which will convert CO to CO2 biologically. Biofilms can be defined as the group of cells firmly united with the help of lipids, protein, DNA and polysaccharides and they can be used for beneficial applications [1] but the main obstacle in the case of biofilm formation is to keep the cell growth under control. We wanted our biofilm to work as a CO filter, due to that reason we designed a quorum sensing (QS) system to control the number of cells and to characterize the activity of biofilm better.
+
                                 Kill switch is a safety system which is necessary to control the release of genetically modified
-
</p>
+
                                organisms (GMOs) into the environment. Since we aimed to construct a biofilm with GMOs which will convert CO to CO2, we needed a kill switch that can prevent our system to cause any harm to the environment. In order to achieve that aim we used the kill switch designed by Berkeley 2008 iGEM Team (Bba_K112808) and also we tried to develop it.
-
 
+
-
<p>Cell population density changes as the bacteria grow and divide in the biofilm and QS can be defined as the regulation of gene expression during this change. As the cell density increases bacteria starts to release molecules named as autoinducers and according to the concentration of autoinducers gene expression is altered[2]. Eventually bacterial growth will be controlled. In our project we aimed to modify E.coli to synthesize its own QS signals, detect the cell density and prevent the cell division.
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                         </span>
                         </span>
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                         <h2 id="how" >How The System Works ?</h2>
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                         <h2>How The System Works ?</h2>
                         <p>
                         <p>
-
At the beginning of our QS system there is a constitutive promoter J23116 which is used for the constitutive production of LuxI, an autoinducer synthetase for AHL (acyl-homoserine lactone). Function of LuxI is to synthesize 3OC6HSL (3-oxohexanoyl-homoserine lactone).
 
-
When 3OC6HSL is produced, it will form a complex with LuxR which is synthesized according to the activity of LuxPL promoter. After that point we aimed LuxR-3OC6HSL complex to increase the activity of LuxPR promoter.
 
-
</p>
 
-
<p>Finally LuxPR activity determines the amount of LasR and when PLasR promoter is activated
 
-
MinC, a cell division inhibitor can be synthesized. Eventually, cell division stops after MinC reaches its critical concentration.</p>
 
 +
                            In order to construct our kill switch system we needed a reagent that E.Coli will grow and in the absence of that reagent all the bacteria should die. For that purpose we prefered to use IPTG (Isopropyl-beta-D-thiogalactopyranoside) because it cannot be metabolised by E.Coli and that’s why  its concentration remains constant in the cell.
 +
                            When IPTG is present in the cell it will bind to LacI which can be produced by the promoter J23116. Basal LacI expression is already present in E.Coli but at low levels and it is hard to control the system with that much LacI. Hence, we prefered to produce it with the help of  J23116.
-
                    </div>
+
                            Our kill switch system includes the expression of endolysin, holin, antiholin and their production is controlled with the initial concentration of IPTG in the environment. Actually
 +
                            cell lysis can occur without IPTG but the lysis rate will be high. By using IPTG we aimed to keep our system under control.
-
                    <div id="Modelling">
+
                             *Endolysin-Holin System; Endolysin and holin are the proteins needed for the cell lysis. Endolysin is a lysozyme and its funtion is to hydrolyze the peptidoglycan bacterial cell wall but it needs holin to form pores on  inner membrane, then it can reach periplasm and cell lysis occur.
-
                        <h2 id="model" >MODELLING[<a href="#table">up</a>]</h2>
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                        <span>
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                             <p>
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</div>
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</div>
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<!-- End Header -->
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<!-- Main -->
 
