Team:Chiba/Project

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{| style="color:#dd0000;background-color:#eeeeee;" cellpadding="3" cellspacing="1" border="1" bordercolor="#cc0303" width="62%" align="center"
 
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!align="center"|[[Team:Chiba|Home]]
 
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!align="center"|[[Team:Chiba/Team|Team]]
 
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!align="center"|[[Team:Chiba/Project|Project]]
 
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!align="center"|[[Team:Chiba/Parts|Parts]]
 
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!align="center"|[[Team:Chiba/Modeling|Modeling]]
 
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!align="center"|[[Team:Chiba/Notebook|Notebook]]
 
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!align="center"|[[Team:Chiba/Safety|Safety]]
 
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<font color="black" size="5.5">Bacterial Double Click</font>
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----
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If there are two-time input for limited time, bacteria shine.
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== '''Overall project''' ==
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|We’re inspired by double-click of computer’s mouse.<br>
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It doesn’t react by first input but react by second input. So we designed bacterial DNA sequence which works like it. Furthermore, we built time-limit in genetic sequence between the first and the second input like a computer mouse. Bacteria which have the DNA sequence can distinguish between the first input and the second one. Once in a while, bacteria might receive input which wasn’t intended. But it was the first input so bacteria don’t react. Even if there was input not intended, bacteria don’t react unless the second input is entered. Moreover, when the limit-time which controlled by the genetic system is passed, the system is going to return to the initial state. So we expect this DNA sequence will work as a safety device.
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== Project Details==
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<li id="current"><a href="https://2010.igem.org/Team:Chiba/Safety"><span>Safety</span></a></li>
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===Requirement of Double Click ===
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1. If there is a click only one-time, nothing happens.<br>
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2. Time of clicking button doesn't matter. It just depends on the number of click. One or two.<br>
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3. After a lot of time pass from the 1st click, it will return to the initial state.<br>
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4. Click two-time in specific limited time(We call this double click.), something occur.
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=== Explanation of DNA sequence ===
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[[Image:Chiba_model_1.jpg‎]]
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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First, we use AHL for input and regard injection of AHL as clicking. <br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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LuxR is generated constitutive.<br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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Injecting AHL to Double Click Bacteria brings dimer of AHL and luxR.&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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We take lux promoter repressed by the dimer.<br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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As the dimer binds Plux, transcription of proteins under the Plux is repressed.<br><br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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So in our project, <br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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<b><font color="red">CLICK controls transcription of overall DNA sequence.</font></b>
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|align="center"| [[Image:Chiba_LuxR.jpg‎|400px|left|frame]]
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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Second, there is a cI operator above gfp DNA sequence.<br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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If cI binds cI operator, transcription of gfp is stopped.<br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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You can see DNA sequence of cI, which a kind of repressor, is under the Plux.<br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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On initial state(there isn't any click), cI is generated normally.<br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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But AHL come into Double Click Bacteria, generation of cI is stopped.<br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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Since repression of Plux starts working by the dimer which is a combination<br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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of AHL and LuxR.<br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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And then, cI is going to disappear,<br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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because all kinds of proteins are bound to be decomposed.<br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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If there isn't cI anymore, bacteria will have chance to shine.<br><br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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We can say<br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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<b><font color="red">cI recognizes the existence of input in this DNA sequence.</font></b>
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|align="center"| [[Image:Chiba_cI.jpg|400px|left|frame]]
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Third, there is an AND Gate with T7ptag and supD.<br>
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As you know, T7ptag becomes T7 polymerase in case of existing supD.<br>
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T7 polymerase is translated to T7 protein which works as activator of <br>
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T7 promoter.<br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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There is a T7 promoter above gfp DNA sequence.<br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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If T7 have been generated once, the T7 promoter starts translating.
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And then we can see bacteria begin to shine.<br><br>
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On initial state, bacteria don't have supD.<br>
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Because, lacI represses the lac promoter above supD DNA sequence.<br>
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So there isn't any T7 polymerase and gfp cannot be genernated,<br>
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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<br>
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under the Plux is repressed.<br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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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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If there is click,<br>
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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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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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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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transcription of T7ptag and lacI are stopped simultaneously.<br>
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But T7ptag are decomposed earlier than lacI because of difference in velocity<br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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of decomposition.<br>
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T7ptag which is a kind of mRNA is decomposed so fast<br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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and lacI which is just a kind of protein is decomposed late.<br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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SupD under the Plac is generated after most of lacI are decomposed.<br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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So, during the clicking, there isn't any T7ptag as far as there is supD.
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After washing AHL(means after 1st click), supD exist.<br>
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SupD is a kind of tRNA and stable which means having long half-life.<br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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ここから下を編集
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If there isn't input anymore, Plux starts activating and T7ptag is generated.<br>
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-------------------------------------------------------------------------------
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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----------------------------------------------------------------------------------- -->
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As result of reacting T7ptag and supD, the AND Gate is going to be available.<br>
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<br><br><br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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In this case, the AND Gate remembers that  <b><font color="red">there was a click.</font></b>
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<br><br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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===<font size="5">Double Click Bacteria</font>===
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Moreover, if there is 2nd click, T7ptag is going to be decomposed again.<br>
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-----
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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We’re inspired by double-click of computer’s mouse. It doesn’t react to the first click but does react when it is accompanied by the second one. This is one of the most accepted, familiarized, and proven mechanism to diminish the erroneous operation. This fail-safe technology should find various uses also in biotechnology.  
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And then, the reaction of And Gate is stopped,<br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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because there isn't T7ptag anymore.<br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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So, T7 which is activator of T7 promoter is also going to be decomposed.<br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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It is time-limit of Double-Click and explain it in next paragraph.
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|align="center"| [[Image:Chiba_third.jpg‎|400px|left|frame]]
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Here is what we envision: once in a while, bacteria might receive input which wasn’t intended. But they are careful enough (they know Homo sapiens always make mistake!) so that they ignore the input for the first time.  However, when they receive the two inputs in succession, they finally judge the input(s) are real, and start taking the action as programmed.  Also, they are smart enough to distinguish double-clicking from the two separate (or erroneous) inputs.
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First, the circuit must distinguish the second input (stimulus) from the first one, even though they are chemically physically the same . To realize this, we need the counting device. Second, the circuit must distinguishes W-click from two separate clicks; the circuit reacts only when the first and the second input are given in short period of time. Thus, the genetic counter should be coupled with the genetic timers. After a certain time after the first input (timeout), the circuit gets back to the initial state so that the  next stimulous is recognized as the first input.
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Actually, there is a hybrid promoter(cI/T7) above gfp DNA sequence.<br>
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It is regulated by T7 and cI which work as activator and repressor, respectively.<br>
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=== Part 3 ===
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===<font size="5">The Bottom Line is...</font>===
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#If there is only an input, nothing happens.
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#Duration of the input is not the matter. The circuit cares only the number of input.
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#However, a certain time after the 1st input, it returns to the initial state.
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#Giving two inputs in the limited time the circuit get activated (gives output).
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== Results ==
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===<font size="5">Systems</font>===
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<a href="https://2010.igem.org/Team:Chiba/System_1"><img src="https://static.igem.org/mediawiki/2010/d/db/Chiba_plan2_1.jpg" border="0" width="300px" /><br>version 1</a>
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<a href="https://2010.igem.org/Team:Chiba/System_2"><img src="https://static.igem.org/mediawiki/2010/1/1a/Chiba_Sys2.jpg" border="0" width="300px" /><br>version 2</a>
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Latest revision as of 03:10, 28 October 2010




