Team:METU/CellLimiter

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<h1>Cell Limiter</h1>
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                    <h1>CELL LIMITER </h1>
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<h2><b> Cell Limiter </b></h2>
<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 class="toclevel-1 tocsection-1"><a href="#overview"><span class="tocnumber"></span> <span class="toctext">Overview</span></a></li>
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<li class="toclevel-1 tocsection-1"><a href="#pcr"><span class="tocnumber">1.</span> <span class="toctext">Overview</span></a></li>
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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>
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<li class="toclevel-1 tocsection-2"><a href="#colony"><span class="tocnumber">2.</span> <span class="toctext">How the System Works?</span></a></li>
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<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-3"><a href="#pcr1"><span class="tocnumber">3.</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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<li class="toclevel-1 tocsection-4"><a href="#cultures"><span class="tocnumber">4.</span> <span class="toctext">References</span></a>
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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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                                 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 <b>[1]</b> 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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<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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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<b>[2]</b>. 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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                         <h2 id="how" >How The System Works ?</h2>
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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).  
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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.
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>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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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.
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                         <h2 id="model" >MODELLING[<a href="#table">up</a>]</h2>
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<h2><b> CELL LIMITER MODEL </b></h2>
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<p><b> Cell Limiter Model </b></p>
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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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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 <a href="https://2012.igem.org/File:METU-quorum_sensing.zip">here</a> should be run in MATLAB to run program. Screenshots from program:</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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<b>Introduction:</b>
<b>Introduction:</b>
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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.
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
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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<b> Equations for Protein Translation </b>
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Equations for protein translation & complex formation
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   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:0cm;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
-
   normal;mso-yfti-cnfc:4"><span class="SpellE"><span lang="EN-US" style="font-size:
+
   normal;mso-yfti-cnfc:4"><span class="SpellE"><span style="font-size:14.0pt;
-
  14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;mso-bidi-font-weight:
+
  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;
-
  bold">P<sub>x</sub></span></span><sub><span lang="EN-US" style="font-size:14.0pt;
+
  mso-bidi-font-weight:bold"><o:p></o:p></span></sub></p>
-
  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:0cm;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
-
   normal"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:11.0pt;
+
   normal"><span style="font-size:14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:
-
  mso-ansi-language:EN-US">Promoter Strength<o:p></o:p></span></p>
+
  EN-US">Promoter Strength<o:p></o:p></span></p>
   </td>
   </td>
  </tr>
  </tr>
Line 404: Line 349:
   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:0cm;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
-
   normal;mso-yfti-cnfc:68"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:
+
   normal;mso-yfti-cnfc:68"><span 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">?</span><sub><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:
+
   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;
-
  EN-US;mso-bidi-font-weight:bold">x<o:p></o:p></span></sub></p>
+
  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 415: Line 360:
   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:0cm;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
-
   normal;mso-yfti-cnfc:64"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:
+
   normal;mso-yfti-cnfc:64"><span 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 424: Line 369:
   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:0cm;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
-
   normal;mso-yfti-cnfc:4"><span class="SpellE"><span lang="EN-US" style="font-size:
+
   normal;mso-yfti-cnfc:4"><span class="SpellE"><span style="font-size:14.0pt;
-
  14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;mso-bidi-font-weight:
+
  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;
-
  bold">K<sub>x</sub></span></span><sub><span lang="EN-US" style="font-size:14.0pt;
+
  mso-bidi-font-weight:bold"><o:p></o:p></span></sub></p>
-
  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:0cm;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
-
   normal"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:11.0pt;
+
