Team:Chiba/AiiA Receiver Phase

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Chiba-U.gif

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AiiA Receiver

Fig. AiiA Receiver


Design

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。（小林）


Fig. AiiA Reaction


Experiment

  • 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

Method

  1. Transformed Sender into E.coli strains(BWΔFliC) and Receivers into E.coli strain(BWΔFliC).
  2. Inoculated them independently in liquid media. Incubated at 37c&deg； 12h.
  3. Inoculated again at 37c&deg； upto about OD600=2.0
  4. Washed them.
  5. Mixed them (Sender:Receiver=1000μl:1000μl).
  6. Incubated at 30°C.
  7. Measured intensity of green fluorescence at regular time intervals.


result

Fig.Time Delay Test

24時間後の蛍光強度を比較すると最大強度は1/4に下がっている。

transfer curveがシグモイドではなく、時間に比例するようになった

AiiAを発現させると、GFPの発現自体が極端に減ってしまったので、AHL自体を減らしてしまうと発現量の最大値が小さくなってしまう。

conclusion

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