Team:METU/KillSwitchOverview

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                    <h1>Kill Switch</h1>
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<h1>Cell Limiter</h1>
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                    <div class="cl">&nbsp;</div>
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<div class="cl">&nbsp;</div>
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<h2><b> Kill Switch </b></h2>
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<h2><b> Cell Limiter </b></h2>
<span class="toctoggle">&nbsp;[<a href="#" class="internal" id="togglelink">gizle</a>]&nbsp;</span></div>
<span class="toctoggle">&nbsp;[<a href="#" class="internal" id="togglelink">gizle</a>]&nbsp;</span></div>
<ul>
<ul>
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<li class="toclevel-2 tocsection-2"><a href="#how"><span class="tocnumber"></span> <span class="toctext">How the System Works?</span></a></li>
<li class="toclevel-2 tocsection-2"><a href="#how"><span class="tocnumber"></span> <span class="toctext">How the System Works?</span></a></li>
<li class="toclevel-2 tocsection-3"><a href="#model"><span class="tocnumber"></span> <span class="toctext">Modelling</span></a></li>
<li class="toclevel-2 tocsection-3"><a href="#model"><span class="tocnumber"></span> <span class="toctext">Modelling</span></a></li>
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<li class="toclevel-1 tocsection-4"><a href="#bio"><span class="tocnumber"></span> <span class="toctext">Biobricks</span></a>
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                             <p>
                             <p>
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                                 Kill switch is a safety system which is necessary to control the release of genetically modified
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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.
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                                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.
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</p>
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<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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                         <p>
                         <p>
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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.
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</p>
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<p>Finally LuxPR activity determines the amount of LasR and when PLasR promoter is activated
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MinC, a cell division inhibitor can be synthesized. Eventually, cell division stops after MinC reaches its critical concentration.</p>
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                            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.
 
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                            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.
 
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                            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
 
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                            cell lysis can occur without IPTG but the lysis rate will be high. By using IPTG we aimed to keep our system under control.
 
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                            *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.
 
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                        </p>
 
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                        <p>
 
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                            The fate of the Gram-negative envelope during holin–endolysin lysis.
 
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                        </p>
 
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                        Pre-Hole Configuration                          Hole Configuration
 
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                        <p>
 
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                            <img src="https://static.igem.org/mediawiki/2012/9/97/Metu-photos-model27.png.jpg" alt=""/>
 
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                            In this figure holins are shown as clear ovals and endolysin is shown as notched circles.
 
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                        </p>
 
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                        <ul>
 
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                            <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>
 
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                            <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>
 
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                        </ul>
 
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                        &nbsp;
 
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                            * Antiholin; It is the protein that blocks the function of holin and it is important for the regulation of cell lysis.
 
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                        </p>
 
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                        <p id="Overview">
 
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                            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.
 
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<p><b> Kill-Swicth Model </b></p>
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<p><b> Cell Limiter Model </b></p>
   <span>
   <span>
&nbsp;
&nbsp;
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<p>
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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>
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<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><img src="https://static.igem.org/mediawiki/2012/3/3d/Metu-photo002.jpg"/> </p>
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<p><img src="https://static.igem.org/mediawiki/2012/d/db/Metu-photo-1.jpg"/> </p>
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<p><img src="https://static.igem.org/mediawiki/2012/7/72/Metu-photo004.jpg"/> </p>
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The aim of kill-switch model is to predict the behavior of kill-switch mechanism in different IPTG concentrations. We first tried to model mechanism by the help of ODEs. Then we used SimBiology toolbox of MATLAB to simulate system.
 
