Team:Chiba/lux promoter

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  <td width="800px"><font size="5" face=verdana>Lux promoter inverter</font><br>
 
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We've charaterized Plux inverter.<br>
 
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There is promoter (HSL-luxR repressor), biobrick number R0061.<br>
 
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We've made Plux inv-GFP and characterized about it.<br><br>
 
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==Abstract==
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LuxR is an AHL-dependent activator.LuxR-AHL complex bind lux box,a 20-bp sequence and activate transcription.  However the lux box is inserted between -35 and -10 and LuxR functions as LuxR-AHL inverter (Plux inv) .The Plux inv was resistered in Biobrick number R0061.We've constructed Plux inv-GFP combinig R0061and E0240 and characterized about it.<br>
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==Experiments==
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===1.Preparing Plux inv-GFP===
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Plasmid:R0061 and E0240<br>
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<font size="3">1,Preparing Plux inx-GFP</font>&nbsp;&nbsp;&nbsp;</font><br><br>
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Strain:XL10-G<br>
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&nbsp;&nbsp;&nbsp;
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protocol<br>
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cloning process is shown in Fig. 1<br>
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Preparing process of Plux inv-GFP  is shown in '''Fig. 1'''.We transformed preparing Plux inv-GFP in XL10-G and confirmed the expression GFP Fluorescence.The sequence is also conformed.
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[[Image:Plux inv-GFP.png|none|frame|right|'''Fig. 1''' 　Evaluation of LuxR Inverter]]
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===2.Characterization Plux inv-GFP===
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[[Image:Plux inv-GFP.png|frame|left|Fig. 1 cloning process]]
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<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
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Plasmid:Plux inv-GFP and Plac-LuxR (LuxR constitutive generator)<br>
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<br><br><br><br><br><br><br><br>
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strain:DH10B<br>
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protocol<br>  
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We cotransformded Plux inv-GFP and LuxR Plac-LuxR in DH10B.
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  <td width="800px"><font size="5" face=verdana>Results</font><br>
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1,LBL
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incubation for12 h at 37゜C.
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<font size="3">1,Preparing Plux inx-GFP</font>&nbsp;&nbsp;&nbsp;</font><br><br>
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&nbsp;&nbsp;&nbsp;
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==Results==
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cloning process is shown in Fig. 1<br>
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</font></td> 
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We confirmed GFP Fluorescence both AHL+ and -sample.<br>
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[[Image:Plux inv results.png|frame|none|Fig. 3 ]]
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[[Image:lux inverter function.png|frame|left|Fig. 2 assay process]]
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==discussion==
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<br><br><br><br><br><br><br><br><br><br><br><br>
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私たちの系では，LuxRの抑制を確認することができなかった。
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<br>1) Egland.K.A, and Greenberg.E.P, Conversion of the ''Vibrio Fischeri'' Transcriptional Activator,LuxR, to a Repressor, ''J. Bacteriol.'', '''182''', P.805-811 (2000)
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<br>2) Cox.R.S.3rd, Surette.M.G, Elowitz.M.B, Programming gene expression with combinatorial promoters, ''Mol Syst Biol'', '''3''' ,145 (2007)
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==Reference==
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#Egland.K.A, and Greenberg.E.P, Conversion of the ''Vibrio Fischeri'' Transcriptional Activator,LuxR, to a Repressor, ''J. Bacteriol.'', '''182''', P.805-811 (2000)
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#Cox.R.S.3rd, Surette.M.G, Elowitz.M.B, Programming gene expression with combinatorial promoters, ''Mol Syst Biol'', '''3''' ,145 (2007)

Latest revision as of 13:29, 24 January 2011




Contents

Abstract

LuxR is an AHL-dependent activator.LuxR-AHL complex bind lux box,a 20-bp sequence and activate transcription. However the lux box is inserted between -35 and -10 and LuxR functions as LuxR-AHL inverter (Plux inv) .The Plux inv was resistered in Biobrick number R0061.We've constructed Plux inv-GFP combinig R0061and E0240 and characterized about it.

Experiments

1.Preparing Plux inv-GFP

Plasmid:R0061 and E0240
Strain:XL10-G
protocol
Preparing process of Plux inv-GFP is shown in Fig. 1.We transformed preparing Plux inv-GFP in XL10-G and confirmed the expression GFP Fluorescence.The sequence is also conformed.

Fig. 1 　Evaluation of LuxR Inverter

2.Characterization Plux inv-GFP

Plasmid:Plux inv-GFP and Plac-LuxR (LuxR constitutive generator)
strain:DH10B

protocol
We cotransformded Plux inv-GFP and LuxR Plac-LuxR in DH10B. 1,LBL incubation for12 h at 37゜C.

Results

We confirmed GFP Fluorescence both AHL+ and -sample.

Fig. 3

discussion

私たちの系では，LuxRの抑制を確認することができなかった。

Reference

  1. Egland.K.A, and Greenberg.E.P, Conversion of the Vibrio Fischeri Transcriptional Activator,LuxR, to a Repressor, J. Bacteriol., 182, P.805-811 (2000)
  2. Cox.R.S.3rd, Surette.M.G, Elowitz.M.B, Programming gene expression with combinatorial promoters, Mol Syst Biol, 3 ,145 (2007)
Retrieved from "http://2010.igem.org/Team:Chiba/lux_promoter"