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<div id="main">
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                        </p>
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<div class="shell">
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<div class="col2">
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-
<p><b> Cell Limiter Model </b></p>
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-
  <span>
+
-
&nbsp;
+
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<p>
+
-
Quorum sensing model aims to determine number of cells and cell density in our system. We used ODEs to model system and write a program in MATLAB. Although the aim was to get certain numbers, we couldn’t succeed it due to lack of parameters found in literature. As a result we decided to include 18 constitutive promoters found in partsregistry in our model, and after the missing parameters are entered, the program will give desired results.
+
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</p>
+
-
<p>You can download zip file from here. deneme22.m should be run in MATLAB to run program. Screenshots from program:
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</p>
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<p><img src="https://static.igem.org/mediawiki/2012/3/3d/Metu-photo002.jpg" width="1054px" height="742px" "/> </p>
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<p><img src="https://static.igem.org/mediawiki/2012/d/db/Metu-photo-1.jpg" width="1054px" height="742px" "/> </p>
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<p><img src="https://static.igem.org/mediawiki/2012/7/72/Metu-photo004.jpg" width="1054px" height="742px" "/> </p>
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<p>
+
                        <p>
-
<b>Introduction:</b>
+
                            The fate of the Gram-negative envelope during holin–endolysin lysis.
-
</p>
+
                        </p>
-
<p><img src="https://static.igem.org/mediawiki/2012/d/d8/Metu-photo006.jpg"/> </p>
+
                        Pre-Hole Configuration                          Hole Configuration
-
<p>
+
-
Our system starts to work with constitutive production of LuxI. After HSL is produced by LuxI, it forms complex with LuxR whoso transcription is controlled by the activity of LuxPL promoter. HSL&LuxR complex negatively regulates LuxPL promoter, as a result when the number of HSL&LuxR complex increase individual cells will drop expression level of LuxR. LasPR promoter is in the control of LasR expression and as HSL&LuxR complex amount increase the LasR production increases. Because the minC expression controlled by LasR promoter, as LasR increases which means as amount of HSL&LuxR complex increases, the expression level of minC increases. Because minC acts as cell division inhibitor after 55 fold of its normal level, we conclude that increase in the HSL&LuxR complex amount to certain level will cause cell division to stop. Since HSL molecules produced by each cell will eventually affect other cells, thanks to this system we can control cell density.
+
-
To understand mechanism correctly and choose constitutive promoter that will control the rate of production of LuxI according to the desired cell density we mathematically modelled system and then create a program that gives user a chance to select starting promoter and to see resulting number of cells graph
+
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</p>
+
                        <p>
 +
                            <img src="https://static.igem.org/mediawiki/2012/9/97/Metu-photos-model27.png.jpg" alt="">
 +
                        </p>
-
</p>
+
                        <p>
 +
                            In this figure holins are shown as clear ovals and endolysin is shown as notched circles.
 +
                        </p>
-
<p>
+
                        <ul>
-
<b>Mathematical Model:</b>
+
                            <li> Accumulation of holin is present in the inner membrane, while endolysin accumulates in the cytosol. Holin action can be prevented with the inhibitors (shown with star and black ovals).</li>
-
</p>
+
                            <li>    When the holes are formed by holin in the inner membrane, endolysin can attack the murein which is important for cell lysis to occur.</li>
 +
                        </ul>
 +
                        &nbsp;
 +
                        <p>
-
In our model, we included all processes shown below.
+
                            * Antiholin; It is the protein that blocks the function of holin and it is important for the regulation of cell lysis.
 +
                        </p>
-
<img src="https://static.igem.org/mediawiki/2012/8/8c/Metu-photo008.jpg" alt="" ">
+
                        <p id="Overview">
 +
                            In our system antiholin is constitutively produced by the promoter J23116 and when antiholin level is in between 1000-3000 molecules, the cell will die. Actually,it is the number of free holin molecules that determines if the cell will die or not.
 +
                        </p>
 +
                    </div>
-
<p><b>Equations for mRNA transcription</b></p>
+
                    <div id="Modelling">
-
&nbsp;
+
                        <h2>Modelling</h2>
-
&nbsp;
+
                        <span>
-
&nbsp;
+
                            <p>
-
<p><img src="https://static.igem.org/mediawiki/2012/b/b7/Metu-photo010.png" alt="" ">
+
                            </p>
-
</p>
+
                        </span>
-
&nbsp;
+
                    </div>
-
<p><img src="https://static.igem.org/mediawiki/2012/8/87/Metu-photo012.png" alt="" ">
+
                    <div id="Results">
-
</p>
+
                        <h2>Results</h2>
-
&nbsp;
+
                        <span>
-
<p><img src="https://static.igem.org/mediawiki/2012/e/e7/Metu-photo014.png" alt="" ">
+
                            <p>
-
</p>
+
-
&nbsp;
+
-
<p><img src="https://static.igem.org/mediawiki/2012/d/d5/Metu-photo016.png" alt="" ">
+
-
</p>
+
-
&nbsp;
+
-
 
