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Contents 1 Abstract 2 Experiments 3 1.Construction of Plux inv-GFP 4 2.Characterization Plux inv-GFP 5 Results 6 Conclusion 7 Reference

Abstract

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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-dependent inverter (Plux inv). Plux inv was resistered in Biobrick number R0061. We've prepared Plux inv-GFP and characterized about it.

Experiments

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1.Construction of 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.

2.Characterization Plux inv-GFP

strain:DH10B
sample
1,Plux inv-GFP and Plac-LuxR(PSB1A3) AHL0 nM and AHL 1000 nM
2,Plux inv-GFP (Positive control)
3,Plux inv (Negative control)

Each sample is incubated for 12 h at 37゜C After incubation,Spin-dawn(14500rpm,1min) and observed the pellet in the UV.

Results

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

Conclusion

We're not able to confirm LuxR repression.
However,iGEMTokyo_tech2010 team also charcterized about Plux inv and succeeded in repressing transcription .

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

Reference

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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)