Team:Chiba/System 2/Result

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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。

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

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)
Cox.R.S.3rd, Surette.M.G, Elowitz.M.B, Programming gene expression with combinatorial promoters, Mol Syst Biol, 3 ,145 (2007)

@@ Line 363: / Line 363: @@
 ===2.Characterization Plux inv-GFP===
-We cotransformed Plux inv-GFP and Plac-LuxR (LuxR generator)by strain of DH10B.
-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,
+strain:DH10B
-スピンダウンしてペレットを観察した。
+sample
-[[Image:lux inverter function.png|frame|center|Fig. 2 characterization]]
+,Plux inv-GFP and Plac-LuxR
+,Plux inv-GFP (Positive control)
+,Plux inv     (Negative control)
+Each sample incubate for 12 h at 37゜C
+Spin-dawn and observed the pellet.
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