Chiba/Quorum Sensing

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Revision as of 20:44, 26 October 2007

Introduction | Project Design ( 1.Sticky Hands | 2.Communication | 3.Size Control ) | Making Marimos | Our Goal || Team Members | メンバ連絡簿

Size Control: Our Aim

Fig1.AHL concentration gradient.

Since produced AHL diffuses all around the bacteria culture, all receivers can ultimately aggregate to one huge marimo with their sticky hands. This final state is not the case of real marimos. Thus our idea for controlling the size of Bacteria Marimos is based on the high performance quorum sensing or AHL-diffusing inhibition.

AHLの届く範囲をコントロールするために以下の３つの方法を考えた：
1. Raise the AHL productivity of Sender
2. Increase the AHL sensitivity of Receiver
3. Use the AHL degrading enzyme aiiA to localize AHL

1.Improving Sender

Design

AHL is synthesized from methionine by the enzyme MetK and LuxI.
We tested in the hope of combined overexpression of these 2 genes will enhance the sender capacity.

Fig.2

Experiment

fig3.

Sender

Ptet-LuxI

Synthesize AHL constantly

metK Sender

Synthesize AHL and express metK constantly

Receiver

Method(Fig3.)

Inoculated sender, MetK sender, and receiver in each liquid medias. Incubated at 37℃ 12h.
Checked OD. Dispensed receiver in each tube equally. Spin downed senders and resuspended with fresh liquid media.
Diluted senders to adjust cell population (5x10⁸,5x10⁷,......, 5x10²).
Mix receivers and senders.
Incubated for 1h to 3h at room tempature
Spindowned and UV checked.

Result

Fig. Methionine 0mM.
metK(+): metK Sender ( pLac-luxI-metK )
metK(-): Sender ( Ptet-luxI )

Fig. Methionine 10mM. metK(+): metK Sender ( pLac-luxI-metK )
metK(-): Sender ( Ptet-luxI )

No difference was seen.

Discussion

No efficiency was observed.
Our Assumption:S-adenosyl methionine was synthesized enough or consumed in other path way.
Sequencing riquired.

2.Improving Receiver

Design

Fig. Data modified from Collins et.al. Mol. Microbiol. 55, 712–723 (2005)

Fig.Data modified from Collins et.al. Mol. Microbiol. 55, 712–723 (2005)

Collins et.al. described the hyper-sensitive variants of luxR to AHL.(Collins, C. H., Arnold, F. H. & Leadbetter, J. R. Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl-homoserine lactones. Mol. Microbiol. 55, 712–723 (2005))
We created two types of sensitive luxR mutants.　理由．

Experiment

sensitive luxR mutants の性能を以下の２つの方法で評価した。

Result

mutant(I45F,S116A)はAHLを添加しなくてもGFPを発現した。

mutant(I45F)はwild typeよりもGFPの発現が多くなった。(AHL 10nM)

mLuxRの論文引用したら？　by tashiro

3.Localizing AHL

Design1

AiiA expression is regulated by pLac, it express constantly and inactivate AHL.

Experiment

Inoculate E.coli carries this aiiA receiver plasmid in Liquid Media.
At the same time, inoculate AHL sender E.coli in Liquid Media.
Dilute receiver cells and spread on agar plate, and spot sender 1μL. Incubate at 37℃
Check plate.(GFP expressed?)

Result

GFP dose not expressed on the plate. File:Aiia conc image

Discussion

Excessive amounts of　aiiA is expressed to generate concentration gradient. We expected aiiA express more moderate to make it.

Design2

図のように，AHLのシグナルが入るとaiiaを合成するような回路を考えた．Marimo全体がAHL quencherとして働く．Marimoの内側でAHLを分解し、Marimoの外側のAHL濃度の上昇を抑える．

Experiment

Result

BBa_T9002との比較ではGFPははじめ発現しないが、しばらくするとGFPが発現するようになる。

(Assumption:GFPは細胞内で分解されにくいからではないだろうか）

Discussion

発現し始めた時が、AHLの濃度がaiiAの分解る能力を超えた時だと考えられる。

Design3

図のようにインバーターをかませた．高い濃度ではaiiAが発現しないためAHLは分解されない．AHLが低い濃度ではaiiAが発現しAHLを分解する．よってマリモの中心付近ではAHLを分解せず，外側へ行くと分解される．

Experiment

このパーツを作ろうとしたが、inverterを組み合わせた時点で終わってしまった。

Subpart:BBa_S03840

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