Team:Chiba/Receiver experiments

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===Result===
===Result===
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==LuxR mutants==
 
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===Design===
 
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[[Image:Location-of-the-mutant-in-LuxR Chiba.gif|frame|right|'''Fig.6''' Location of the LuxR mutant in LuxR]]
 
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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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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'''
 
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**[http://partsregistry.org/Part:BBa_S03623 BBa_S03623 (AHL auto inucer)]
 
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[[Image:LuxI-sender Chiba.gif]]
 
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*'''Receivers'''
 
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**mutant LuxR Receiver
 
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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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[[Image:T9002_Chiba.gif]]
 
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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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# Transformed sender (Ptet-luxI), mutant LuxR Receiver (Ptet-mLuxR-Plux-GFP) and WT LuxR Receiver ()into E coli strains (BW⊿FliC)
 
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# 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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# Inoculated again in liquid media upto about OD600=2 at 37℃
 
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# Washed Senders and receiver.
 
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# Mixed them. (Sender:Receiver=1000μL:1000μL)
 
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# Incubated at 30°C. 
 
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# Measured intensity of green fluorescence at regular time intervals.
 
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===Result===
 
'''>[[Team:Chiba/Project#Receiver|Back to the project page]]'''
'''>[[Team:Chiba/Project#Receiver|Back to the project page]]'''

Latest revision as of 19:28, 29 October 2008

Chiba-U.gif

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Contents

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

>Back to the project page

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