Team:Chiba/Experiments:LuxR mutant

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Revision as of 02:58, 30 October 2008

LuxR mutant (Under Planning)

Design

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 I45F mutation

Fig. 4 L42S mutation

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.

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.

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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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