Team:Chiba/Project

From 2008.igem.org

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(Sender Phase )
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==Introduction==
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==Abstruct==
[[Image:Project design chiba QS.gif|frame|right|'''Fig.1''' Project desgin]]
[[Image:Project design chiba QS.gif|frame|right|'''Fig.1''' Project desgin]]
::'''"Team : Chiba - E.coli time manager"'''
::'''"Team : Chiba - E.coli time manager"'''
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===Motivation===
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==Introduction==
[[‎Image:Chiba-logo.gif‎||frame|'''Fig.2''' Team logo]]
[[‎Image:Chiba-logo.gif‎||frame|'''Fig.2''' Team logo]]
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<br>
<br>
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===Comparison with clock gene===
 
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時間を測る仕組みとしてあげられるのが、サーカディアンリズムを担う時計遺伝子である。iGEM Harbard 2006, Chiba 2006 がcyanobacteriaの時計遺伝子であるKaiに関わるprojectを進めていた。時計遺伝子と私たちの考えるシステムでのメリットとデメリットを比較してみる。
 
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== Project Design==
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==Our project==
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AHL濃度が上がって、スレッショルド超えるまでの時間を遅くして、発現を遅らせる。
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==System design==
Our project uses two classes of bacteria: senders and receivers.Senders produce signaling molecules, and receivers are activated only after a particular concentration of this molecule is reached.The communication using non-endogenous molecules is less sensitive,and it requires higher signal concentration to take effect.This results in slower activation of receivers.
Our project uses two classes of bacteria: senders and receivers.Senders produce signaling molecules, and receivers are activated only after a particular concentration of this molecule is reached.The communication using non-endogenous molecules is less sensitive,and it requires higher signal concentration to take effect.This results in slower activation of receivers.
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Communication using non-endogenous molecules is less sensitive, and requires a higher signal concentration to take effect.This results in slower activation of receivers.
Communication using non-endogenous molecules is less sensitive, and requires a higher signal concentration to take effect.This results in slower activation of receivers.
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====Sender Phase ====
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===Sender ===
*Quorum-Sensing Cross-talk
*Quorum-Sensing Cross-talk
[[Image:AHL variety chiba.gif|frame|'''Fig.4''' AHL varieties]]
[[Image:AHL variety chiba.gif|frame|'''Fig.4''' AHL varieties]]
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日本語:異なる生物は、アシル鎖の長さ、あるいはC-3位の置換基が異なる種類のAHLを合成する。''Vibrio fischeri''は3OC6HSLを、''Pseudomonas aeruginosa''はC4HSL、C6HSL、および3OC12HSLを合成する(Fig.4).AHLを受け取り、遺伝子発現を活性化するLuxRは''Vibrio fischeri''由来であり、低濃度の3OC6HSLに対して(~5nM)、応答する。しかし、他種生物由来のAHLに対しては、より高い濃度でないと応答しないことが分かっている[http://partsregistry.org/Part:BBa_F2620:Specificity <sup>(2)</sup>].AHL合成速度が十分に遅いならば、LuxRは、3OC6HSLに対して最も早く応答し、他のAHLに対してはそれよりも遅く応答する。
日本語:異なる生物は、アシル鎖の長さ、あるいはC-3位の置換基が異なる種類のAHLを合成する。''Vibrio fischeri''は3OC6HSLを、''Pseudomonas aeruginosa''はC4HSL、C6HSL、および3OC12HSLを合成する(Fig.4).AHLを受け取り、遺伝子発現を活性化するLuxRは''Vibrio fischeri''由来であり、低濃度の3OC6HSLに対して(~5nM)、応答する。しかし、他種生物由来のAHLに対しては、より高い濃度でないと応答しないことが分かっている[http://partsregistry.org/Part:BBa_F2620:Specificity <sup>(2)</sup>].AHL合成速度が十分に遅いならば、LuxRは、3OC6HSLに対して最も早く応答し、他のAHLに対してはそれよりも遅く応答する。
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====Receiver Phase====
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[[Team:Chiba/Sender_Experiments|Sender experiments detail]]
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===Receiver ===
English:
English:
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#レシーバータンパク質であるLuxRに変異を入れることで、AHLに対する応答感度を上下させること
#レシーバータンパク質であるLuxRに変異を入れることで、AHLに対する応答感度を上下させること
#AHL分解も同時に起こすことで、AHL合成速度を遅くする
#AHL分解も同時に起こすことで、AHL合成速度を遅くする
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*Quorum-Sensing Cross-talk
*Quorum-Sensing Cross-talk
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日本語:
日本語:
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*LuxR/Plux mutants show
*LuxR/Plux mutants show
#a greater response to 3OC6HSL[http://authors.library.caltech.edu/5553/ <sup>(3)</sup>]
#a greater response to 3OC6HSL[http://authors.library.caltech.edu/5553/ <sup>(3)</sup>]
#a increase in sensitivity to 3OC12HSL[http://mic.sgmjournals.org/cgi/content/abstract/151/11/3589  <sup>(4)</sup>].
#a increase in sensitivity to 3OC12HSL[http://mic.sgmjournals.org/cgi/content/abstract/151/11/3589  <sup>(4)</sup>].
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[[Team:Chiba/Receiver_experiments|Receiver experiments detail]]
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===AiiA===
*AHL reporter with aiiA
*AHL reporter with aiiA
:Express LuxR and aiiA constantly. AiiA degrades AHL as signaling molecule. Express GFP when the AHL concentration exceed the capacity of aiiA.
:Express LuxR and aiiA constantly. AiiA degrades AHL as signaling molecule. Express GFP when the AHL concentration exceed the capacity of aiiA.
:This enables the delay of the activation time of receiver.
:This enables the delay of the activation time of receiver.
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[[Team:Chiba/AiiA_experiments|AiiA experiments detail]]
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==Demo Experiments==
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実験内容とdataかるく
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[[Team:Chiba/Demo_experiments|Demo experiments detail]]
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==Conclusion==
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けつろん
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==Future Work==
===references===
===references===

