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Team:Chiba/Experiments:/LuxR mutant
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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)]] | ||
| - | [[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''']] | + | [[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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#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>]] | #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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===Experiment=== | ===Experiment=== | ||
Revision as of 20:36, 29 October 2008
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LuxR mutant (Under Planning
Design
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
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)
- Ile45PheとするとAHLに対する感受性が約10倍上昇する(1)
- Leu42AlaとするとAHLに対する感受性が1/15に減少する(2)
- Leu42SerとするとAHLに対する感受性が1/1000に減少する(2)
Experiment
- Sender
- [http://partsregistry.org/Part:BBa_S03623 BBa_S03623 (AHL auto inucer)]
- Receivers
- mutant LuxR Receiver
- WT LuxR Receiver ([http://partsregistry.org/Part:BBa_S03623 BBa_T9002 ])
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.
Reference
- 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)
- 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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