<p>
<p>
<b>Introduction:</b>
<b>Introduction:</b>
</p>
</p>
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<p><img src="https://static.igem.org/mediawiki/2012/d/d8/Metu-photo006.jpg"/> </p>
<p>
<p>
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As can be seen from the diagram our circuit involves constitutive production of LacI. LacI Promoter is negatively regulated by LacI. The control mechanism of our system is inhibition of LacI by IPTG molecule. Since LacI is inhibited by IPTG its effect on LacI promoter changes with respect to IPTG concentration.
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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.
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Holin and Endolysin expression is controlled by cI promoter which is negatively regulated by cI.  
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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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Our first assumption in the model is that when the free Holin molecules, which means number of Holin molecules minus number of Antiholin molecules, reach 3000[ref] cell lysis occurs.
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Because cI concentration depends on the activity of LacI promoter, we can conclude that in the presence of IPTG the concentration of cI will be high and expression of Holin and Endolysin will be lowered. Due to the constitutive production of Antiholin, we can say that free Holin molecules will be lowered and chance of cell lysis to occur will drop. However, to understand the overall behaviour of system with respect to IPTG concentration we need to model and simulate the system.
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In our model, we included all processes shown below.
In our model, we included all processes shown below.
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<img src="https://static.igem.org/mediawiki/2012/8/8c/Metu-photo008.jpg" alt="" ">
<p><b>Equations for mRNA transcription</b></p>
<p><b>Equations for mRNA transcription</b></p>
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<b>  
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Equations for protein translation & complex formation
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<b> Equations for Protein Translation </b>
 
