Team:Chiba/Project

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Abstract

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

Fig. Our system

従来:消えていく物質の存在時間によって時間調節する。一つ一つが機械となる。

千葉:常に物質が一定の割合で蓄積されるとき、ある閾値超えるまでの時間を調節する。

調節する方法には、二つが挙げられる。

・蓄積するときの割合を変える。

・閾値自体を変える。

全体が機械となる。









System design

Fig.Project 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

　　Quorum sensing is a cell-to-cell signaling action of bacteria. Species of Gram-Negative signaling transfer molecules (so-called autoinducer) is a series of acyl homoserine lactone (AHL). The signals are synthesized 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.

シグナル分子に対する応答速度を変えるためには３つの方法が考えられる。

  1. シグナル分子を作るSenderを変える
  2. シグナルを受け取るReceiverを変える
  3. シグナル分子自体を分解して、邪魔をする

Signal Molecule Sender

Fig.4 AHL varieties
Sender switch Chiba.jpg

　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.LuxR,which is original for Vibrio fischeri,is activated by its cognate autoinducer,3OC6HSL.However,LuxR is also activated by non-endogenous molecules,C4HSL,C6HSL,and 3OC12HSL.Activation by non-endogenous molecules requires a higher signal concentration[http://partsregistry.org/Part:BBa_F2620:Specificity (2)].This results in slower activation of receivers,when AHL concentration is increasing.

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に対しても応答することが知られており、より高い濃度のAHLが必要となる[http://partsregistry.org/Part:BBa_F2620:Specificity (2)].AHLがゆっくり溜まっていく時、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

Signal Molecule Receiver

Chiba project design Receiver.jpg

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、その応答時間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. レシーバーのコピーナンバーを変える.

（福冨、小林）


Quorum-Sensing Cross-talk

Receiver switch chiba.jpg

English:

日本語:





Copy nomber of Receiver Plasmid

レシーバーのコピーナンバーを減らすことで、thresholdに達するまでの時間を短縮する。

LuxR/Plux mutants show

私たちはmutated Receiverを用いることで、AHL感受性の違う２種類（WTと変異体）のレシーバーを用意し、delay-timeのバリエーションを増やした。

  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

Signal Molecule Quencher

Chiba project design.jpg

AiiAによるAHL分解を同時に起こすことで、AHL供給速度を遅くする.

  • 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]
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