 

 




Double Click Bacteria

We’re inspired by double-click of computer’s mouse. It doesn’t react to the first click but does react when it is accompanied by the second one. This is one of the most accepted, familiarized, and proven mechanism to diminish the erroneous operation. This fail-safe technology should find various uses also in biotechnology.

Here is what we envision: once in a while, bacteria might receive input which wasn’t intended. But they are careful enough (they know Homo sapiens always make mistake!) so that they ignore the input for the first time. However, when they receive the two inputs in succession, they finally judge the input(s) are real, and start taking the action as programmed. Also, they are smart enough to distinguish double-clicking from the two separate (or erroneous) inputs.

First, the circuit must distinguish the second input (stimulus) from the first one, even though they are chemically physically the same . To realize this, we need the counting device. Second, the circuit must distinguishes W-click from two separate clicks; the circuit reacts only when the first and the second input are given in short period of time. Thus, the genetic counter should be coupled with the genetic timers. After a certain time after the first input (timeout), the circuit gets back to the initial state so that the next stimulous is recognized as the first input.




The Bottom Line is...

  1. If there is only an input, nothing happens.
  2. Duration of the input is not the matter. The circuit cares only the number of input.
  3. However, a certain time after the 1st input, it returns to the initial state.
  4. Giving two inputs in the limited time the circuit get activated (gives output).



Systems


version 1
version 2

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