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<p>C is HSL&LuxR Complex</p>
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   normal;mso-yfti-cnfc:1"><b><span style="font-size:14.0pt;mso-bidi-font-size:
+
   normal;mso-yfti-cnfc:1"><b><span lang="EN-US" style="font-size:14.0pt;
-
  11.0pt;color:white;mso-themecolor:background1;mso-ansi-language:EN-US">Description<o:p></o:p></span></b></p>
+
  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>
   </td>
  </tr>
  </tr>
Line 341: Line 366:
   mso-border-bottom-alt:solid black .5pt;mso-border-bottom-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">
   background:white;mso-background-themecolor:background1;padding:1.45pt 5.4pt 1.45pt 5.4pt">
-
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-
   normal;mso-yfti-cnfc:68"><span style="font-size:14.0pt;mso-bidi-font-size:
+
   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>
   11.0pt;mso-ansi-language:EN-US;mso-bidi-font-weight:bold">N<o:p></o:p></span></p>
   </td>
   </td>
Line 350: Line 375:
   text1;mso-border-right-alt:solid black .5pt;mso-border-right-themecolor:text1;
   text1;mso-border-right-alt:solid black .5pt;mso-border-right-themecolor:text1;
   padding:1.45pt 5.4pt 1.45pt 5.4pt">
   padding:1.45pt 5.4pt 1.45pt 5.4pt">
-
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-
   normal;mso-yfti-cnfc:64"><span style="font-size:14.0pt;mso-bidi-font-size:
+
   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>
   11.0pt;mso-ansi-language:EN-US">Plasmid copy number<o:p></o:p></span></p>
   </td>
   </td>
Line 359: Line 384:
   text1;mso-border-left-alt:solid black .5pt;mso-border-left-themecolor:text1;
   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">
   background:white;mso-background-themecolor:background1;padding:1.45pt 5.4pt 1.45pt 5.4pt">
-
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-
   normal;mso-yfti-cnfc:4"><span class="SpellE"><span style="font-size:14.0pt;
+
   normal;mso-yfti-cnfc:4"><span class="SpellE"><span lang="EN-US" style="font-size:
-
  mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;mso-bidi-font-weight:bold">P<sub>x</sub></span></span><sub><span style="font-size:14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;
+
  14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;mso-bidi-font-weight:
-
  mso-bidi-font-weight:bold"><o:p></o:p></span></sub></p>
+
  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>
   <td valign="top" style="border:none;border-right:solid black 1.0pt;mso-border-right-themecolor:
   <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;
   text1;mso-border-right-alt:solid black .5pt;mso-border-right-themecolor:text1;
   padding:1.45pt 5.4pt 1.45pt 5.4pt">
   padding:1.45pt 5.4pt 1.45pt 5.4pt">
-
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-
   normal"><span style="font-size:14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:
+
   normal"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:11.0pt;
-
  EN-US">Promoter Strength<o:p></o:p></span></p>
+
  mso-ansi-language:EN-US">Promoter Strength<o:p></o:p></span></p>
   </td>
   </td>
  </tr>
  </tr>
Line 378: Line 404:
   mso-border-bottom-alt:solid black .5pt;mso-border-bottom-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">
   background:white;mso-background-themecolor:background1;padding:1.45pt 5.4pt 1.45pt 5.4pt">
-
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-
   normal;mso-yfti-cnfc:68"><span style="font-size:14.0pt;mso-bidi-font-size:
+
   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;
   11.0pt;mso-bidi-font-family:Calibri;mso-bidi-theme-font:minor-latin;
-
   mso-ansi-language:EN-US;mso-bidi-font-weight:bold">a</span><sub><span style="font-size:14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;
+
   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:
-
  mso-bidi-font-weight:bold">x<o:p></o:p></span></sub></p>
+
  EN-US;mso-bidi-font-weight:bold">x<o:p></o:p></span></sub></p>
   </td>
   </td>
   <td valign="top" style="border:solid black 1.0pt;mso-border-themecolor:text1;
   <td valign="top" style="border:solid black 1.0pt;mso-border-themecolor:text1;
Line 389: Line 415:
   text1;mso-border-right-alt:solid black .5pt;mso-border-right-themecolor:text1;
   text1;mso-border-right-alt:solid black .5pt;mso-border-right-themecolor:text1;
   padding:1.45pt 5.4pt 1.45pt 5.4pt">
   padding:1.45pt 5.4pt 1.45pt 5.4pt">
-
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-
   normal;mso-yfti-cnfc:64"><span style="font-size:14.0pt;mso-bidi-font-size:
+
   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>
   11.0pt;mso-ansi-language:EN-US">Degradation Rate<o:p></o:p></span></p>
   </td>
   </td>
Line 398: Line 424:
   text1;mso-border-left-alt:solid black .5pt;mso-border-left-themecolor:text1;
   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">
   background:white;mso-background-themecolor:background1;padding:1.45pt 5.4pt 1.45pt 5.4pt">
-
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-
   normal;mso-yfti-cnfc:4"><span class="SpellE"><span style="font-size:14.0pt;
+
   normal;mso-yfti-cnfc:4"><span class="SpellE"><span lang="EN-US" style="font-size:
-
  mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;mso-bidi-font-weight:bold">K<sub>x</sub></span></span><sub><span style="font-size:14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;
+
  14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;mso-bidi-font-weight:
-
  mso-bidi-font-weight:bold"><o:p></o:p></span></sub></p>
+
  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>
   <td valign="top" style="border:none;border-right:solid black 1.0pt;mso-border-right-themecolor:
   <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;
   text1;mso-border-right-alt:solid black .5pt;mso-border-right-themecolor:text1;
   padding:1.45pt 5.4pt 1.45pt 5.4pt">
   padding:1.45pt 5.4pt 1.45pt 5.4pt">
-
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-
   normal"><span style="font-size:14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:
+
   normal"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:11.0pt;
-
  EN-US">Disassociation (Equilibrium) Constant<o:p></o:p></span></p>
+
  mso-ansi-language:EN-US">Disassociation (Equilibrium) Constant<o:p></o:p></span></p>
   </td>
   </td>
  </tr>
  </tr>
Line 417: Line 444:
