Team:Chiba/Experiments:/LuxR　mutant

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[[Image:Receiver switch mLuxR chiba.jpg|left]]
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<br>English:
<br>English:
<br>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。
<br>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。
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[[Image:Mutant-LuxR Chiba.gif|frame|right|'''Table.1''' Seisitivity of LuxR mutants. Data modified from Collins et.al. Mol. Microbiol. 55, 712–723 (2005)]]
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[[Image:AHL-activation-of-mutagenizenized LuxR molecules Chiba.gif|frame|right|'''Table.Ahl activation of mutagenized LuxR molecules. Data modfied from B.Kock et al. Microbiology (2005), 151, 3589-3602''']]
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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
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LuxRに変異を入れることでLuxRのAHLに対する感受性を変えることができると報告されている。[[Team:Chiba/AHL Receiver Phase#References|<sup>(1),(2)</sup>]]
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#Ile45PheとするとAHLに対する感受性が約10倍上昇する[[Team:Chiba/AHL Receiver Phase#References|<sup>(1)</sup>]]
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#Leu42AlaとするとAHLに対する感受性が1/15に減少する[[Team:Chiba/AHL Receiver Phase#References|<sup>(2)</sup>]]
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#Leu42SerとするとAHLに対する感受性が1/1000に減少する[[Team:Chiba/AHL Receiver Phase#References|<sup>(2)</sup>]]
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====[[Team:Chiba/Receiver_experiments|Receiver experiments details]]====
====[[Team:Chiba/Receiver_experiments|Receiver experiments details]]====
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[[Image:Receiver switch mLuxR chiba.jpg|frame|left|'''Fig.''' LuxR mutant]]
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== LuxR mutant (Under Plannning) ==
== LuxR mutant (Under Plannning) ==
===Design===
===Design===

Revision as of 20:22, 29 October 2008

Chiba-U.gif

Home The Team The Project Parts Submitted to the Registry Reference Notebook Acknowledgements


LuxR mutant (Under Planning

Design

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)].


Table.1 Seisitivity of LuxR mutants. Data modified from Collins et.al. Mol. Microbiol. 55, 712–723 (2005)
Table.Ahl activation of mutagenized LuxR molecules. Data modfied from B.Kock et al. Microbiology (2005), 151, 3589-3602

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

LuxRに変異を入れることでLuxRのAHLに対する感受性を変えることができると報告されている。(1),(2)


  1. Ile45PheとするとAHLに対する感受性が約10倍上昇する(1)
  2. Leu42AlaとするとAHLに対する感受性が1/15に減少する(2)
  3. Leu42SerとするとAHLに対する感受性が1/1000に減少する(2)



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


LuxR mutant (Under Plannning)

Design

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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