Team:Chiba/Experiments:/LuxR　mutant

From 2008.igem.org

(Difference between revisions)
(→Design)
(→Experiment)
Line 33: Line 33:
===Experiment===
===Experiment===
 +
{| class="tbl" |
 +
|-
 +
|
*'''Sender'''
*'''Sender'''
-
**[http://partsregistry.org/Part:BBa_S03623 BBa_S03623 (AHL auto inucer)]
+
|
 +
*'''Receivers'''
 +
|-
 +
| rowspan="2" | **[http://partsregistry.org/Part:BBa_S03623 BBa_S03623 (AHL autoinucer)]
[[Image:LuxI-sender Chiba.gif]]
[[Image:LuxI-sender Chiba.gif]]
-
 
+
|
-
*'''Receivers'''
+
*mutant LuxR Receiver |
-
**mutant LuxR Receiver
+
[[Image:Mutant-LuxR-genetic-circuit_Chiba.gif]]f]]
-
[[Image:Mutant-LuxR-genetic-circuit_Chiba.gif]]
+
|-
-
 
+
|
-
**WT LuxR Receiver ([http://partsregistry.org/Part:BBa_S03623 BBa_T9002 ])
+
*WT LuxR Receiver ([http://partsregistry.org/Part:BBa_S03623 BBa_T9002 ])
[[Image:T9002_Chiba.gif]]
[[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。
+
===Method===
-
 
+
# Transformed sender (Ptet-luxI), mutant LuxR Receiver (Ptet-mLuxR-Plux-GFP) and WT LuxR Receiver ()into E coli strains (BW⊿FliC)
# 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 Sender, WT Receiver (wild type luxR/BW⊿FliC) and mutated Receiver (1point mutation/BW⊿FliC) in liquid media for 12 h at 37℃.  

Revision as of 20:38, 29 October 2008

Chiba-U.gif

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


LuxR mutant (Under Planning

Design

Receiver switch mLuxR chiba.jpg


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


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
  • Receivers
**[http://partsregistry.org/Part:BBa_S03623 BBa_S03623 (AHL autoinucer)]

LuxI-sender Chiba.gif

  • mutant LuxR Receiver |

Mutant-LuxR-genetic-circuit Chiba.giff]]

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

T9002 Chiba.gif


Method

  1. Transformed sender (Ptet-luxI), mutant LuxR Receiver (Ptet-mLuxR-Plux-GFP) and WT LuxR Receiver ()into E coli strains (BW⊿FliC)
  2. Inoculated Sender, WT Receiver (wild type luxR/BW⊿FliC) and mutated Receiver (1point mutation/BW⊿FliC) in liquid media for 12 h at 37℃.
  3. Inoculated again in liquid media upto about OD600=2 at 37℃
  4. Washed Senders and receiver.
  5. Mixed them. (Sender:Receiver=1000μL:1000μL)
  6. Incubated at 30°C.
  7. Measured intensity of green fluorescence at regular time intervals.

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
Retrieved from "http://2008.igem.org/Team:Chiba/Experiments:/LuxR%E3%80%80mutant"