Chiba/Quorum Sensing

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Revision as of 20:16, 26 October 2007

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 thought combined overexpression of these 2 genes enhances the sender capacity.

AHLはFig.2のpathwayで合成されている．AHLを増加させるためにMetKを過剰発現させ，methionineを加えてみる。

Fig.2

Experiment

fig3.

Sender

Ptet-LuxI

• Synthesize AHL constantly

metK Sender

• Synthesize AHL and express metK constantly

Receiver

Method(Fig3.)

1. Inoculated sender, MetK sender, and receiver in each liquid medias. Incubated at 37℃ 12h.
2. Checked OD. Dispensed receiver in each tube equally. Spin downed senders and resuspended with fresh liquid media.
3. Diluted senders to adjust cell population (5x10⁸,5x10⁷,......, 5x10²).
4. Mix receivers and senders.
5. Incubated for 1h to 3h at room tempature
6. 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

• 合成されたS-adenosyl Methionineがほかの代謝経路に使われて、AHL合成に使われなかったのだろうか．

2.Improving Receiver

Design

Fig. Data modified from Collins et.al.(Ref. 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))

Fig.

Collins et.al. described the hyper-sensitive variants of luxR to AHL :(ref).
We created two types of sensitive luxR mutants.

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

（なんかいいタイトルないかな）
Controlling outlineとか･･･ダサい？by tashiro
最後に梅野さん達がつくったpptがあったように、Localizing AHL とかでは？by toyotaro

Design1

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

Experiment

1. Inoculate E.coli carries this aiiA receiver plasmid in Liquid Media.
2. At the same time, inoculate AHL sender E.coli in Liquid Media.
3. Dilute receiver cells and spread on agar plate, and spot sender 1μL. Incubate at 37℃
4. 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