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Team:Chiba/Receiver experiments
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==LuxR mutants== | ==LuxR mutants== | ||
[[Image:Location-of-the-mutant-in-LuxR Chiba.gif|frame|right|'''Fig.6''' Location of the LuxR mutant in LuxR]] | [[Image:Location-of-the-mutant-in-LuxR Chiba.gif|frame|right|'''Fig.6''' Location of the LuxR mutant in LuxR]] | ||
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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。 | ||
===method=== | ===method=== | ||
| + | LuxR mutantsとWT LuxRのディレイタイムはfollowing procedureによってanalyzeした。 | ||
| + | # 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 again in liquid media upto about OD600=2 at 37℃ | ||
| + | # Washed Senders and receiver. | ||
| + | # Mixed them. (Sender:Receiver=1000μL:1000μL) | ||
| + | # Incubated at 30°C. | ||
| + | # Measured intensity of green fluorescence at regular time intervals. | ||
===result=== | ===result=== | ||
Revision as of 05:42, 25 October 2008
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Project Design ( Sender experiments | Receiver experiments ) | Our Goal |
Contents |
LuxR mutants
Fig.6 Location of the LuxR mutant in LuxR
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。
method
LuxR mutantsとWT LuxRのディレイタイムはfollowing procedureによってanalyzeした。
- 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 again in liquid media upto about OD600=2 at 37℃
- Washed Senders and receiver.
- Mixed them. (Sender:Receiver=1000μL:1000μL)
- Incubated at 30°C.
- Measured intensity of green fluorescence at regular time intervals.
result
AiiA Receiver
Fig.6 AiiA Receiver
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。
method
- Sender
- [http://partsregistry.org/Part:BBa_S03623 BBa_S03623 (AHL auto inucer)]
- Receivers
- AiiA Receiver
- non-AiiA Receiver
result
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