Team:Chiba/Experiments:LuxR mutant
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LuxR mutant (Under Planning)
Design
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)
- 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
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**[http://partsregistry.org/Part:BBa_S03623 BBa_S03623 (AHL autoinucer)] |
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Method
- 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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