Team:Chiba/Receiver experiments

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<span style="font-size:120%;font-weight:bold;">[[Team:Chiba/Project|Project Design]] ( [[Team:Chiba/Sender_experiments|Sender experiments]] | [[Team:Chiba/Receiver_experiments|Receiver experiments]] )  |  [[Team:Chiba/Goal|Our Goal]]</span><br>
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==LuxR mutants==
==LuxR mutants==

Revision as of 11:25, 25 October 2008

Chiba-U.gif

Home The Team The Project Parts Submitted to the Registry Notebook


Contents

LuxR mutants

Fig.6 Location of the LuxR mutant in LuxR
Table.1 Seisitivity of LuxR mutants. 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))

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

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

LuxI-sender Chiba.gif

  • Receivers
    • mutant LuxR Receiver

Mutant-LuxR-genetic-circuit Chiba.gif

    • WT LuxR Receiver ([http://partsregistry.org/Part:BBa_S03623 BBa_T9002 ])

T9002 Chiba.gif


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 autoinucer)]

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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