   mso-border-bottom-alt:solid black .5pt;mso-border-bottom-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">
   background:white;mso-background-themecolor:background1;padding:1.45pt 5.4pt 1.45pt 5.4pt">
-
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-
   normal;mso-yfti-cnfc:68"><span class="SpellE"><span style="font-size:14.0pt;
+
   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-size:11.0pt;mso-ansi-language:EN-US;mso-bidi-font-weight:bold">n<sub>x</sub></span></span><sub><span style="font-size:14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;
+
  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>
   mso-bidi-font-weight:bold"><o:p></o:p></span></sub></p>
   </td>
   </td>
Line 427: Line 454:
   text1;mso-border-right-alt:solid black .5pt;mso-border-right-themecolor:text1;
   text1;mso-border-right-alt:solid black .5pt;mso-border-right-themecolor:text1;
   padding:1.45pt 5.4pt 1.45pt 5.4pt">
   padding:1.45pt 5.4pt 1.45pt 5.4pt">
-
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-
   normal;mso-yfti-cnfc:64"><span style="font-size:14.0pt;mso-bidi-font-size:
+
   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">Hill Coefficient<o:p></o:p></span></p>
   11.0pt;mso-ansi-language:EN-US">Hill Coefficient<o:p></o:p></span></p>
   </td>
   </td>
Line 436: Line 463:
   text1;mso-border-left-alt:solid black .5pt;mso-border-left-themecolor:text1;
   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">
   background:white;mso-background-themecolor:background1;padding:1.45pt 5.4pt 1.45pt 5.4pt">
-
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-
   normal;mso-yfti-cnfc:4"><span class="SpellE"><span style="font-size:14.0pt;
+
   normal;mso-yfti-cnfc:4"><span class="SpellE"><span lang="EN-US" style="font-size:
-
  mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;mso-bidi-font-weight:bold">t<sub>x</sub></span></span><sub><span style="font-size:14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;
+
  14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;mso-bidi-font-weight:
-
  mso-bidi-font-weight:bold"><o:p></o:p></span></sub></p>
+
  bold">t<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>
   <td valign="top" style="border:none;border-right:solid black 1.0pt;mso-border-right-themecolor:
   <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;
   text1;mso-border-right-alt:solid black .5pt;mso-border-right-themecolor:text1;
   padding:1.45pt 5.4pt 1.45pt 5.4pt">
   padding:1.45pt 5.4pt 1.45pt 5.4pt">
-
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-
   normal"><span style="font-size:14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:
+
   normal"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:11.0pt;
-
  EN-US">Translation Rate<o:p></o:p></span></p>
+
  mso-ansi-language:EN-US">Translation Rate<o:p></o:p></span></p>
   </td>
   </td>
  </tr>
  </tr>
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   mso-border-bottom-alt:solid black .5pt;mso-border-bottom-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">
   background:white;mso-background-themecolor:background1;padding:1.45pt 5.4pt 1.45pt 5.4pt">
-
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-
   normal;mso-yfti-cnfc:68"><span class="SpellE"><span style="font-size:14.0pt;
+
   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-size:11.0pt;mso-ansi-language:EN-US;mso-bidi-font-weight:bold">k<sub>x</sub></span></span><sub><span 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>
   mso-bidi-font-weight:bold"><o:p></o:p></span></sub></p>
   </td>
   </td>
Line 465: Line 493:
   text1;mso-border-right-alt:solid black .5pt;mso-border-right-themecolor:text1;
   text1;mso-border-right-alt:solid black .5pt;mso-border-right-themecolor:text1;
   padding:1.45pt 5.4pt 1.45pt 5.4pt">
   padding:1.45pt 5.4pt 1.45pt 5.4pt">
-
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-
   normal;mso-yfti-cnfc:64"><span style="font-size:14.0pt;mso-bidi-font-size:
+
   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">Binding on rate<o:p></o:p></span></p>
   11.0pt;mso-ansi-language:EN-US">Binding on rate<o:p></o:p></span></p>
   </td>
   </td>
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   text1;mso-border-left-alt:solid black .5pt;mso-border-left-themecolor:text1;
   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">
   background:white;mso-background-themecolor:background1;padding:1.45pt 5.4pt 1.45pt 5.4pt">
-
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-
   normal;mso-yfti-cnfc:4"><span style="font-size:14.0pt;mso-bidi-font-size:
+
   normal;mso-yfti-cnfc:4"><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<o:p></o:p></sub></span></p>
   11.0pt;mso-ansi-language:EN-US;mso-bidi-font-weight:bold">k<sub>-x<o:p></o:p></sub></span></p>
   </td>
   </td>
Line 481: Line 509:
   text1;mso-border-right-alt:solid black .5pt;mso-border-right-themecolor:text1;
   text1;mso-border-right-alt:solid black .5pt;mso-border-right-themecolor:text1;
   padding:1.45pt 5.4pt 1.45pt 5.4pt">
   padding:1.45pt 5.4pt 1.45pt 5.4pt">
-
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-
   normal"><span style="font-size:14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:
+
   normal"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:11.0pt;
-
  EN-US">Binding off rate<o:p></o:p></span></p>
+
  mso-ansi-language:EN-US">Binding off rate<o:p></o:p></span></p>
   </td>
   </td>
  </tr>
  </tr>
-
  <tr style="mso-yfti-irow:8;mso-yfti-lastrow:yes">
+
  <tr style="mso-yfti-irow:8">
   <td valign="top" style="border:solid black 1.0pt;mso-border-themecolor:text1;
   <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:
   border-right:none;mso-border-top-alt:solid black .5pt;mso-border-top-themecolor:
Line 492: Line 520:
   mso-border-bottom-alt:solid black .5pt;mso-border-bottom-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">
   background:white;mso-background-themecolor:background1;padding:1.45pt 5.4pt 1.45pt 5.4pt">
-
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-
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  mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;mso-bidi-font-weight:bold">D<sub>x</sub></span></span><sub><span style="font-size:14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;
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<p> <b> Simulation Results </b> </p>
 
