Team:Chiba/lux promoter

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Revision as of 22:44, 27 October 2010

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. Plux inv-GFPおよびLuxR generatorであるPlac-LuxR (PSB1A3)を共形質転換した。

得られたコロニーをpre cultureしたあと，AHL100 nM(AHL+)およびAHLなしのLB液体培地 (Amp,Kan) に加えた。

37℃で12 時間培養した

sampleをspindawnし，ペレットの蛍光を確認した。

Results

AHLの有無に関わらず蛍光が観察された。
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)