+
-
<b>
+
-
Equations for protein translation & complex formation
+
-
</b>
+
-
&nbsp;
+
-
&nbsp;
+
-
&nbsp;
+
-
<p><img src="https://static.igem.org/mediawiki/2012/7/73/Metu-photo019.png" alt="" />
+
-
</p>
+
-
&nbsp;
+
-
<p><img src="https://static.igem.org/mediawiki/2012/6/64/Metu-photo020.png" alt="" />
+
-
</p>
+
-
&nbsp;
+
-
<p><img src="https://static.igem.org/mediawiki/2012/6/6a/Metu-photo022.png" alt="" />
+
-
</p>
+
-
&nbsp;
+
-
<p><img src="https://static.igem.org/mediawiki/2012/6/64/Metu-photo024.png" alt="" />
+
-
</p>
+
-
&nbsp;
+
-
<p><img src="https://static.igem.org/mediawiki/2012/9/95/Metu-photo026.png" alt="" />
+
-
</p>
+
-
&nbsp;
+
-
<p><img src="https://static.igem.org/mediawiki/2012/d/da/Metu-photo028.png" alt="" />
+
-
</p>
+
-
&nbsp;
+
-
<p><img src="https://static.igem.org/mediawiki/2012/b/b8/Metu-photo030.png" alt="" />
+
-
</p>
+
-
&nbsp;
+
-
<p>C is HSL&LuxR Complex</p>
+
-
 
