Team:Chiba/System 2/Result

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(2.Characterization Plux inv-GFP)
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===2.Characterization Plux inv-GFP===
===2.Characterization Plux inv-GFP===
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We cotransformed Plux inv-GFP and Plac-LuxR (LuxR generator)by strain of DH10B.
 
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ロニーをつついてLB液体培地(AHL0および1000 nM)で12 h培養し,
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strain:DH10B
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スピンダウンしてペレットを観察した。
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sample
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[[Image:lux inverter function.png|frame|center|Fig. 2 characterization]]
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1,Plux inv-GFP and Plac-LuxR
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2,Plux inv-GFP (Positive control)
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3,Plux inv    (Negative control)
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Each sample incubate for 12 h at 37゜C
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Spin-dawn and observed the pellet.
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Revision as of 00:19, 28 October 2010




 

 

 







Version 2 :

Contents

Abstract


LuxR is AHL-dependent activator.LuxR-AHL complex binds lux box, 20-bp sequence centered at position -42.5 from starting site and activates transcription.However lux box is inserted between -35 and -10,LuxR functions as AHL-inverter (Plux inv). Plux inv is resistered in Biobrick number R0061.We've prepared Plux inv-GFP and characterized about it.


Experiments


1.Construction Plux inv-GFP

Construction process is shown in Fig. 1.We constructed Plux inv-GFP conbining R0061 and E0240 and transformed by strain of XL10-G.

GFP fluorescence and sequence is confirmed.

Fig. 1  Evaluation of LuxR Inverter

2.Characterization Plux inv-GFP

strain:DH10B sample 1,Plux inv-GFP and Plac-LuxR 2,Plux inv-GFP (Positive control) 3,Plux inv (Negative control)


Each sample incubate for 12 h at 37゜C Spin-dawn and observed the pellet.




Results


細胞のペレット写真をFig. 3に示す。両方のサンプルから蛍光が確認された。

Fig. 3

Discussion

We can't confirm LuxR repression.私たちは,LuxRの抑制を確認することができなかった。 原因として,私たちの実験レポータ遺伝子 Coxらの論文でもlux box を-35および-10の間に挿入しても,抑制効果がないことが示されている。


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)