   normal"><span style="font-size:14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:
-
  mso-ansi-language:EN-US">Disassociation (Equilibrium) Constant<o:p></o:p></span></p>
+
  EN-US">Disassociation (Equilibrium) Constant<o:p></o:p></span></p>
   </td>
   </td>
  </tr>
  </tr>
Line 444: Line 388:
   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:0cm;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0in;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;
+
   normal;mso-yfti-cnfc:68"><span class="SpellE"><span style="font-size:14.0pt;
-
  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-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"><o:p></o:p></span></sub></p>
   mso-bidi-font-weight:bold"><o:p></o:p></span></sub></p>
   </td>
   </td>
Line 454: Line 398:
   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:0cm;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
-
   normal;mso-yfti-cnfc:64"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:
+
   normal;mso-yfti-cnfc:64"><span 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>
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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:0cm;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
-
   normal;mso-yfti-cnfc:4"><span class="SpellE"><span lang="EN-US" style="font-size:
+
   normal;mso-yfti-cnfc:4"><span class="SpellE"><span style="font-size:14.0pt;
-
  14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:EN-US;mso-bidi-font-weight:
+
  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;
-
  bold">t<sub>x</sub></span></span><sub><span lang="EN-US" style="font-size:14.0pt;
+
  mso-bidi-font-weight:bold"><o:p></o:p></span></sub></p>
-
  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:0cm;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
-
   normal"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:11.0pt;
+
   normal"><span style="font-size:14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:
-
  mso-ansi-language:EN-US">Translation Rate<o:p></o:p></span></p>
+
  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:0cm;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0in;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;
+
   normal;mso-yfti-cnfc:68"><span class="SpellE"><span style="font-size:14.0pt;
-
  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-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"><o:p></o:p></span></sub></p>
   mso-bidi-font-weight:bold"><o:p></o:p></span></sub></p>
   </td>
   </td>
Line 493: Line 436:
   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:0cm;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
-
   normal;mso-yfti-cnfc:64"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:
+
   normal;mso-yfti-cnfc:64"><span 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:0cm;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
-
   normal;mso-yfti-cnfc:4"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:
+
   normal;mso-yfti-cnfc:4"><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<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 509: Line 452:
   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:0cm;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
-
   normal"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:11.0pt;
+
   normal"><span style="font-size:14.0pt;mso-bidi-font-size:11.0pt;mso-ansi-language:
-
  mso-ansi-language:EN-US">Binding off rate<o:p></o:p></span></p>
+
  EN-US">Binding off 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:0cm;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0in;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;
+
   normal;mso-yfti-cnfc:68"><span class="SpellE"><span style="font-size:14.0pt;
-
  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;
+
  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;
   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 530: Line 473:
   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:0cm;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
-
   normal;mso-yfti-cnfc:64"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:
+
   normal;mso-yfti-cnfc:64"><span style="font-size:14.0pt;mso-bidi-font-size:
   11.0pt;mso-ansi-language:EN-US">Diffusion rate<o:p></o:p></span></p>
   11.0pt;mso-ansi-language:EN-US">Diffusion 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:0cm;margin-bottom:.0001pt;line-height:
+
   <p class="MsoNormal" style="margin-bottom:0in;margin-bottom:.0001pt;line-height:
-
   normal;mso-yfti-cnfc:4"><span lang="EN-US" style="font-size:14.0pt;mso-bidi-font-size:
+
   normal;mso-yfti-cnfc:4"><span style="font-size:14.0pt;mso-bidi-font-size:
   11.0pt;mso-ansi-language:EN-US;mso-bidi-font-weight:bold">d<o:p></o:p></span></p>
   11.0pt;mso-ansi-language:EN-US;mso-bidi-font-weight:bold">d<o:p></o:p></span></p>
   </td>
   </td>
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<h2 id="cultures">References</h2>
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<p><b>[1]</b> Hong S.H., Hedge M., Kim J., Wang X., Jayaraman A., Wood T.K. (2012).  Synthetic quorum-sensing circuit to control consortial biofilm formation and dispersal in amicrofluidic device. Nature Communications, 3 (613). doi: 10.1038/ncomms1616
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<b>[2]</b> Melissa B. Miller and Bonnie L. Bassler. (2001). Quorum sensing in bacteria. Annual Review of Microbiology, 55, 165-199. doi: 10.1146/annurev.micro.55.1.165
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Latest revision as of 22:44, 26 September 2012

CELL LIMITER

 

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

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 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      

 

 

 

 

 

 

 

C is HSL&LuxR Complex

Parameter

Description

N

Plasmid copy number

Px

Promoter Strength

ax

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

References

[1] Hong S.H., Hedge M., Kim J., Wang X., Jayaraman A., Wood T.K. (2012). Synthetic quorum-sensing circuit to control consortial biofilm formation and dispersal in amicrofluidic device. Nature Communications, 3 (613). doi: 10.1038/ncomms1616

[2] Melissa B. Miller and Bonnie L. Bassler. (2001). Quorum sensing in bacteria. Annual Review of Microbiology, 55, 165-199. doi: 10.1146/annurev.micro.55.1.165

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