+
-
<table class="ListTable31" border="1" cellspacing="0" cellpadding="0" style="border-collapse:
+
-
collapse;border:none;mso-border-alt:solid black .5pt;mso-border-themecolor:
+
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text1;mso-yfti-tbllook:1184;mso-padding-alt:1.45pt 5.4pt 1.45pt 5.4pt">
+
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<tbody><tr style="mso-yfti-irow:-1;mso-yfti-firstrow:yes">
+
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  <td valign="top" style="border-top:solid black 1.0pt;mso-border-top-themecolor:
+
-
  text1;border-left:solid black 1.0pt;mso-border-left-themecolor:text1;
+
-
  border-bottom:none;border-right:none;mso-border-top-alt:solid black .5pt;
+
-
  mso-border-top-themecolor:text1;mso-border-left-alt:solid black .5pt;
+
-
  mso-border-left-themecolor:text1;background:black;mso-background-themecolor:
+
-
  text1;padding:1.45pt 5.4pt 1.45pt 5.4pt">
+
-
  <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
+
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  normal;mso-yfti-cnfc:517"><b><span lang="EN-US" style="font-size:14.0pt;
+
-
  mso-bidi-font-size:11.0pt;color:white;mso-themecolor:background1;mso-ansi-language:
+
-
  EN-US">Parameter<o:p></o:p></span></b></p>
+
-
  </td>
+
-
  <td valign="top" style="border-top:solid black 1.0pt;mso-border-top-themecolor:
+
-
  text1;border-left:none;border-bottom:none;border-right:solid black 1.0pt;
+
-
  mso-border-right-themecolor:text1;mso-border-top-alt:solid black .5pt;
+
-
  mso-border-top-themecolor:text1;mso-border-right-alt:solid black .5pt;
+
-
  mso-border-right-themecolor:text1;background:black;mso-background-themecolor:
+
-
  text1;padding:1.45pt 5.4pt 1.45pt 5.4pt">
+
-
  <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
+
-
  normal;mso-yfti-cnfc:1"><b><span lang="EN-US" style="font-size:14.0pt;
+
-
  mso-bidi-font-size:11.0pt;color:white;mso-themecolor:background1;mso-ansi-language:
+
-
  EN-US">Description<o:p></o:p></span></b></p>
+
-
  </td>
+
-
</tr>
+
-
<tr style="mso-yfti-irow:0">
+
-
  <td valign="top" style="border:solid black 1.0pt;mso-border-themecolor:text1;
+
-
  border-right:none;mso-border-top-alt:solid black .5pt;mso-border-top-themecolor:
+
-
  text1;mso-border-left-alt:solid black .5pt;mso-border-left-themecolor:text1;
+
-
  mso-border-bottom-alt:solid black .5pt;mso-border-bottom-themecolor:text1;
+
-
  background:white;mso-background-themecolor:background1;padding:1.45pt 5.4pt 1.45pt 5.4pt">
+
-
  <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
+
-
  normal;mso-yfti-cnfc:68"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:
+
-
  11.0pt;mso-ansi-language:EN-US;mso-bidi-font-weight:bold">N<o:p></o:p></span></p>
+
-
  </td>
+
-
  <td valign="top" style="border:solid black 1.0pt;mso-border-themecolor:text1;
+
-
  border-left:none;mso-border-top-alt:solid black .5pt;mso-border-top-themecolor:
+
-
  text1;mso-border-bottom-alt:solid black .5pt;mso-border-bottom-themecolor:
+
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  text1;mso-border-right-alt:solid black .5pt;mso-border-right-themecolor:text1;
+
-
  padding:1.45pt 5.4pt 1.45pt 5.4pt">
+
-
  <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
+
-
  normal;mso-yfti-cnfc:64"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:
+
-
  11.0pt;mso-ansi-language:EN-US">Plasmid copy number<o:p></o:p></span></p>
+
-
  </td>
+
-
</tr>
+
-
<tr style="mso-yfti-irow:1">
+
-
  <td valign="top" style="border:none;border-left:solid black 1.0pt;mso-border-left-themecolor:
+
-
  text1;mso-border-left-alt:solid black .5pt;mso-border-left-themecolor:text1;
+
-
  background:white;mso-background-themecolor:background1;padding:1.45pt 5.4pt 1.45pt 5.4pt">
+
-
  <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
+
-
  normal;mso-yfti-cnfc:4"><span class="SpellE"><span lang="EN-US" style="font-size:
+
-
  14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;mso-bidi-font-weight:
+
-
  bold">P<sub>x</sub></span></span><sub><span lang="EN-US" style="font-size:14.0pt;
+
-
  mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;mso-bidi-font-weight:bold"><o:p></o:p></span></sub></p>
+
-
  </td>
+
-
  <td valign="top" style="border:none;border-right:solid black 1.0pt;mso-border-right-themecolor:
+
-
  text1;mso-border-right-alt:solid black .5pt;mso-border-right-themecolor:text1;
+
-
  padding:1.45pt 5.4pt 1.45pt 5.4pt">
+
-
  <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
+
-
  normal"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:11.0pt;
+
-
  mso-ansi-language:EN-US">Promoter Strength<o:p></o:p></span></p>
+
-
  </td>
+
-
</tr>
+
-
<tr style="mso-yfti-irow:2">
+
-
  <td valign="top" style="border:solid black 1.0pt;mso-border-themecolor:text1;
+
-
  border-right:none;mso-border-top-alt:solid black .5pt;mso-border-top-themecolor:
+
-
  text1;mso-border-left-alt:solid black .5pt;mso-border-left-themecolor:text1;
+
-
  mso-border-bottom-alt:solid black .5pt;mso-border-bottom-themecolor:text1;
+
-
  background:white;mso-background-themecolor:background1;padding:1.45pt 5.4pt 1.45pt 5.4pt">
+
-
  <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
+
-
  normal;mso-yfti-cnfc:68"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:
+
-
  11.0pt;mso-bidi-font-family:Calibri;mso-bidi-theme-font:minor-latin;
+
-
  mso-ansi-language:EN-US;mso-bidi-font-weight:bold">?</span><sub><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:
+
-
  EN-US;mso-bidi-font-weight:bold">x<o:p></o:p></span></sub></p>
+
-
  </td>
+
-
  <td valign="top" style="border:solid black 1.0pt;mso-border-themecolor:text1;
+
-
  border-left:none;mso-border-top-alt:solid black .5pt;mso-border-top-themecolor:
+
-
  text1;mso-border-bottom-alt:solid black .5pt;mso-border-bottom-themecolor:
+
-
  text1;mso-border-right-alt:solid black .5pt;mso-border-right-themecolor:text1;
+
-
  padding:1.45pt 5.4pt 1.45pt 5.4pt">
+
-
  <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
+
-
  normal;mso-yfti-cnfc:64"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:
+
-
  11.0pt;mso-ansi-language:EN-US">Degradation Rate<o:p></o:p></span></p>
+
-
  </td>
+
-
</tr>
+
-
<tr style="mso-yfti-irow:3">
+
-
  <td valign="top" style="border:none;border-left:solid black 1.0pt;mso-border-left-themecolor:
+
-
  text1;mso-border-left-alt:solid black .5pt;mso-border-left-themecolor:text1;
+
-
  background:white;mso-background-themecolor:background1;padding:1.45pt 5.4pt 1.45pt 5.4pt">
+
-
  <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
+
-
  normal;mso-yfti-cnfc:4"><span class="SpellE"><span lang="EN-US" style="font-size:
+
-
  14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;mso-bidi-font-weight:
+
-
  bold">K<sub>x</sub></span></span><sub><span lang="EN-US" style="font-size:14.0pt;
+
-
  mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;mso-bidi-font-weight:bold"><o:p></o:p></span></sub></p>
+
-
  </td>
+
-
  <td valign="top" style="border:none;border-right:solid black 1.0pt;mso-border-right-themecolor:
+
-
  text1;mso-border-right-alt:solid black .5pt;mso-border-right-themecolor:text1;
+
-
  padding:1.45pt 5.4pt 1.45pt 5.4pt">
+
-
  <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
+
-
  normal"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:11.0pt;
+
-
  mso-ansi-language:EN-US">Disassociation (Equilibrium) Constant<o:p></o:p></span></p>
+
-
  </td>
+
-
</tr>
+
-
<tr style="mso-yfti-irow:4">
+
-
  <td valign="top" style="border:solid black 1.0pt;mso-border-themecolor:text1;
+
-
  border-right:none;mso-border-top-alt:solid black .5pt;mso-border-top-themecolor:
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-
  text1;mso-border-left-alt:solid black .5pt;mso-border-left-themecolor:text1;
+
-
  mso-border-bottom-alt:solid black .5pt;mso-border-bottom-themecolor:text1;
+
-
  background:white;mso-background-themecolor:background1;padding:1.45pt 5.4pt 1.45pt 5.4pt">
+
-
  <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
