Team:Chiba/System 2/Result
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+ | <li><a href="https://2010.igem.org/Team:Chiba/Project"><span>Double Click System</span></a></li> | ||
+ | <li><a href="https://2010.igem.org/Team:Chiba/System_1"><span>Version 1</span></a></li> | ||
+ | <li><a href="https://2010.igem.org/Team:Chiba/System_2"><span>Version 2</span></a></li> </ul> | ||
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+ | <!--- 2nd tab End---> | ||
+ | <!---Main Contents Start ---> | ||
+ | <br><br> | ||
+ | [[Team:Chiba/System 2|<<Back]]<br><br> | ||
+ | <font size=6>Version 2 :</font> | ||
+ | ===<font size="5">Abstract</font>=== | ||
+ | ----- | ||
+ | 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>=== | ||
+ | ----- | ||
+ | |||
+ | ===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. | ||
[[Image:Plux inv-GFP.png|none|frame|right|'''Fig. 1''' Evaluation of LuxR Inverter]] | [[Image:Plux inv-GFP.png|none|frame|right|'''Fig. 1''' Evaluation of LuxR Inverter]] | ||
===2.Characterization Plux inv-GFP=== | ===2.Characterization Plux inv-GFP=== | ||
- | |||
- | |||
- | |||
- | ==Results== | + | strain:DH10B<br> |
- | [[Image:Plux inv results.png| | + | sample<br> |
+ | 1,Plux inv-GFP and Plac-LuxR(PSB1A3) AHL0 nM and AHL 1000 nM<br> | ||
+ | 2,Plux inv-GFP (Positive control)<br> | ||
+ | 3,Plux inv (Negative control)<br> | ||
+ | |||
+ | |||
+ | |||
+ | 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]] | ||
+ | <br><br><br> | ||
+ | |||
+ | ===<font size="5">Results</font>=== | ||
+ | ----- | ||
+ | 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|thumb|left|Fig. 3 ]] | ||
+ | |||
+ | ===Conclusion=== | ||
+ | |||
+ | We're not able to confirm LuxR repression.<br> | ||
+ | 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> | ||
- | ==Reference== | + | ===<font size="5">Reference</font>=== |
+ | ----- | ||
#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) | #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) | #Cox.R.S.3rd, Surette.M.G, Elowitz.M.B, Programming gene expression with combinatorial promoters, ''Mol Syst Biol'', '''3''' ,145 (2007) |
Latest revision as of 08:00, 18 November 2010
<<Back
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)