Team:Chiba/System 2/Result

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<li><a href="https://2010.igem.org/Team:Chiba/System_2/Testing_individual_parts"><span>Testing</span></a></li>
 
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<li><a href="https://2010.igem.org/Team:Chiba/System_2/Construction_Process"><span>Construction</span></a></li>
 
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<li><a href="https://2010.igem.org/Team:Chiba/System_2/Evaluation_subsystem"><span>Evaluation</span></a></li>
 
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<li><a href="https://2010.igem.org/Team:Chiba/System_2/Result"><span>Result</span></a></li>
 
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<li><a href="https://2010.igem.org/Team:Chiba/System_2/Remarks"><span>Remarks</span></a></li>
 
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<br><br><br><br><br><br>
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<br><br>
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[[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-inverter (Plux inv). Plux inv is resistered in Biobrick number R0061.We've prepared Plux inv-GFP and characterized about it.<br><br><br>
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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>
===<font size="5">Experiments</font>===
===<font size="5">Experiments</font>===
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===1.Construction Plux inv-GFP===
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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.
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===
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strain:DH10B
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strain:DH10B<br>
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sample
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sample<br>
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1,Plux inv-GFP and Plac-LuxR <br>
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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>
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add AHL1000 nM (AHL+)and
 
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Each sample incubated for 12 h at 37゜C  
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Spin-dawn and observed the pellet.
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Each sample is incubated for 12 h at 37゜C  
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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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===<font size="5">Results</font>===
===<font size="5">Results</font>===
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Pellet Photos is shown in'''Fig. 3'''
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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.
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[[Image:Plux inv results.png|none|right|Fig. 3 Pellet photos of the incubeted sample]]
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[[Image:Plux inv results.png|thumb|left|Fig. 3 ]]
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===Discussion===
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===Conclusion===
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We can't confirm LuxR repression.私たちは，LuxRの抑制を確認することができなかった。
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We're not able to confirm LuxR repression.<br>
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原因として，私たちの実験レポータ遺伝子
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However,''iGEMTokyo_tech2010'' team also charcterized about '''Plux inv''' and succeeded in repressing transcription .<br>
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Coxらの論文でもlux box を-35および-10の間に挿入しても，抑制効果がないことが示されている。
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<br><br><br>
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It does not completely repress transcription, Plasmids, strains ,culture conditions,and detection method LuxR suppression can not be confirmed (Cox et al,2007).<br>
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<br>
===<font size="5">Reference</font>===
===<font size="5">Reference</font>===

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.

Fig. 1 　Evaluation of LuxR Inverter

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.

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

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

  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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