Team:Chiba/Receiver experiments
From 2008.igem.org
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*High Copy Receiver[http://partsregistry.org/Part:BBa_T9002 BBa_T9002 (AHL Reporter)] | *High Copy Receiver[http://partsregistry.org/Part:BBa_T9002 BBa_T9002 (AHL Reporter)] | ||
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[[Image:T9002.copy-number-change Chiba.gif]] | [[Image:T9002.copy-number-change Chiba.gif]] | ||
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*Medium Copy Receiver | *Medium Copy Receiver | ||
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[[Image:Low-copy-Receiver Chiba.gif]] | [[Image:Low-copy-Receiver Chiba.gif]] | ||
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===Result=== | ===Result=== |
Revision as of 06:36, 26 October 2008
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Contents |
Receiver crosstalk
Method
Result
Change Receiver's Copy Number
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短縮する。 (小林)
Method
- High Copy Receiver[http://partsregistry.org/Part:BBa_T9002 BBa_T9002 (AHL Reporter)]
- Medium Copy Receiver
Result
LuxR mutants
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。 (小林)
Method
- 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.
Result
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