Team:Chiba/System 1/Result

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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_1/Testing_components"><span>Testing</span></a></li>
 
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<li><a href="https://2010.igem.org/Team:Chiba/System_1/Construction_Process"><span>Construction</span></a></li>
 
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<li><a href="https://2010.igem.org/Team:Chiba/System_1/Evaluation_subsystem"><span>Evaluation</span></a></li>
 
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<li><a href="https://2010.igem.org/Team:Chiba/System_1/Result"><span>Result</span></a></li>
 
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<li><a href="https://2010.igem.org/Team:Chiba/System_1/Remarks"><span>Remarks</span></a></li>
 
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__NOTOC__
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<br><br>
 +
[[Team:Chiba/System 1|<<Back]]
 +
<br><br>
 +
<font size=5>Version 1 :Evaluating Core Devices</font><br>
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<br><br><br>
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==<font size="4">Successful operation of this system requires</font>==
-
<br><br><br>
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===1. Generator of T7 RNA Polymerase pulse===
-
 
+
In response to both the 1st and the 2nd input, T7 RNAP has to be effective for a moment then get silent. To fulfill this task,
 +
*Lux/CI434 hybrid promoter:
 +
**must be activated by LuxR in the presence of AHLs<br>
 +
**must be strictly repressed by CI434<br>
 +
**must NOT be repressed by CI (another repressor components in the circuit)<br>
 +
*CI434
 +
**should be expressed upon AHL input<br>
 +
**must become effective after a certain time (delayed repression)<br>
 +
**should tightly repress <br>
 +
*T7RNAP
 +
**must be cleared off as soon as possible when its expression is stopped <br>
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===<font size="5">Requirement of realizing genetic double click system</font>===
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===2. Output module (AND gates of T7RNAP &  (NOT CI)) ===
-
-----
+
*T7/CI promoter should be activated by T7 RNAP
-
1. T7 RNAP pulse in response to 1st and 2nd input<br>
+
*T7/CI promoter should be tightly repressed by CI
-
In response to 1st and 2nd input, T7 RNAP has to express as pulse.<br>
+
*CI should be degraded off after expression shutted off(upon AHL input)<br>
-
To accomplish this, Lux/CI434 hybrid promoter should be activated or repressed stringently.<br>
+
-
So, we checked this part (Data shown below).<br>
+
-
<br>
+
-
2. CI repression<br>
+
-
CI needs to work as a repressor of T7/CI-OR1 hybrid promoter at 1st input and needs to
+
-
have degradated before 2nd input not to work as a repressor at 2nd input.
+
-
To accomplish this, T7/CI-OR1 hybrid promoter should be activated or repressed stringently.<br>
+
-
So, we checked this part (Data shown below).<br>
+
-
<br>
+
-
3. Setting the fixed time<br>
+
-
There is the fixed time between 1st and 2nd input.
+
-
To accomplish this, CI has to re-express after washout. (Data not shown)
+
-
===<font size="4">1-1. Lux/CI434 hybrid promoter</font>===
+
===3. Tuning the timing.===
-
-----
+
*During the T7 RNAP pulse, CI should keep its repression power. ( T7RNAP pulse should be gone before repression by CI start decreasing)
 +
*'Invertimer' should create enough time to clear off the repression by CI<br>
-
===<font size="4">1-2. Pulse generator</font>===
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==<font size="4"> GFP Pulse Generator Result</font>==
-
-----
+
*[http://partsregistry.org/wiki/index.php?title=Part:BBa_K396006 BBa_K396006]
 +
*[http://partsregistry.org/wiki/index.php?title=Part:BBa_K396011 BBa_K396011]
 +
[[Image:名称未設定.png]]
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===<font size="4">2. T7/CI-OR1 hybrid promoter </font>===
+
===Protocol===
-
-----
+
E. coli strain DH10B was used for the pulse-generator experiments.
-
*[http://partsregistry.org/wiki/index.php?title=Part:BBa_K396000 BBa_K396000]
+
Co-transformed cell were pre-cultured in test tubes for overnight at 37C, 200rpm.
-
T7/CI-OR1 hybrid promoter was designed to be activated by T7 RNA Polymerase and repressed by lambda CI protein. In sequence design, lambda CI operator site 1 (OR1, Part of BBa_R1051) was directly attached to the downstream of T7 promoter sequence (BBa_I719005). This characterization was implemented to validate the promoter function. In results, the activation and repression was actually observed.
+
Dilute the culture 1:100 into 2ml of fresh medium and grow for an additional 5 hours in Supplemented M9 medium .
 +
For the liquid experiments, expression was induced at the log phase (OD600 of 0.4)  
 +
by the addition of AHL (3OC6HSL)at the appropriate concentration.  
 +
One-milliliter samples were taken every 5 min.
-
===<font size="3"> Checking the parts</font>===
 
