Team:Chiba/Experiments:/LuxR　mutant

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==LuxR mutant (Under Planning==
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==LuxR mutant (Under Planning)==
===Design===
===Design===
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{| style="border:0px;" cellpadding="5px"
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[[Image:Receiver switch mLuxR chiba.jpg|left]]
[[Image:Receiver switch mLuxR chiba.jpg|left]]
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<br clear=all>
 
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<br>English:
 
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<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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#a greater response to 3OC6HSL[http://authors.library.caltech.edu/5553/ <sup>(3)</sup>]
 
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#a increase in sensitivity to 3OC12HSL[http://mic.sgmjournals.org/cgi/content/abstract/151/11/3589  <sup>(4)</sup>].
 
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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)]]
[[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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[[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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Collins et.al. described the hyper-sensitive variants of luxR to AHL.(1)
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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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CollonsらとKockらによって、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/Experiments:/LuxR　mutant (section)#Reference|<sup>(1),(2)</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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#Ile45PheとするとAHLに対する感受性が約10倍上昇する[[Team:Chiba/Experiments:/LuxR　mutant (section)#Reference|<sup>(1)</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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#Leu42AlaとするとAHLに対する感受性が1/15に減少する[[Team:Chiba/Experiments:/LuxR　mutant (section)#Reference|<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/Experiments:/LuxR　mutant (section)#Reference|<sup>(2)</sup>]]
<br clear=all>
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===Experiment===
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====[[Team:Chiba/Receiver_experiments|Receiver experiments details]]====
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{| class="tbl" |
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-->
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====[[Team:Chiba/AHL Receiver Phase#LuxR　mutant|LuxR mutant (Under construction)]]====
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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[[Team:Chiba/Project#references|<sup>(5),(6)</sup>]]
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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 (2005))
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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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(小林)
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===Method===
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*'''Sender'''
*'''Sender'''
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**[http://partsregistry.org/Part:BBa_S03623 BBa_S03623 (AHL auto inucer)]
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|
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*'''Receivers'''
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| rowspan="2" | **[http://partsregistry.org/Part:BBa_S03623 BBa_S03623 (AHL autoinucer)]
[[Image:LuxI-sender Chiba.gif]]
[[Image:LuxI-sender Chiba.gif]]
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*'''Receivers'''
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*mutant LuxR Receiver |
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**mutant LuxR Receiver
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[[Image:Mutant-LuxR-genetic-circuit_Chiba.gif]]f]]
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[[Image:Mutant-LuxR-genetic-circuit_Chiba.gif]]
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**WT LuxR Receiver ([http://partsregistry.org/Part:BBa_S03623 BBa_T9002 ])
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*WT LuxR Receiver ([http://partsregistry.org/Part:BBa_S03623 BBa_T9002 ])
[[Image:T9002_Chiba.gif]]
[[Image:T9002_Chiba.gif]]
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|}
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LuxR mutantsとWT LuxRのディレイタイムはfollowing procedureによってanalyzeした。
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===Method===
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# Transformed sender (Ptet-luxI), mutant LuxR Receiver (Ptet-mLuxR-Plux-GFP) and WT LuxR Receiver ()into E coli strains (BW⊿FliC)
# Transformed sender (Ptet-luxI), mutant LuxR Receiver (Ptet-mLuxR-Plux-GFP) and WT LuxR Receiver ()into E coli strains (BW⊿FliC)
# Inoculated Sender, WT Receiver (wild type luxR/BW⊿FliC) and mutated Receiver (1point mutation/BW⊿FliC) in liquid media for 12 h at 37℃.  
# Inoculated Sender, WT Receiver (wild type luxR/BW⊿FliC) and mutated Receiver (1point mutation/BW⊿FliC) in liquid media for 12 h at 37℃.  
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# Incubated at 30°C.   
# Incubated at 30°C.   
# Measured intensity of green fluorescence at regular time intervals.
# Measured intensity of green fluorescence at regular time intervals.
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===Result===
 
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'''>[[Team:Chiba/Project#Receiver|Back to the project page]]'''
 
===Reference===
===Reference===
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#B.Koch et.al.:The LuxR receptor: the sites of interaction with quorum-sensing signals and inhibitors. Microbiol. 2005 Nov;151(Pt 11):3589-602.
#B.Koch et.al.:The LuxR receptor: the sites of interaction with quorum-sensing signals and inhibitors. Microbiol. 2005 Nov;151(Pt 11):3589-602.
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'''>[[Team:Chiba/Project#Receiver|Back to the project page]]'''
{| style="color:white;background-color:Maroon" cellpadding="3" cellspacing="3" border="1" bordercolor="white" width="100%" align="center"
{| style="color:white;background-color:Maroon" cellpadding="3" cellspacing="3" border="1" bordercolor="white" width="100%" align="center"

Latest revision as of 21:50, 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
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.(1) CollonsらとKockらによって、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)


Experiment

  • Sender
  • Receivers
**[http://partsregistry.org/Part:BBa_S03623 BBa_S03623 (AHL autoinucer)]

LuxI-sender Chiba.gif

  • mutant LuxR Receiver |

Mutant-LuxR-genetic-circuit Chiba.giff]]

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

T9002 Chiba.gif


Method

  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.

Reference

  1. Collins et.al. Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl-homoserine lactones. Mol. Microbiol. 55, 712–723 (2005)
  2. B.Koch et.al.:The LuxR receptor: the sites of interaction with quorum-sensing signals and inhibitors. Microbiol. 2005 Nov;151(Pt 11):3589-602.

>Back to the project page

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