Team:Chiba/Receiver experiments

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Contents

Copy number of Receiver Plasmid

Receiver copy number chiba.jpg


English:
日本語:レシーバーのコピーナンバーを減らすことで、GFPが確認できるまでの時間を延長する。 コピーナンバーが少なくなれば、LuxRの合成量は減少する。 LuxRの合成量が減少すれば、AHLを受け取るLuxRは従来より減ってしまう。 そのため、プロモーターの活性化は遅くなり、GFPの発現量は減る。 したがって、コピーナンバーが多いレシーバーより、GFPが確認できるまでの時間を延長させることができる。 （杉山）


LuxR/Plux mutants show

Receiver switch mLuxR chiba.jpg


English:
日本語: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。

  1. a greater response to 3OC6HSL[http://authors.library.caltech.edu/5553/ (3)]
  2. a increase in sensitivity to 3OC12HSL[http://mic.sgmjournals.org/cgi/content/abstract/151/11/3589 (4)].


Receiver experiments details

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LuxR mutant (Under construction)

レシーバータンパク質であるLuxRに変異を入れることで応答感度を上下させる(5),(6)

Fig. LuxR mutant


Receiver crosstalk

Design

Table.1 LuxR family gene

センダーとレシーバーを元来とは異なる組み合わせにすることによって、thresholdに達する時間を遅くする。（杉山）

Method

Result

Change Receiver's Copy Number

Design

レシーバーのコピーナンバーを減らすことで、thresholdに達するまでの時間を伸ばす。 （小林）

Method

  • High Copy Receiver[http://partsregistry.org/Part:BBa_T9002 BBa_T9002 (AHL Reporter)]

T9002.copy-number-change Chiba.gif


  • Medium Copy Receiver

Low-copy-Receiver Chiba.gif


High Copy ReceiverとMedium Copy Receiverのディレイタイムはfollowing procedureによってanalyzeした。

  1. Transformed sender(Ptet-LuxI), high copy receiver(Ptet-LuxR-Plux-GFP-colE1) and Medium copy receiver(Ptet-LuxR-Plux-GFP-p15A) respectively into E coli strains(BD⊿FliC).
  2. Inoculated them independently in liquid media. Incubated at 37℃ 12h.
  3. Inoculated again in Fresh liquid media upto about OD600=2 at 37℃
  4. Washed sender and receivers.
  5. Mixed them. (Sender:Receiver=1000μL:1000μL)
  6. Incubated at 30°C.
  7. Measured intensity of green fluorescence at regular time intervals.(Fluoroskan AscentR FL＆Fluoroskan AscentR　Thermo ELECTRON CORPORATION)

Result

LuxR mutants

Design

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。 (小林)

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

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