-
----
 
-
[[Image:T7-CI-1.png|frame|center|Fig. How to check the parts]]
 
-
[[Image:CI-2.png|frame|center|Fig. Data: Under the situation of CI non-expressed , T7 RNAP causes GFP expression by activating T7/CI hybrid promoter (Fig.1). On the other hand, under the situation of CI expressed, T7 RNAP does not cause GFP expression (Fig.2). The reason of this is considered as following. Under the situation of CI expressed, even though T7 RNAP tries to activate T7/CI promoter, the promoter Is repressed by CI because the ability of CI repression is superior to the one of T7 activation for this promoter. So it is concluded that T7/CI hybrid promoter is to be activated by T7 RNA Polymerase and repressed by lambda CI protein, following the rules of CI repression > T7 activation.]]
 
-
===<font size="3">Reference</font>===
+
===Result===
-
----
+
By now, there are no obvious pulse observed.The construction we use for measuring GFP is lux/cI434 hybrid promoter following by destabilized GFP. We also check GFP with the same
-
Dubendorf, J.W. & Studier, F.W. Controlling basal expression in an
+
degradation tag following by hybrid promoter as a positive control, it neither generate any fluorescent.Maybe because the GFP expression is too low to be investigate because we use
-
inducible T7 expression system by blocking the target T7 promoter with
+
weak RBS and GFP with LVA.We will continue with investigating GFP pulse. Except that, we
-
lac repressor. Journal of Molecular Biology 219, 45-59 (1991).
+
want to western blot T7 RNA Polymerase pulse and output GFP in plasmid 1 whose transcription
 +
factors are T7 RNA Polymerase and cI protein.We hope there will be GFP pulse generated when co-transform plasmid 1 and 2 because of T7 RNA Polymerase amplifier.

Latest revision as of 03:27, 28 October 2010




 

 



<<Back

Version 1 :Evaluating Core Devices

Successful operation of this system requires

1. Generator of T7 RNA Polymerase pulse

In response to both the 1st and the 2nd input, T7 RNAP has to be effective for a moment then get silent. To fulfill this task,

  • Lux/CI434 hybrid promoter:
    • must be activated by LuxR in the presence of AHLs
    • must be strictly repressed by CI434
    • must NOT be repressed by CI (another repressor components in the circuit)
  • CI434
    • should be expressed upon AHL input
    • must become effective after a certain time (delayed repression)
    • should tightly repress
  • T7RNAP
    • must be cleared off as soon as possible when its expression is stopped

2. Output module (AND gates of T7RNAP & (NOT CI))

  • T7/CI promoter should be activated by T7 RNAP
  • T7/CI promoter should be tightly repressed by CI
  • CI should be degraded off after expression shutted off(upon AHL input)

3. Tuning the timing.

  • During the T7 RNAP pulse, CI should keep its repression power. ( T7RNAP pulse should be gone before repression by CI start decreasing)
  • 'Invertimer' should create enough time to clear off the repression by CI

GFP Pulse Generator Result

  • [http://partsregistry.org/wiki/index.php?title=Part:BBa_K396006 BBa_K396006]
  • [http://partsregistry.org/wiki/index.php?title=Part:BBa_K396011 BBa_K396011]

名称未設定.png

Protocol

E. coli strain DH10B was used for the pulse-generator experiments. Co-transformed cell were pre-cultured in test tubes for overnight at 37C, 200rpm. Dilute the culture 1:100 into 2ml of fresh medium and grow for an additional 5 hours in Supplemented M9 medium . For the liquid experiments, expression was induced at the log phase (OD600 of 0.4) by the addition of AHL (3OC6HSL)at the appropriate concentration. One-milliliter samples were taken every 5 min.


Result

By now, there are no obvious pulse observed.The construction we use for measuring GFP is lux/cI434 hybrid promoter following by destabilized GFP. We also check GFP with the same degradation tag following by hybrid promoter as a positive control, it neither generate any fluorescent.Maybe because the GFP expression is too low to be investigate because we use weak RBS and GFP with LVA.We will continue with investigating GFP pulse. Except that, we want to western blot T7 RNA Polymerase pulse and output GFP in plasmid 1 whose transcription factors are T7 RNA Polymerase and cI protein.We hope there will be GFP pulse generated when co-transform plasmid 1 and 2 because of T7 RNA Polymerase amplifier.

Retrieved from "http://2010.igem.org/Team:Chiba/System_1/Result"