Team:Chiba/Modeling

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                         <li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Plasmid1">Plasmid 1</a></li>
                         <li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Plasmid1">Plasmid 1</a></li>
<li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Plasmid2">Plasmid 2</a></li>
<li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Plasmid2">Plasmid 2</a></li>
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<li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/luxinvert">lux Promoter</a></li><br>
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<li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/lux_promoter">lux Promoter</a></li><br>
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<li id="current"><a href="https://2010.igem.org/Team:Chiba/Notebook"><span>Notebook</span></a></li>
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<li><a href="https://2010.igem.org/Team:Chiba/Notebook"><span>Notebook</span></a>
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                        <li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Notebook">Notebook 1</a></li>
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<li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Notebook_2">Notebook 2</a></li>
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<li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Notebook_3">Notebook 3</a></li><br>
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<li><a href="https://2010.igem.org/Team:Chiba/Support"><span>Support</span></a></li>
<li><a href="https://2010.igem.org/Team:Chiba/Support"><span>Support</span></a></li>
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<li><a href="https://2010.igem.org/Team:Chiba/Safety"><span>Safety</span></a></li>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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Our second design of genetic double-click system consists of a pulse-generator and two inverters which can be seemed as a slow pulse.<br>
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In this system, we also use AHL input and GFP output.<br>
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This time, we use AHL as an activate signal, so when there is AHL added, Lux promoter will be activated.<br>
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The transcription factors of output are T7 RNA Polymerase and cI repressor. <br>
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<center>[[Image:Chiba_plan2_1.jpg]]</center><br><br>
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At initial state, LuxR and cI protein are constitutively generated.<br>
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cI binds to the operator site of PT7/cI.<br>
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When 1st input is injected, LuxR-AHL dimmer binds to the Lux-box of the lux promoters so that T7 RNA Polymerase,  cI434 and tetR protein are generated<br>
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at the same time.<br>
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cI434 gradually accumulates, and gradually repress the transcription of T7 RNA Polymerase so that the expression of T7 RNA Polymerase can be shown<br>
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as a pulse.<br>
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At the same time, Transcription of cI is stopped by tetR, cI decomposes and the PT7/cI promoter is unbound.<br>
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This derepression occurs after the pulse of T7 RNAP has passed.<br>
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In other words, the operator sites of PT7/cI is repressed by cI when there is pulse of T7 RNA Polymerase.<br>
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So, it cannot transcribe GFP. TetR creates a time delay here from input to derepression.<br>
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<center>[[Image:Chiba_planB_2.jpg]]</center><br><br>
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Because of this time delay and one-time pulse, bacteria can never work by one input. <br>
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<center>[[Image:Chiba_planB_3.jpg]]</center><br><br>
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Before injecting 2nd input, we must create none-input-environment.<br>
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So we choose to wash the 1st input. By washing , tetR protein and cI434 protein will degrade.<br>
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cI434 protein should disappear so that when there is 2nd input, T7 RNA Polymerase will be shown as a pulse which is the same as the 1st time.<br>
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cI will begin to generate if tetR protein gets lost.<br>
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We recognize it as time-limit, when there is enough cI generated (this means cI repression is stronger than T7 RNA Polymerase activation),<br>
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there will be no GFP output.<br>
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On the contrary, when there is less cI protein or no cI protein at the moment, T7 RNA Polymerase pulse can accumulate GFP output. <br><br>
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By the second injected AHL before the inhibition by cI, T7 RNA Polymerase binds to the PT7/cI promoter and transcribes the downstream GFP.</font><br>
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<center>[[Image:Chiba_planB_4.jpg]]</center><br><br>
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Latest revision as of 04:44, 25 October 2010




 




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