Team:Chiba/Experiments:LuxR mutant

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::::[[Image:Receiver switch mLuxR chiba.jpg|left|frame|'''Fig.　1 LuxR mutant''']]
::::[[Image:Receiver switch mLuxR chiba.jpg|left|frame|'''Fig.　1 LuxR mutant''']]
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[[Image:LuxR I46F chiba.png|thumb|'''Fig. 3''' I45F mutation]]
[[Image:LuxR I46F chiba.png|thumb|'''Fig. 3''' I45F mutation]]
[[Image:LuxR L42S chiba.png|thumb|right|'''Fig. 4''' L42S mutation]]
[[Image:LuxR L42S chiba.png|thumb|right|'''Fig. 4''' L42S mutation]]
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#L42S(Fig. 4)とするとAHLに対する感受性が1/1000に減少する[[Team:Chiba/Experiments:LuxR mutant#Reference|<sup>(2)</sup>]]
#L42S(Fig. 4)とするとAHLに対する感受性が1/1000に減少する[[Team:Chiba/Experiments:LuxR mutant#Reference|<sup>(2)</sup>]]
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Fig. 3, Fig. 4　
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Fig. 3, Fig. 4　
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Hypothetical positions of residues in TraR corresponding to those found to modulate  
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Hypothetical positions of residues in TraR corresponding to those found to modulate  
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acyl-HSL specificity in LuxR.　The crystal　structure of the LuxR homologue TraR  
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acyl-HSL specificity in LuxR.　The crystal　structure of the LuxR homologue TraR  
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(PDB 1L3L) has been determined　(Zhang et al., 2002).
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(PDB 1L3L) has been determined　(Zhang et al., 2002).
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Revision as of 02:55, 30 October 2008

Chiba-U.gif

!align="center"|Home !align="center"|The Team !align="center"|The Project !align="center"|Parts Submitted to the Registry !align="center"|Reference !align="center"|Notebook !align="center"|Acknowledgements |}


LuxR mutant (Under Planning)

Design

Fig.　1 LuxR mutant
Fig. 3 I45F mutation
Fig. 4 L42S mutation

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. I45F(Fig. 3)とするとAHLに対する感受性が約10倍上昇する(1)
  2. L42AとするとAHLに対する感受性が1/15に減少する(2)
  3. L42S(Fig. 4)とするとAHLに対する感受性が1/1000に減少する(2)

Fig. 3, Fig. 4　 Hypothetical positions of residues in TraR corresponding to those found to modulate acyl-HSL specificity in LuxR.　The crystal　structure of the LuxR homologue TraR (PDB 1L3L) has been determined　(Zhang et al., 2002).

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

Mutant-LuxR-genetic-circuit Chiba.gif

  • 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

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