Team:Chiba/Project

From 2008.igem.org

(Difference between revisions)

Revision as of 13:49, 25 October 2008

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

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(LuxI-type　proteins) and once they have reached a threshold　concentration,they bound to a transcriptional regulatory　protein(LuxR-type proteins) 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

Fig.4 AHL varieties

　English:Each species has their own LuxI-type proteins,which synthesize their specific autoinducers,AHLs.AHLs produced by different LuxI-type proteins differ only in the length of the acyl-chain moiety and substitution at position C-3.Vibrio fischeri has LuxI,which synthesize 3OC6HSL.Pseudomonas aeruginosa has RhlI and LasI,which synthesize C4HSL、C6HSL,respectively.

日本語:異なる生物はそれぞれに異なるLuxIタイプのタンパク質を持ち、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LuxIにより3OC6HSLを、Pseudomonas aeruginosaはRhlIにより、C4HSL、C6HSLを、LasIにより3OC12HSLを合成する(Fig.4).AHLを受け取り応答するLuxRタンパク質は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。

(冨永)

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。

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。
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

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

a greater response to 3OC6HSL[http://authors.library.caltech.edu/5553/ ⁽³⁾]
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

[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]
[http://partsregistry.org/Part:BBa_F2620:Specificity BBa_F2620:Specificity]
[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]
[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

@@ Line 56: / Line 56: @@
 ===Sender ===
 [[Image:AHL variety chiba.gif|frame|'''Fig.4''' AHL varieties]]
-　English:Bacterias have their own LuxI-type proteins.Each proteins synthesize their specific autoinducers,AHLs.AHLs produced by different LuxI-type proteins differ only in the length of the acyl-chain moiety and substitution at position C-3.''Vibrio fischeri'' has LuxI,which synthesize 3OC6HSL.''Pseudomonas aeruginosa'' has RhlI and LasI,which synthesize C4HSL、C6HSL,respectively.
+　English:Each species has their own LuxI-type proteins,which synthesize their specific autoinducers,AHLs.AHLs produced by different LuxI-type proteins differ only in the length of the acyl-chain moiety and substitution at position C-3.''Vibrio fischeri'' has LuxI,which synthesize 3OC6HSL.''Pseudomonas aeruginosa'' has RhlI and LasI,which synthesize C4HSL、C6HSL,respectively.
 日本語:異なる生物はそれぞれに異なるLuxIタイプのタンパク質を持ち、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LuxIにより3OC6HSLを、''Pseudomonas aeruginosa''はRhlIにより、C4HSL、C6HSLを、LasIにより3OC12HSLを合成する(Fig.4).AHLを受け取り応答するLuxRタンパク質は''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。