Team:Chiba/Receiver experiments

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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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