Team:Chiba/Receiver experiments

From 2008.igem.org

(Difference between revisions)

Latest revision as of 19:28, 29 October 2008

Home	The Team	The Project	Parts Submitted to the Registry	Reference	Notebook	Acknowledgements

Receiver crosstalk

Design

Table.1 LuxR family gene

センダーとレシーバーを元来とは異なる組み合わせにすることによって、thresholdに達する時間を遅くする。（杉山）

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

>Back to the project page

Home	The Team	The Project	Parts Submitted to the Registry	Notebook

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 !align="center"|[[Team:Chiba/Project|The Project]]
 !align="center"|[[Team:Chiba/Parts|Parts Submitted to the Registry]]
+!align="center"|[[Team:Chiba/Reference|Reference]]
 !align="center"|[[Team:Chiba/Notebook|Notebook]]
+!align="center"|[[Team:Chiba/Acknowledgements|Acknowledgements]]
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-{| style="border:0;width:100%;font-family:'Trebuchet MS'" cellpadding="20px" cellspacing="0"
+==Receiver crosstalk==
-| align="center" |
-<span style="font-size:120%;font-weight:bold;">[[Team:Chiba/Project|Project Design]] ( [[Team:Chiba/Sender_experiments|Sender experiments]] | [[Team:Chiba/Receiver_experiments|Receiver experiments]] )  |  [[Team:Chiba/Goal|Our Goal]]</span><br>
-|}
-==LuxR mutants==
-[[Image:Location-of-the-mutant-in-LuxR Chiba.gif|frame|right|'''Fig.6''' Location of the LuxR mutant in LuxR]]
-[[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)]]
+===Design===
+[[Image:Table R chiba.gif|frame|left|Table.1 LuxR family gene]]
+センダーとレシーバーを元来とは異なる組み合わせにすることによって、thresholdに達する時間を遅くする。（杉山）
-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))
+===Method===
-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。
+===Result===
-===method===
+==Change Receiver's Copy Number==
-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==
+===Design===
-[[Image:AiiA-Receiver Chiba.gif|frame|right|'''Fig.6''' AiiA Receiver]]
+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===
-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。
+*High Copy Receiver[http://partsregistry.org/Part:BBa_T9002 BBa_T9002 (AHL Reporter)]
+[[Image:T9002.copy-number-change Chiba.gif]]
+*Medium Copy Receiver
-===method===
+[[Image:Low-copy-Receiver Chiba.gif]]
-*'''Sender'''
-**[http://partsregistry.org/Part:BBa_S03623 BBa_S03623 (AHL auto inucer)]
-[[Image:LuxI-sender Chiba.gif]]
+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。
-*'''Receivers'''
+#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).
-**AiiA Receiver
+#Inoculated them independently in liquid media. Incubated at 37℃ 12h.
-[[Image:AiiA-Receiver-genetic-circu Chiba.gif]]
+#Inoculated again in Fresh liquid media upto about OD600=2 at 37℃
-**non-AiiA Receiver
+#Washed sender and receivers.
-[[Image:Non-AiiA-Receiver Chiba.gif]]
+#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===
+===Result===
+'''>[[Team:Chiba/Project#Receiver|Back to the project page]]'''
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Team:Chiba/Receiver experiments

From 2008.igem.org

Latest revision as of 19:28, 29 October 2008

Contents

Receiver crosstalk

Design

Method

Result

Change Receiver's Copy Number

Design

Method

Result