Team:Chiba/System 2/Result
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- | [[Team:Chiba/System 2|<<Back]]<br> | + | [[Team:Chiba/System 2|<<Back]]<br><br> |
<font size=6>Version 2 :</font> | <font size=6>Version 2 :</font> | ||
===<font size="5">Abstract</font>=== | ===<font size="5">Abstract</font>=== | ||
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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.<br><br><br> | + | 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.<br><br><br> |
===<font size="5">Experiments</font>=== | ===<font size="5">Experiments</font>=== | ||
----- | ----- | ||
- | ===1.Construction Plux inv-GFP=== | + | ===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. | Construction process is shown in '''Fig. 1'''.We constructed Plux inv-GFP conbining R0061 and E0240 and transformed by strain of XL10-G. | ||
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===2.Characterization Plux inv-GFP=== | ===2.Characterization Plux inv-GFP=== | ||
- | strain:DH10B | + | strain:DH10B<br> |
- | sample | + | sample<br> |
- | 1,Plux inv-GFP and Plac-LuxR <br> | + | 1,Plux inv-GFP and Plac-LuxR(PSB1A3) AHL0 nM and AHL 1000 nM<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> | ||
- | |||
- | Each sample incubated for 12 h at 37゜C | + | |
- | Spin-dawn and observed the pellet. | + | |
+ | Each sample is incubated for 12 h at 37゜C | ||
+ | After incubation,Spin-dawn(14500rpm,1min) and observed the pellet in the UV. | ||
[[Image:Plux inv function.png|none|frame|Fig. 2 characterization of Plux inv-GFP]] | [[Image:Plux inv function.png|none|frame|Fig. 2 characterization of Plux inv-GFP]] | ||
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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. | Pellet Photos is shown in '''Fig. 3'''.We confirmed GFP fluorescence both AHL+ and AHL-.So We're not able to confirm LuxR repression. | ||
- | [[Image:Plux inv results.png| | + | [[Image:Plux inv results.png|thumb|left|Fig. 3 ]] |
- | === | + | ===Conclusion=== |
We're not able to confirm LuxR repression.<br> | We're not able to confirm LuxR repression.<br> | ||
- | However,''iGEMTokyo_tech2010'' also | + | However,''iGEMTokyo_tech2010'' team also charcterized about '''Plux inv''' and succeeded in repressing transcription .<br> |
- | + | It does not completely repress transcription, Plasmids, strains ,culture conditions,and detection method LuxR suppression can not be confirmed (Cox et al,2007).<br> | |
- | <br> | + | <br> |
===<font size="5">Reference</font>=== | ===<font size="5">Reference</font>=== |
Latest revision as of 08:00, 18 November 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) 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
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)