Team:Chiba/Receiver experiments

From 2008.igem.org

(Difference between revisions)
(Method)
(LuxR mutants)
 
(19 intermediate revisions not shown)
Line 10: Line 10:
!align="center"|[[Team:Chiba/Acknowledgements|Acknowledgements]]
!align="center"|[[Team:Chiba/Acknowledgements|Acknowledgements]]
|}
|}
-
 
==Receiver crosstalk==
==Receiver crosstalk==
 +
 +
===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)
===Method===
===Method===
Line 19: Line 22:
==Change Receiver's Copy Number==
==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。
+
 
 +
===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)
(小林)
===Method===
===Method===
-
**[http://partsregistry.org/Part:BBa_T9002 BBa_T9002 (AHL Reporter)]
+
*High Copy Receiver[http://partsregistry.org/Part:BBa_T9002 BBa_T9002 (AHL Reporter)]
 +
 
[[Image:T9002.copy-number-change Chiba.gif]]
[[Image:T9002.copy-number-change Chiba.gif]]
-
[[Image:Low-copy-Receiver Chiba.gif]]
 
-
===Result===
+
*Medium Copy Receiver
-
==LuxR mutants==
+
[[Image:Low-copy-Receiver Chiba.gif]]
-
[[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)]]
+
-
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))
+
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。
-
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。
+
#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)
-
===Method===
+
===Result===
-
*'''Sender'''
+
-
**[http://partsregistry.org/Part:BBa_S03623 BBa_S03623 (AHL auto inucer)]
+
-
[[Image:LuxI-sender Chiba.gif]]
+
-
*'''Receivers'''
 
-
**mutant LuxR Receiver
 
-
[[Image:Mutant-LuxR-genetic-circuit_Chiba.gif]]
 
-
 
-
**WT LuxR Receiver ([http://partsregistry.org/Part:BBa_S03623 BBa_T9002 ])
 
-
[[Image:T9002_Chiba.gif]]
 
-
 
-
 
-
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===
 
'''>[[Team:Chiba/Project#Receiver|Back to the project page]]'''
'''>[[Team:Chiba/Project#Receiver|Back to the project page]]'''

Latest revision as of 19:28, 29 October 2008

Chiba-U.gif

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

Contents

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)]

T9002.copy-number-change Chiba.gif


  • Medium Copy Receiver

Low-copy-Receiver 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。

  1. 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).
  2. Inoculated them independently in liquid media. Incubated at 37℃ 12h.
  3. Inoculated again in Fresh liquid media upto about OD600=2 at 37℃
  4. Washed sender and receivers.
  5. Mixed them. (Sender:Receiver=1000μL:1000μL)
  6. Incubated at 30°C.
  7. 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