Team:Chiba/Project

From 2008.igem.org

Introduction

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.

Motivation

Fig.2 Team logo

Comparison with clock gene

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。

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

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 Phase　

• 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://www3.interscience.wiley.com/journal/119124142/abstract ⁽¹⁾][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。

Receiver Phase

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⁾].

• 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.

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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