Team:Chiba/System 2

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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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Our second circiut of double click system is called <b>Recognize circuit</b>.<br>
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It consists of <b>recognizing input</b> system and <b>memorizing input</b> system.
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   <td width="965px"><font size="4" face="verdana">Control of circuit</font><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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We use AHL for input and regard injection of AHL as clicking. LuxR is generated constitutive. Injecting AHL to Double Click Bacteria brings dimer of AHL and luxR. We take lux promoter repressed by the dimer. As the dimer binds Plux, transcription of proteins under the Plux is repressed.<br>
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So in our project, <br>
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<b><font color="red">CLICK controls transcription of overall DNA sequence.</font></b>
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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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In recognizing input system, there is a cI operator above gfp DNA sequence. If cI binds cI operator, transcription of gfp is stopped. You can see DNA sequence of cI, which a kind of repressor, is under the Plux. On initial state(there isn't any click), cI is generated normally. But AHL come into Double Click Bacteria, generation of cI is stopped. Since repression of Plux starts working by the dimer which is a combination of AHL and LuxR. And then, cI is going to disappear, because all kinds of proteins are bound to be decomposed. If there isn't cI anymore, bacteria will have chance to shine.<br>
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We can say<br>
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<b><font color="red">cI recognizes the existence of input</font></b> in this DNA sequence.</td>
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In memorizing system, there is an AND Gate with T7ptag and supD. As you know, T7ptag becomes T7 polymerase in case of existing supD. T7 polymerase is translated to T7 protein which works as activator of T7 promoter. There is a T7 promoter above gfp DNA sequence. If T7 have been generated once, the T7 promoter starts translating. And then we can see bacteria begin to shine.<br><br>
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On initial state, bacteria don't have supD. Because, lacI represses the lac promoter above supD DNA sequence.So there isn't any T7 polymerase and gfp cannot be genernated, in spite of being T7ptag in bacteria.<br><br>
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As written above, when AHL is entered into bacteria, transcription of proteins under the Plux is repressed. If there is click, transcription of T7ptag and lacI are stopped simultaneously. But T7ptag are decomposed earlier than lacI because of difference in velocity of decomposition. T7ptag which is a kind of mRNA is decomposed so fast and lacI which is just a kind of protein is decomposed late. SupD under the Plac is generated after most of lacI are decomposed. So, during the clicking, there isn't any T7ptag as far as there is supD. <br><br>
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After washing AHL(means after 1st click), supD exist. SupD is a kind of tRNA and stable which means having long half-life. If there isn't input anymore, Plux starts activating and T7ptag is generated. As result of reacting T7ptag and supD, the AND Gate is going to be available. In this case, <b><font color="red">the AND Gate remembers that there was a click.</font></b><br><br>
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Moreover, if there is 2nd click, T7ptag is going to be decomposed again. And then, the reaction of And Gate is stopped, because there isn't T7ptag anymore. So, T7 which is activator of T7 promoter is also going to be decomposed. It is time-limit of Double-Click and explain it in next paragraph. </td>
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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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Actually, 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.
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On this promoter, repression is stronger than activation. And also, it is low unregulated activation. <font color="red"><b>When 1st input</b></font> is come in, cI recognizes the input and going to be decomposed.
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But there isn't T7 yet, <font color="red"><b>gfp cannot be generated</b></font>, despite there isn't repressor. <font color="red"><b>After 1st input,</b></font> AND gate remembers there was a click, and generation of T7 is stared. But, <font color="red"><b>gfp cannot be generated</b></font>, because there is cI already. <font color="red"><b>When 2nd input</b></font> is come, cI also recognizes the input and going to be decomposed. If the 2nd input comes within time that the AND is working, T7 exists in bacteria and decomposition of cI is finished, <font color="red"><b>gfp is going to be generated.</b></font>As a result, bacteria shines with gfp and Double Click is completed.</td>
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Revision as of 17:52, 27 October 2010




 

 

 




Overall Circuit


Our second circiut of double click system is called Recognize circuit.
It consists of recognizing input system and memorizing input system.
Control of circuit
We use AHL for input and regard injection of AHL as clicking. LuxR is generated constitutive. Injecting AHL to Double Click Bacteria brings dimer of AHL and luxR. We take lux promoter repressed by the dimer. As the dimer binds Plux, transcription of proteins under the Plux is repressed.
So in our project,
CLICK controls transcription of overall DNA sequence.
Recognizing Input System
In recognizing input system, there is a cI operator above gfp DNA sequence. If cI binds cI operator, transcription of gfp is stopped. You can see DNA sequence of cI, which a kind of repressor, is under the Plux. On initial state(there isn't any click), cI is generated normally. But AHL come into Double Click Bacteria, generation of cI is stopped. Since repression of Plux starts working by the dimer which is a combination of AHL and LuxR. And then, cI is going to disappear, because all kinds of proteins are bound to be decomposed. If there isn't cI anymore, bacteria will have chance to shine.
We can say
cI recognizes the existence of input in this DNA sequence.
Memorizing Input System
In memorizing system, there is an AND Gate with T7ptag and supD. As you know, T7ptag becomes T7 polymerase in case of existing supD. T7 polymerase is translated to T7 protein which works as activator of T7 promoter. There is a T7 promoter above gfp DNA sequence. If T7 have been generated once, the T7 promoter starts translating. And then we can see bacteria begin to shine.

On initial state, bacteria don't have supD. Because, lacI represses the lac promoter above supD DNA sequence.So there isn't any T7 polymerase and gfp cannot be genernated, in spite of being T7ptag in bacteria.

As written above, when AHL is entered into bacteria, transcription of proteins under the Plux is repressed. If there is click, transcription of T7ptag and lacI are stopped simultaneously. But T7ptag are decomposed earlier than lacI because of difference in velocity of decomposition. T7ptag which is a kind of mRNA is decomposed so fast and lacI which is just a kind of protein is decomposed late. SupD under the Plac is generated after most of lacI are decomposed. So, during the clicking, there isn't any T7ptag as far as there is supD.

After washing AHL(means after 1st click), supD exist. SupD is a kind of tRNA and stable which means having long half-life. If there isn't input anymore, Plux starts activating and T7ptag is generated. As result of reacting T7ptag and supD, the AND Gate is going to be available. In this case, the AND Gate remembers that there was a click.

Moreover, if there is 2nd click, T7ptag is going to be decomposed again. And then, the reaction of And Gate is stopped, because there isn't T7ptag anymore. So, T7 which is activator of T7 promoter is also going to be decomposed. It is time-limit of Double-Click and explain it in next paragraph.
Co-working of two systems


Actually, 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. When 1st input is come in, cI recognizes the input and going to be decomposed. But there isn't T7 yet, gfp cannot be generated, despite there isn't repressor. After 1st input, AND gate remembers there was a click, and generation of T7 is stared. But, gfp cannot be generated, because there is cI already. When 2nd input is come, cI also recognizes the input and going to be decomposed. If the 2nd input comes within time that the AND is working, T7 exists in bacteria and decomposition of cI is finished, gfp is going to be generated.As a result, bacteria shines with gfp and Double Click is completed.

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