Team:Chiba/Experiments:LuxR mutant

From 2008.igem.org

(Difference between revisions)
(Method)
(LuxR mutant (Under Planning))
Line 21: Line 21:
| width="50%" valign="top"|
| width="50%" valign="top"|
-
[[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)]]
+
[[Image:Mutant-LuxR Chiba.gif|frame|right|'''Fig.1''' Seisitivity of LuxR mutants. Data modified from Collins et.al. Mol. Microbiol. 55, 712–723 (2005)]]
-
[[Image:AHL-activation-of-mutagenizenized LuxR molecules Chiba.gif|frame|right|'''Table. '''Ahl activation of mutagenized LuxR molecules. '''Data modfied from B.Kock et al. Microbiology (2005), 151, 3589-3602''']]
+
[[Image:AHL-activation-of-mutagenizenized LuxR molecules Chiba.gif|frame|right|'''Fig.2''' Ahl activation of mutagenized LuxR molecules. '''Data modfied from B.Kock et al. Microbiology (2005), 151, 3589-3602''']]
| width="25%" valign="top"|
| width="25%" valign="top"|
Line 57: Line 57:
|
|
*mutant LuxR Receiver  
*mutant LuxR Receiver  
-
[[Image:Mutant-LuxR-genetic-circuit_Chiba.gif]]
+
[[Image:Mutant-LuxR-genetic-circuit_Chiba.gif|frame|'''Fig.3''' ]]
|-
|-
|  
|  

Revision as of 01:46, 30 October 2008

Chiba-U.gif


LuxR mutant (Under Planning)

Design

Receiver switch mLuxR chiba.jpg
Fig.1 Seisitivity of LuxR mutants. Data modified from Collins et.al. Mol. Microbiol. 55, 712–723 (2005)
Fig.2 Ahl activation of mutagenized LuxR molecules. Data modfied from B.Kock et al. Microbiology (2005), 151, 3589-3602

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)

  1. Ile45PheとするとAHLに対する感受性が約10倍上昇する(1)
  2. Leu42AlaとするとAHLに対する感受性が1/15に減少する(2)
  3. Leu42SerとするとAHLに対する感受性が1/1000に減少する(2)


Experiment

Sender; LuxI plasmid transformed into E.coli strains (JW1908) Receiver; LuxR-gfp plasmid transformed into E.coli strains (JW1908)

Culture/ Cndn.
  1. Both Sender and Receiver (+/- Mutation) were inoculated into small (2mL) culture and was shaken separately for 12h (at 37C)
  2. Inoculated into flesh media, shaken until cell density hit 2.0 in OD600
  3. Washed the cell and re-suspended. Cell density checked.
  4. Mixed Sender and Receiver (Sender/Receiver 1000μl/1000μl).
  5. Incubated at 30°C.
  6. Time-chased the fluorescence (485nm(excitation) and 527nm(emission)) by gfp.
  • Sender
  • Receivers
[http://partsregistry.org/Part:BBa_S03623 BBa_S03623]

LuxI-sender Chiba.gif

  • mutant LuxR Receiver
Fig.3
  • WT LuxR Receiver ([http://partsregistry.org/Part:BBa_S03623 BBa_T9002 ])

High-Copy-Receiver Chiba.gif


Reference

  1. 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)
  2. 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.

>Back to the project page

Home The Team The Project Parts Submitted to the Registry Notebook