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When the IPTG concentration is 0;
 
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Because the critical point in kill-switch is “Holin-Antiholin” molecules, we ran simulation in different IPTG concentrations and to understand result easier we select the outputs as Holin and Antiholin. Since translation rate of endolysin is more than Holin and Antiholin we can assume that there will be enough endolysin to make cell lysis occur. Also from the graph above we can understand that endolysin will be in sufficient amount.
 
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<p>By the help of our model, we can conclude that when there is no IPTG molecule, cell lysis occur in 275 s.  However when there is 100000 IPTG molecules, cell needs 2290 s to lyse. As can be seen from graphs until a very high IPTG concentration like in the Run14, the increase in Holin can eventually cause lysis. As a result of our model we concluded that after an IPTG concentration of 700000 molecule cell lysis will not occur. We are not considering the generation time of cell in this model since it can later be used for different organisms. If we consider e.coli with a generation time of 20 min, even in the medium with IPTG of 100000 molecules, it will divide before it can lyse.
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METU IGEM 2012
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<p>When IPTG concentration is 700000;</p>
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<p><img src="https://static.igem.org/mediawiki/2012/3/34/Metu-photos-model20.png.jpg" alt="" height="371px" width="727px" /></p>
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<p>Individual graphs can be accessed here.</p>
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<p><b> Sensivity Analysis </b></p>
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<p>Since the challenging part of modelling is choice of parameters, we conducted a sensitivity analysis to see how much result of our model depends on parameters. By the help of those, the critical values to be measured through experiments can be understood easily.
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Revision as of 19:04, 23 September 2012

Cell Limiter

 

Overview

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.

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.

How The System Works ?

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.

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.

MODELLING[up]

Cell Limiter Model

 

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.

You can download zip file from here. deneme22.m should be run in MATLAB to run program. Screenshots from program:

Introduction:

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

Mathematical Model:

In our model, we included all processes shown below.

Equations for mRNA transcription

     

 

 

 

  Equations for protein translation & complex formation      

 

 

 

 

 

 

 

C is HSL&LuxR Complex

Parameter

Description

N

Plasmid copy number

Px

Promoter Strength

?x

Degradation Rate

Kx

Disassociation (Equilibrium) Constant

nx

Hill Coefficient

tx

Translation Rate

kx

Binding on rate

k-x

Binding off rate

Dx

Diffusion rate

d

cell density

µ

production rate



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