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-dependent inverter (Plux inv). Plux inv was 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're not able to confirm LuxR repression.
However,iGEMTokyo_tech2010 also charcterize about Plux inv and succeeded in repressing transcription .

Because it does not completely repress transcription, Plasmids, strains ,culture conditions,and detection method LuxR suppression can not be confirmed(Cox et al,2007).

We use more

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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