Team:Chiba/Receiver experiments

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(LuxR/Plux mutants)
(LuxR mutants)
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==LuxR mutants==
==LuxR mutants==
[[Image:Location-of-the-mutant-in-LuxR Chiba.gif|frame|right|'''Fig.6''' Location of the LuxR mutant in LuxR]]
[[Image:Location-of-the-mutant-in-LuxR Chiba.gif|frame|right|'''Fig.6''' Location of the LuxR mutant in LuxR]]
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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))
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私たちはmutated Receiverを用いることで、AHL感受性の違う2種類(WTと変異体)のレシーバーを用意し、delay-timeのバリエーションを増やした。
===method===
===method===
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LuxR mutantsとWT LuxRのディレイタイムはfollowing procedureによってanalyzeした。
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# Transformed sender (Ptet-luxI), mutant LuxR Receiver (Ptet-mLuxR-Plux-GFP) and WT LuxR Receiver ()into E coli strains (BW⊿FliC)
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# Inoculated Sender, WT Receiver (wild type luxR/BW⊿FliC) and mutated Receiver (1point mutation/BW⊿FliC) in liquid media for 12 h at 37℃.
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# Inoculated again in liquid media upto about OD600=2 at 37℃
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# Washed Senders and receiver.
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# Mixed them. (Sender:Receiver=1000μL:1000μL)
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# Incubated at 30°C. 
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# Measured intensity of green fluorescence at regular time intervals.
===result===
===result===

Revision as of 05:42, 25 October 2008

Chiba-U.gif

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Project Design ( Sender experiments | Receiver experiments ) | Our Goal

Contents

LuxR mutants

Fig.6 Location of the LuxR mutant in LuxR

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))

私たちはmutated Receiverを用いることで、AHL感受性の違う2種類(WTと変異体)のレシーバーを用意し、delay-timeのバリエーションを増やした。

method

LuxR mutantsとWT LuxRのディレイタイムはfollowing procedureによってanalyzeした。

  1. Transformed sender (Ptet-luxI), mutant LuxR Receiver (Ptet-mLuxR-Plux-GFP) and WT LuxR Receiver ()into E coli strains (BW⊿FliC)
  2. Inoculated Sender, WT Receiver (wild type luxR/BW⊿FliC) and mutated Receiver (1point mutation/BW⊿FliC) in liquid media for 12 h at 37℃.
  3. Inoculated again in liquid media upto about OD600=2 at 37℃
  4. Washed Senders and receiver.
  5. Mixed them. (Sender:Receiver=1000μL:1000μL)
  6. Incubated at 30°C.
  7. Measured intensity of green fluorescence at regular time intervals.

result

AiiA Receiver

Fig.6 AiiA Receiver


オートインデューサー不活性化酵素であるAiiAをレシーバー菌で発現させることにより、レシーバー菌内でのAHL濃度の増加が遅くなるため、AiiAを発現しないレシーバー菌に比べて、GFP発現が遅くなる。



method

  • Sender
    • [http://partsregistry.org/Part:BBa_S03623 BBa_S03623 (AHL auto inucer)]

LuxI-sender Chiba.gif


  • Receivers
    • AiiA Receiver

AiiA-Receiver-genetic-circu Chiba.gif

    • non-AiiA Receiver

Non-AiiA-Receiver Chiba.gif


result

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