Team:Chiba/System 2/Result

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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)
add AHL1000 nM (AHL+)and

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

Fig. 2 characterization of Plux inv-GFP




Results

Pellet Photos is shown in Fig. 3.We confirmed GFP fluorescence both AHL+ and AHL-.So We're not able to confirm LuxR repression.

Fig. 3 Pellet photos of the incubeted sample

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)
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