Team:Chiba/Project/Experiments:Crosstalk

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Crosstalk

Design

Sender switch Chiba.jpg

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.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(2).This results in slower activation of receivers,when AHL concentration is increasing.

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。それぞれの生物種のLuxIタイプのタンパク質、それが合成する分子名は以下の表のようである。 (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、3OC6HSLに応答する。しかし、他種生物由来のAHLにも応答することが知られており、このとき、より高い濃度のAHLが必要となる(1),(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)

Fig.4 AHL varieties


Experiments

センダーの違いによるディレイをみることをめざし，次の表に示すセンダーとレシーバを別々の培地で培養@37℃,12h後（stationaryに達したら？対数増殖機の細胞の分裂によるsender量増加を防ぐため？），それぞれを混ぜ，３０℃で培養し，一定時間ごとの蛍光強度（__nm）を測定した。

senders (cell: XL10-Gold) receiver (cell: JW1908)
*[http://partsregistry.org/Part:BBa_K084007 plac+rbs+LasI] (plasmid?)

Tet-lasi chiba.gif

*[http://partsregistry.org/Part:BBa_T9002 BBa_T9002 (Express GFP in response to AHL)] (plasmid?)

Reporter 9002 chiba.gif

*[http://partsregistry.org/Part:BBa_K084008 plac+rbs+RhlI] (plasmid?)

Tet-rhli chiba.gif

*[http://partsregistry.org/Part:BBa_K084012 plac+rbs+LuxI] (plasmid?)

Tet-luxi chiba.gif

Results&Discussion

Fig. senders crosstalk test.senders strain XL10Gold,Receiver strain JW1908.Reaction temparature was 30°C.All measurements are averages from three replicate cultures with error bars representing standard deviations.Labeling:LuxI,RhlI,LasI means fluorescence induced by AHLs synthesized by LuxI,RhlI,LasI respectively.

3OC6HSL,3OC12HSLに対する、LuxRの応答時間に、二時間の差ができた（3OC12HSLの場合、3OC6HSLに比べて二時間遅れて応答する）。 (そのときの条件は、培養温度30°C、genelatorの株はXL10Gold,Receiverの株はJW1908のとき、であった。) <??

Future plans

Details

Method

1.Crosstalk test

To characterize quorum sensing crosstalk, constitutive AHL senders were mixed with constitutive receivers and measure fluorescence intensity.

  1. Transformed Senders into E.coli strains(XL10GOLD) and Receiver into E.coli strain(JW1908).
  2. Inoculated them independently in liquid media. Incubated at 37°C 12h
  3. Mixed them.
  4. Incubated at 30°C.
  5. Measured intensity of green fluorescence at regular time intervals.

more details...

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