Revision as of 11:20, 25 October 2008

Chiba-U.gif

Home The Team The Project Parts Submitted to the Registry Reference Notebook Acknowledgements

Project Design ( Sender experiments | Receiver experiments ) | Our Goal

Abstruct

Fig.1 Project desgin
"Team : Chiba - E.coli time manager"

  We control the timing of gene expression by using multiple signaling devices.To this end,we utilize molecules associated with Quorum sensing, a phenomenon that allows bacteria to communicate with each other.Our project uses two classes of bacteria: senders and receivers. Senders produce signaling molecules, and Receivers are activated only after a particular concentration of this molecule is reached.Although different quorum sensing species have slightly different signaling molecules, these molecules are not completely specific to their hosts and cross-species reactivity is observed [http://www3.interscience.wiley.com/journal/119124142/abstract (1)],[http://partsregistry.org/Part:BBa_F2620:Specificity (2)]. Communication using non-endogenous molecules is less sensitive, and requires a higher signal concentration to take effect.This results in slower activation of receivers.


Introduction

Fig.2 Team logo








Our project

AHL濃度が上がって、スレッショルド超えるまでの時間を遅くして、発現を遅らせる。


System design

Our project uses two classes of bacteria: senders and receivers.Senders produce signaling molecules, and receivers are activated only after a particular concentration of this molecule is reached.The communication using non-endogenous molecules is less sensitive,and it requires higher signal concentration to take effect.This results in slower activation of receivers.

About Quorum Sensing

Fig.3 Constructed circuit of Quorum sensing(Vibrio fischeri model).

  Quorum sensing is a cell-to-cell signaling action of bacteria. They detect the cell density of the same species and coordinate the expression behavior of their cells. Species of Gram-Negative signaling transfer molecules (so-called autoinducer) is a series of acyl homoserine lactone (AHL). The signals are synthesized from S-adenosylmethionine(SAM) by a synthase protein and once they have reached a threshold concentration,they bound to a transcriptional regulatory protein to induce expression of target genes.

