Team:Chiba/System 2

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<li><a href="https://2010.igem.org/Team:Chiba/Project"><span>Project</span></a></li>
<li><a href="https://2010.igem.org/Team:Chiba/Project"><span>Project</span></a></li>
<li><a href="https://2010.igem.org/Team:Chiba/Parts"><span>Parts</span></a></li>
<li><a href="https://2010.igem.org/Team:Chiba/Parts"><span>Parts</span></a></li>
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<li><a href="https://2010.igem.org/Team:Chiba/Result"><span>Result</span></a></li>
 
<li><a href="https://2010.igem.org/Team:Chiba/Notebook"><span>Notebook</span></a></li>
<li><a href="https://2010.igem.org/Team:Chiba/Notebook"><span>Notebook</span></a></li>
<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/Project"><span>Overall project</span></a></li>
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<li><a href="https://2010.igem.org/Team:Chiba/Project"><span>Double Click System</span></a></li>
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<li><a href="https://2010.igem.org/Team:Chiba/System_1"><span>System 1</span></a></li>
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<li><a href="https://2010.igem.org/Team:Chiba/System_1"><span>Version 1</span></a></li>
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<li><a href="https://2010.igem.org/Team:Chiba/System_2"><span>System 2</span></a></li>                       </ul>
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<li><a href="https://2010.igem.org/Team:Chiba/System_2"><span>Version 2</span></a></li>                       </ul>
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<li><a href="https://2010.igem.org/Team:Chiba/System_2/Testing_individual_parts"><span>Testing</span></a></li>
 
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<li><a href="https://2010.igem.org/Team:Chiba/System_2/Construction_Process"><span>Construction</span></a></li>
 
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<li><a href="https://2010.igem.org/Team:Chiba/System_2/Operating_the_W-click_system"><span>Operating</span></a></li>
 
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<li><a href="https://2010.igem.org/Team:Chiba/System_2/Evaluation_subsystem"><span>Evaluation</span></a></li>
 
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<li><a href="https://2010.igem.org/Team:Chiba/System_2/Remarks"><span>Remarks</span></a></li>
 
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=Version 2=
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<table border="0" cellpadding="30" cellspacing="0" align=”center”>
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  <td width="800px"><font size="4" face="verdana">Overall Circuit</font><br>
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Our first design of genetic double-click system is based on a combination of a Fast-pulse and a Slow-pulse. This system consists of a pulse-generator and two inverters which can be seemed as a slow pulse.In this system, we also use AHL input and GFP output. This time, we use AHL as an activate signal, so when there is AHL added, Lux promoter will be activated. The transcription factors of output are T7 RNA Polymerase and cI repressor.<br>
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==Overview==
 +
{|-
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|Our second circiut of double click system is called <b>Recognize circuit</b>.<br>
 +
It consists of <b>recognizing</b> system and <b>memorizing</b> system.<br>
 +
Recognizing system can recognize the existence of input.<br>
 +
And Memorizing system can memorize the existence of input in the past for a while.<br>
 +
When the two system work together, bacteria is going to shine.<br>
 +
We hope this circuit works as double click system.
 +
|[[Image:Chiba_cI.jpg]]
 +
|}
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<table border="0" cellpadding="30" cellspacing="0" align=”center”>
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We use AHL for input and regard injection of AHL as clicking. In our project, CLICK controls transcription of overall DNA sequence. <br>
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<tr>
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In recognizing input system, there is a cI operator above gfp DNA sequence. cI recognizes the existence of input in this DNA sequence.<br>
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  <td width="965px"><font size="4" face="verdana">Fast Pulse</font><br>
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In memorizing system, there is an AND Gate with T7ptag and supD. In this case, the AND Gate remembers that there was a click.<br>
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  <hr width="500" size="1" align="left"><br>
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There is a hybrid promoter(cI/T7) above gfp DNA sequence. It is regulated by T7 and cI which work as activator and repressor, respectively. On this promoter, repression is stronger than activation. And also, it is low unregulated activation. Working of this promoter depends on recognizing and memorizing system.<br>
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[[Team:Chiba/System_2/Overall|details...]]
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<img src="https://static.igem.org/mediawiki/2010/4/41/Chiba_planB_2.jpg" width="800px"><br>
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At initial state, LuxR and cI protein are constitutively generated. cI binds to the operator site of PT7/cI. 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 at the same time. cI434 gradually accumulates, and gradually repress the transcription of T7 RNA Polymerase so that the expression of T7 RNA Polymerase can be shown as a pulse. At the same time, Transcription of cI is stopped by tetR, cI decomposes and the PT7/cI promoter is unbound. This derepression occurs after the pulse of T7 RNAP has passed. In other words, the operator sites of PT7/cI is repressed by cI when there is pulse of T7 RNA Polymerase. So, it cannot transcribe GFP. TetR creates a time delay here from input to derepression. Because of this time delay and one-time pulse, bacteria can never work by one input.  
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==Circuit Construction==
-
 
