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Team:Chiba/Receiver experiments
From 2008.igem.org
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Receiver crosstalk
Design
[[Image:Table R chiba.gif|frame|left|Table.1 LuxR family gene
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)]
- Medium Copy Receiver
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。
- 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).
- Inoculated them independently in liquid media. Incubated at 37℃ 12h.
- Inoculated again in Fresh liquid media upto about OD600=2 at 37℃
- Washed sender and receivers.
- Mixed them. (Sender:Receiver=1000μL:1000μL)
- Incubated at 30°C.
- Measured intensity of green fluorescence at regular time intervals.(Fluoroskan AscentR FL＆Fluoroskan AscentR Thermo ELECTRON CORPORATION)
Result
LuxR mutants
Fig.6 Location of the LuxR mutant in LuxR
Table.1 Seisitivity of LuxR mutants. Data modified from Collins et.al. Mol. Microbiol. 55, 712–723 (2005)
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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