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Team:Chiba/Project
From 2008.igem.org
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Project Design ( Sender experiments | Receiver experiments ) | Our Goal |
Introduction
- "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 (M.K Winson et al.FEMS Microbiology Letters,1998)]. 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
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. 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
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.5).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)、遺伝子発現が起こる。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遺伝子発現が起こらないことが分かっている([http://partsregistry.org/Part:BBa_F2620:Specificity BBa_F2620:Specificity])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
AHLを合成するSenderだけではなく、AHLを受け取る側のReceiverにもさまざまな種類があれば、時間調節により幅を持たせることができる。そこで私たちは、いくつかの方法を考えた。
- 由来生物の違うレシーバータンパク質を発現させ、Receiver側でクロストークさせること
- 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を分解し、より多くのAHLが合成されなければ遺伝子発現が起こらないようにすること
- LuxR/Plux mutants show
- a greater response to 3OC6HSL ([http://authors.library.caltech.edu/5553/ C. H. Collins.et al.Mol.Microbiol.2005.])
- a increase in sensitivity to 3OC12HSL ([http://mic.sgmjournals.org/cgi/content/abstract/151/11/3589 B. Koch.et al.Microbiology (2005)]).
- 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.
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