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Team:Chiba/System 2/Result
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strain:DH10B<br> | strain:DH10B<br> | ||
sample<br> | sample<br> | ||
| - | 1,Plux inv-GFP and Plac-LuxR <br> | + | 1,Plux inv-GFP and Plac-LuxR(PSB1A3) <br> |
2,Plux inv-GFP (Positive control)<br> | 2,Plux inv-GFP (Positive control)<br> | ||
3,Plux inv (Negative control)<br> | 3,Plux inv (Negative control)<br> | ||
Revision as of 03:29, 28 October 2010
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Version 2 :
Contents |
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 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)
2,Plux inv-GFP (Positive control)
3,Plux inv (Negative control)
add AHL1000 nM (AHL+)and
Each sample is incubated for 12 h at 37゜C After incubation,Spin-dawn(14500rpm,1min) and observed the pellet in the UV.
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.
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
- 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)
