Team:Chiba/System 1

From 2010.igem.org

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==Circuit Construction==
==Circuit Construction==
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[[Image:Chiba_plan2_1.jpg|right|500px]]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.
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[[Image:Chiba_plan2_1.jpg|right|500px]]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.[[Team:Chiba/System 1/Design|detail...]]
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==Results==
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===Testing individual parts===
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To realize the genetic double click system, we made two plasmids. First one is “GFP generator”, which generates the output (GFP expression) on condition that T7 RNAP activates the GFP upstream promoter and also CI protein does not repress that promoter. Second one is “Pulse generator”, which generate the pulse of T7 RNAP in response to the 1st and 2nd input. The subparts of those plasmids are shown below.
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[[Image:Chiba_icon_1.jpg]]
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Regarding with those two plasmids, we tested the function of each parts in the plasmids.
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For GFP generator, the parts shown below were checked.
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#Constitutive promoter
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#LuxR
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#T7/CI-OR1 hybrid promoter
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For Pulse generator, the parts shown below were checked.
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#Lux/CI434 hybrid promoter
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#Pulse generator
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Also, we checked if there were no crosstalk between CI/CI434 and CI promoter/CI434 promoter.
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===Construction===
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===Evaluating Core Devices===
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===Whole System===
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<html>
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<br><br><br><br><br><br>
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<font size=6>Version 1 :</font>
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<table border="0" cellpadding="30" cellspacing="0" align=”center”>
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<tr>
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  <td width="800px"><font size="4" face="verdana">Overall Circuit</font><br>
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  <hr width="500" size="1" align="left"><br>
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<font size=2 face=verdana>
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“Genetic double click system” is based on the AND-gate which is switched only when same inputs are given two times in quick succession.
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This system requires the operations which never happen naturally to turn the switch.   
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So, we believe that this is very safety device.
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This system requires that a first input is transient in order to allow a second input.
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Unlike chemical inputs, light inputs are very suitable for this system because it does not remain after inputting.  But, there is not light sensor with good sensitivity.  So, we produced artificially transient input by AHL inputting and washing. 
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Next, this system is required to memorize the transient first input since this system is based on AND gate.  In addition, this system requires fixed time between the first and the second input like a computer mouse to recognize double-click and two separated single-clicks as different, so, this system is required to memorize the transient first input temporarily.<br>
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In our first design, we decided a pulse-generator to memorize the input temporarily. And, we designed the system which is based on combination of a Fast-pulse and a Slow-pulse in principle as shown in the following figure. <br><br>
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<center>
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<font size=2 face=verdana>
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<img src="https://static.igem.org/mediawiki/2010/6/60/Chiba_ani3.gif"><br>
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</center>
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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><br>
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<center>
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<img src="https://static.igem.org/mediawiki/2010/d/db/Chiba_plan2_1.jpg"><br>
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</center>
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<br><br>
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</td>
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</tr>
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</table>
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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">Fast Pulse</font><br>
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  <hr width="500" size="1" align="left"><br>
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<center>
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<font size=2 face=verdana>
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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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</center>
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</td>
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</table>
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<table border="0" cellpadding="30" cellspacing="0" margin-top="-50">
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  <td width="800px"><font size=2 face=verdana>
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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. <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>
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<font size=2 face=verdana>
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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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</center>
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</td>
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</table>
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<table border="0" cellpadding="30" cellspacing="0" margin-top="-50">
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  <td width="800px"><font size=2 face=verdana>
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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. <br>
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<center>
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<br><br><img src="https://static.igem.org/mediawiki/2010/9/91/Chiba_planB_4.jpg" width="500px"><br>
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</center>
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</td>
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</table>
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</html>
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<!-------- Main -------->
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Revision as of 01:02, 28 October 2010




 

 

 


Contents

Version 1

Overview

Chiba ani3.gif
“Genetic double click system” - version 1 is based on the AND-gate which is switched only when same inputs are given two times in quick succession. This system requires the operations which never happen naturally to turn the switch. So, we believe that this is very safety device. This system requires that a first input is transient in order to allow a second input. Unlike chemical inputs, light inputs are very suitable for this system because it does not remain after inputting. But, there is not light sensor with good sensitivity. So, we produced artificially transient input by AHL inputting and washing. Next, this system is required to memorize the transient first input since this system is based on AND gate. In addition, this system requires fixed time between the first and the second input like a computer mouse to recognize double-click and two separated single-clicks as different, so, this system is required to memorize the transient first input temporarily.

In our first design, we decided a pulse-generator to memorize the input temporarily. And, we designed the system which is based on combination of a Fast-pulse and a Slow-pulse in principle as shown in the following figure. detail...


Circuit Construction

Chiba plan2 1.jpg
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.detail...


Results

Testing individual parts

To realize the genetic double click system, we made two plasmids. First one is “GFP generator”, which generates the output (GFP expression) on condition that T7 RNAP activates the GFP upstream promoter and also CI protein does not repress that promoter. Second one is “Pulse generator”, which generate the pulse of T7 RNAP in response to the 1st and 2nd input. The subparts of those plasmids are shown below. Chiba icon 1.jpg Regarding with those two plasmids, we tested the function of each parts in the plasmids. For GFP generator, the parts shown below were checked.

  1. Constitutive promoter
  2. LuxR
  3. T7/CI-OR1 hybrid promoter

For Pulse generator, the parts shown below were checked.

  1. Lux/CI434 hybrid promoter
  2. Pulse generator

Also, we checked if there were no crosstalk between CI/CI434 and CI promoter/CI434 promoter.

Construction

Evaluating Core Devices

Whole System

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