More about Quorum Sensing

  • [http://parts.mit.edu/registry/index.php/Featured_Parts:Cell-Cell-Signaling Cell-Cell-Signaling]
  • [http://www.che.caltech.edu/groups/fha/quorum.html About Quorum sensing]

Controlling the time of a cell-to-cell signaling action

Communication using non-endogenous molecules is less sensitive, and requires a higher signal concentration to take effect.This results in slower activation of receivers.

Sender

  • Quorum-Sensing Cross-talk
Fig.4 AHL varieties

 English:AHLs produced by different bacteria differ only in the length of the acyl-chain moiety and substitution at position C-3.

日本語:異なる生物は、アシル鎖の長さ、あるいはC-3位の置換基が異なる種類のAHLを合成する。Vibrio fischeriは3OC6HSLを、Pseudomonas aeruginosaはC4HSL、C6HSL、および3OC12HSLを合成する(Fig.4).AHLを受け取り、遺伝子発現を活性化するLuxRはVibrio fischeri由来であり、低濃度の3OC6HSLに対して(~5nM)、応答する。しかし、他種生物由来のAHLに対しては、より高い濃度でないと応答しないことが分かっている[http://partsregistry.org/Part:BBa_F2620:Specificity (2)].AHL合成速度が十分に遅いならば、LuxRは、3OC6HSLに対して最も早く応答し、他のAHLに対してはそれよりも遅く応答する。

Sender experiments detail

Receiver

English:

日本語:AHLを合成するSenderだけではなく、AHLを受け取る側のReceiverを変えれば、その応答感度を変えることができる。そこで私たちは、以下のいくつかの方法を考えた。

  1. 一種類のSender(AHL<--LuxI)に対して、由来生物の異なるレシーバタンパク質でそれを受信する。
  2. レシーバータンパク質であるLuxRに変異を入れることで、AHLに対する応答感度を上下させること
  3. AHL分解も同時に起こすことで、AHL合成速度を遅くする


  • Quorum-Sensing Cross-talk
Table.1 LuxR family gene Data modified from Fekete et.al. Anal Bioanal Chem (2007)

English:

日本語:

  • LuxR/Plux mutants show
  1. a greater response to 3OC6HSL[http://authors.library.caltech.edu/5553/ (3)]
  2. a increase in sensitivity to 3OC12HSL[http://mic.sgmjournals.org/cgi/content/abstract/151/11/3589 (4)].


Receiver experiments detail

AiiA

  • AHL reporter with aiiA
Express LuxR and aiiA constantly. AiiA degrades AHL as signaling molecule. Express GFP when the AHL concentration exceed the capacity of aiiA.
This enables the delay of the activation time of receiver.


AiiA experiments detail

Demo Experiments

実験内容とdataかるく

Demo experiments detail

Conclusion

けつろん

Future Work

references

  1. [http://www3.interscience.wiley.com/journal/119124142/abstract M.K Winson et al.:Construction and analysis of luxCDABE-based plasmid sensors for investigating N-acyl homoserine lactone-mediated quorum sensing.FEMS Microbiology Letters 163 (1998) 185-192]
  2. [http://partsregistry.org/Part:BBa_F2620:Specificity BBa_F2620:Specificity]
  3. [http://authors.library.caltech.edu/5553/ C. H. Collins.et al.:Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl-homoserine lactones.Mol.Microbiol.2005.55(3).712–723]
  4. [http://mic.sgmjournals.org/cgi/content/abstract/151/11/3589 B. Koch. et al.:The LuxR receptor: the sites of interaction with quorum-sensing signals and inhibitors.Microbiology 151 (2005),3589-3602]
Home The Team The Project Parts Submitted to the Registry Reference Notebook Acknowledgements