Team:Chiba/Experiments:LuxR mutant

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Revision as of 02:48, 30 October 2008

LuxR mutant (Under Planning)

Design

Fig.　1 LuxR mutant

Fig. 2 Seisitivity of LuxR mutants. Data modified from Collins et.al. Mol. Microbiol. 55, 712–723 (2005)

Fig.4.3 Ahl activation of mutagenized LuxR molecules. Data modfied from B.Kock et al. Microbiology (2005), 151, 3589-3602

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)

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

Both Sender and Receiver (+/- Mutation) were inoculated into small (2mL) culture and was shaken separately for 12h (at 37C)
Inoculated into flesh media, shaken until cell density hit 2.0 in OD600
Washed the cell and re-suspended. Cell density checked.
Mixed Sender and Receiver (Sender/Receiver 1000μl/1000μl).
Incubated at 30°C.
Time-chased the fluorescence (485nm(excitation) and 527nm(emission)) by gfp.

Sender

Receivers

[http://partsregistry.org/Part:BBa_S03623 BBa_S03623]

mutant LuxR Receiver

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

Reference

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)
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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@@ Line 18: / Line 18: @@
 | width="50%" valign="top" |
-::::[[Image:Receiver switch mLuxR chiba.jpg|left|frame|'''Fig.4.1 LuxR mutant''']]
+::::[[Image:Receiver switch mLuxR chiba.jpg|left|frame|'''Fig.　1 LuxR mutant''']]
 | width="50%" valign="top"|
-[[Image:Mutant-LuxR Chiba.gif|frame|right|'''Fig. 4.2'''  Seisitivity of LuxR mutants. Data modified from Collins et.al. Mol. Microbiol. 55, 712–723 (2005)]]
+[[Image:Mutant-LuxR Chiba.gif|frame|right|'''Fig. 2'''  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|'''Fig.4.3''' Ahl activation of mutagenized LuxR molecules. '''Data modfied from B.Kock et al. Microbiology (2005), 151, 3589-3602''']]
@@ Line 27: / Line 27: @@
 |}
-[[Image:LuxR I46F chiba.png|thumb|right|'''Fig. 4.3''' Ile45Phe mutation]]
+[[Image:LuxR I46F chiba.png|thumb|right|'''Fig. 3''' I45F mutation]]
-[[Image:LuxR L42S chiba.png|thumb|right|'''Fig. 4.4''' Leu45Ser mutation]]
+[[Image:LuxR L42S chiba.png|thumb|right|'''Fig. 4''' L42S mutation]]
 Collins et.al. described the hyper-sensitive variants of luxR to AHL.[[Team:Chiba/Experiments:LuxR mutant#Reference|<sup>(1)</sup>]]
 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。[[Team:Chiba/Experiments:LuxR mutant#Reference|<sup>(1),(2)</sup>]]
-#Ile45Phe(Fig. 4.3)とするとAHLに対する感受性が約10倍上昇する[[Team:Chiba/Experiments:LuxR mutant#Reference|<sup>(1)</sup>]]
+#I45F(Fig. 3)とするとAHLに対する感受性が約10倍上昇する[[Team:Chiba/Experiments:LuxR mutant#Reference|<sup>(1)</sup>]]
-#Leu42AlaとするとAHLに対する感受性が1/15に減少する[[Team:Chiba/Experiments:LuxR mutant#Reference|<sup>(2)</sup>]]
+#L42AとするとAHLに対する感受性が1/15に減少する[[Team:Chiba/Experiments:LuxR mutant#Reference|<sup>(2)</sup>]]
-#Leu42Ser(Fig. 4.4)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>]]
+ 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).
 <br clear=all>