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We've designed DNA sequence like following Figure. <br>
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<table border="0" cellpadding="30" cellspacing="0" align=”center”>
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<tr>
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  <td width="965px"><font size="4" face="verdana">Slow Pulse</font><br>
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  <hr width="500" size="1" align="left"><br>
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<center>
<center>
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<font size=2 face=verdana>
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[[Image:Chiba_Sys2.jpg]]</center>
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<img src="https://static.igem.org/mediawiki/2010/2/2f/Chiba_planB_3.jpg" width="800px"><br>
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Based on this sequence, we've also designed vector.<br>
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Before injecting 2nd input, we must create none-input-environment. So we choose to wash the 1st input. By washing , tetR protein and cI434 protein will degrade. 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. cI will begin to generate if tetR protein gets lost. We recognize it as time-limit, when there is enough cI generated (this means cI repression is stronger than T7 RNA Polymerase activation), there will be no GFP output. On the contrary, when there is less cI protein or no cI protein at the moment, T7 RNA Polymerase pulse can accumulate GFP output. By the second injected AHL before the inhibition by cI, T7 RNA Polymerase binds to the PT7/cI promoter and transcribes the downstream GFP.  
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<center>
<center>
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<img src="https://static.igem.org/mediawiki/2010/9/91/Chiba_planB_4.jpg" width="500px"><br>
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[[Image:Chiba_ver2.jpg]]</center>
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</center>
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Prom-luxR&PT7/cI-GFP vector is constructed collaboration with Double Click System Version 1.<br><br><br>
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</td>
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</table>
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 +
==Result==
 +
===Testing===
 +
In this system, lux promoter inverter is sensor of input.
 +
We've researched Plux inverter and got some results.<br>
 +
We need lux promoter repressed by AHL-luxR dimer. But Plux inverter of biobrick didn't work. So we've tried to charaterize it and make Plux inverter.<br>
 +
LuxR is AHL-dependent activator. LuxR-AHL complex binds lux box, 20-bp sequence centered at position -42.5 from starting site and activates transcription. However lux box is inserted between -35 and -10,LuxR functions as AHL-inverter (Plux inv). Plux inv is resistered in Biobrick number R0061. We've prepared Plux inv-GFP and characterized about it. [[Team:Chiba/System_2/Result|details...]]
 +
===Evaluation===
 +
<br>
 +
===Remark&Notes===
 +
<br>
-
 
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==Conclusion==
-
 
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It does not completely repress transcription, Plasmids, strains ,culture conditions,and detection method LuxR suppression can not be confirmed(Cox et al,2007).<br>
-
 
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[[Team:Chiba/System_2/Result|details...]]
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Before injecting 2nd input, we must create none-input-environment. So we choose to wash the 1st input. By washing , tetR protein and cI434 protein will degrade. 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. cI will begin to generate if tetR protein gets lost. We recognize it as time-limit, when there is enough cI generated (this means cI repression is stronger than T7 RNA Polymerase activation), there will be no GFP output. On the contrary, when there is less cI protein or no cI protein at the moment, T7 RNA Polymerase pulse can accumulate GFP output. By the second injected AHL before the inhibition by cI, T7 RNA Polymerase binds to the PT7/cI promoter and transcribes the downstream GFP.
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<center>
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<img src="https://static.igem.org/mediawiki/2010/9/91/Chiba_planB_4.jpg" width="500px"><br>
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Latest revision as of 03:34, 28 October 2010




 

 

Contents

Version 2

Overview

Our second circiut of double click system is called Recognize circuit.

It consists of recognizing system and memorizing system.
Recognizing system can recognize the existence of input.
And Memorizing system can memorize the existence of input in the past for a while.
When the two system work together, bacteria is going to shine.
We hope this circuit works as double click system.

Chiba cI.jpg

We use AHL for input and regard injection of AHL as clicking. In our project, CLICK controls transcription of overall DNA sequence.
In recognizing input system, there is a cI operator above gfp DNA sequence. cI recognizes the existence of input in this DNA sequence.
In memorizing system, there is an AND Gate with T7ptag and supD. In this case, the AND Gate remembers that there was a click.
There is a hybrid promoter(cI/T7) above gfp DNA sequence. It is regulated by T7 and cI which work as activator and repressor, respectively. On this promoter, repression is stronger than activation. And also, it is low unregulated activation. Working of this promoter depends on recognizing and memorizing system.
details...


Circuit Construction

We've designed DNA sequence like following Figure.

Chiba Sys2.jpg

Based on this sequence, we've also designed vector.

Chiba ver2.jpg

Prom-luxR&PT7/cI-GFP vector is constructed collaboration with Double Click System Version 1.


Result

Testing

In this system, lux promoter inverter is sensor of input. We've researched Plux inverter and got some results.
We need lux promoter repressed by AHL-luxR dimer. But Plux inverter of biobrick didn't work. So we've tried to charaterize it and make Plux inverter.
LuxR is AHL-dependent activator. LuxR-AHL complex binds lux box, 20-bp sequence centered at position -42.5 from starting site and activates transcription. However lux box is inserted between -35 and -10,LuxR functions as AHL-inverter (Plux inv). Plux inv is resistered in Biobrick number R0061. We've prepared Plux inv-GFP and characterized about it. details...

Evaluation


Remark&Notes


Conclusion

It does not completely repress transcription, Plasmids, strains ,culture conditions,and detection method LuxR suppression can not be confirmed(Cox et al,2007).
details...


Retrieved from "http://2010.igem.org/Team:Chiba/System_2"