+
-
  normal;mso-yfti-cnfc:68"><span class="SpellE"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;
+
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  mso-bidi-font-weight:bold">n<sub>x</sub></span></span><sub><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;
+
-
  mso-bidi-font-weight:bold"><o:p></o:p></span></sub></p>
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  </td>
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  <td valign="top" style="border:solid black 1.0pt;mso-border-themecolor:text1;
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  <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
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  normal;mso-yfti-cnfc:64"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:
+
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  11.0pt;mso-ansi-language:EN-US">Hill Coefficient<o:p></o:p></span></p>
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-
  </td>
+
-
</tr>
+
-
<tr style="mso-yfti-irow:5">
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  <td valign="top" style="border:none;border-left:solid black 1.0pt;mso-border-left-themecolor:
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  background:white;mso-background-themecolor:background1;padding:1.45pt 5.4pt 1.45pt 5.4pt">
+
-
  <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
+
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  normal;mso-yfti-cnfc:4"><span class="SpellE"><span lang="EN-US" style="font-size:
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  14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;mso-bidi-font-weight:
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  bold">t<sub>x</sub></span></span><sub><span lang="EN-US" style="font-size:14.0pt;
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  mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;mso-bidi-font-weight:bold"><o:p></o:p></span></sub></p>
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  </td>
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  <td valign="top" style="border:none;border-right:solid black 1.0pt;mso-border-right-themecolor:
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  text1;mso-border-right-alt:solid black .5pt;mso-border-right-themecolor:text1;
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  padding:1.45pt 5.4pt 1.45pt 5.4pt">
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  <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
+
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  normal"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:11.0pt;
+
-
  mso-ansi-language:EN-US">Translation Rate<o:p></o:p></span></p>
+
-
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+
-
</tr>
+
-
<tr style="mso-yfti-irow:6">
+
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  border-right:none;mso-border-top-alt:solid black .5pt;mso-border-top-themecolor:
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  text1;mso-border-left-alt:solid black .5pt;mso-border-left-themecolor:text1;
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  mso-border-bottom-alt:solid black .5pt;mso-border-bottom-themecolor:text1;
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  background:white;mso-background-themecolor:background1;padding:1.45pt 5.4pt 1.45pt 5.4pt">
+
-
  <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
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  normal;mso-yfti-cnfc:68"><span class="SpellE"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;
+
-
  mso-bidi-font-weight:bold">k<sub>x</sub></span></span><sub><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;
+
-
  mso-bidi-font-weight:bold"><o:p></o:p></span></sub></p>
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  <td valign="top" style="border:solid black 1.0pt;mso-border-themecolor:text1;
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  border-left:none;mso-border-top-alt:solid black .5pt;mso-border-top-themecolor:
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  padding:1.45pt 5.4pt 1.45pt 5.4pt">
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  <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
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  normal;mso-yfti-cnfc:64"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:
+
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  11.0pt;mso-ansi-language:EN-US">Binding on rate<o:p></o:p></span></p>
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-
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+
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</tr>
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<tr style="mso-yfti-irow:7">
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  <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
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  normal;mso-yfti-cnfc:4"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:
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  11.0pt;mso-ansi-language:EN-US;mso-bidi-font-weight:bold">k<sub>-x<o:p></o:p></sub></span></p>
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  <td valign="top" style="border:none;border-right:solid black 1.0pt;mso-border-right-themecolor:
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  <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
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  normal"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:11.0pt;
+
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  mso-ansi-language:EN-US">Binding off rate<o:p></o:p></span></p>
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+
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</tr>
+
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<tr style="mso-yfti-irow:8">
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  <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
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  normal;mso-yfti-cnfc:68"><span class="SpellE"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;
+
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  mso-bidi-font-weight:bold">D<sub>x</sub></span></span><sub><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;
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  mso-bidi-font-weight:bold"><o:p></o:p></span></sub></p>
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  <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
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  <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
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  normal;mso-yfti-cnfc:4"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:
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  11.0pt;mso-ansi-language:EN-US;mso-bidi-font-weight:bold">d<o:p></o:p></span></p>
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  <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
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  normal"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:11.0pt;
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  mso-ansi-language:EN-US">cell density<o:p></o:p></span></p>
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<tr style="mso-yfti-irow:10;mso-yfti-lastrow:yes">
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  <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
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  11.0pt;mso-bidi-font-family:Calibri;mso-bidi-theme-font:minor-latin;
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  mso-ansi-language:EN-US;mso-bidi-font-weight:bold">µ</span><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;
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  mso-bidi-font-weight:bold"><o:p></o:p></span></p>
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Revision as of 08:44, 24 September 2012

Kill Switch

 

Overview

Kill switch is a safety system which is necessary to control the release of genetically modified organisms (GMOs) into the environment. Since we aimed to construct a biofilm with GMOs which will convert CO to CO2, we needed a kill switch that can prevent our system to cause any harm to the environment. In order to achieve that aim we used the kill switch designed by Berkeley 2008 iGEM Team (Bba_K112808) and also we tried to develop it.

How The System Works ?

In order to construct our kill switch system we needed a reagent that E.Coli will grow and in the absence of that reagent all the bacteria should die. For that purpose we prefered to use IPTG (Isopropyl-beta-D-thiogalactopyranoside) because it cannot be metabolised by E.Coli and that’s why its concentration remains constant in the cell. When IPTG is present in the cell it will bind to LacI which can be produced by the promoter J23116. Basal LacI expression is already present in E.Coli but at low levels and it is hard to control the system with that much LacI. Hence, we prefered to produce it with the help of J23116. Our kill switch system includes the expression of endolysin, holin, antiholin and their production is controlled with the initial concentration of IPTG in the environment. Actually cell lysis can occur without IPTG but the lysis rate will be high. By using IPTG we aimed to keep our system under control. *Endolysin-Holin System; Endolysin and holin are the proteins needed for the cell lysis. Endolysin is a lysozyme and its funtion is to hydrolyze the peptidoglycan bacterial cell wall but it needs holin to form pores on inner membrane, then it can reach periplasm and cell lysis occur.

The fate of the Gram-negative envelope during holin–endolysin lysis.

Pre-Hole Configuration Hole Configuration

In this figure holins are shown as clear ovals and endolysin is shown as notched circles.

  • Accumulation of holin is present in the inner membrane, while endolysin accumulates in the cytosol. Holin action can be prevented with the inhibitors (shown with star and black ovals).
  • When the holes are formed by holin in the inner membrane, endolysin can attack the murein which is important for cell lysis to occur.
 

* Antiholin; It is the protein that blocks the function of holin and it is important for the regulation of cell lysis.

In our system antiholin is constitutively produced by the promoter J23116 and when antiholin level is in between 1000-3000 molecules, the cell will die. Actually,it is the number of free holin molecules that determines if the cell will die or not.

Modelling

Results

Biobricks

Retrieved from "http://2012.igem.org/Team:METU/KillSwitchOverview"