Team:Chiba/System 2/Result

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<li><a href="https://2010.igem.org/Team:Chiba/Project"><span>Overall project</span></a></li>
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<li><a href="https://2010.igem.org/Team:Chiba/Project"><span>Double Click System</span></a></li>
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<li><a href="https://2010.igem.org/Team:Chiba/System_1"><span>System 1</span></a></li>
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<li><a href="https://2010.igem.org/Team:Chiba/System_1"><span>Version 1</span></a></li>
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<li><a href="https://2010.igem.org/Team:Chiba/System_2"><span>System 2</span></a></li>                       </ul>
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<li><a href="https://2010.igem.org/Team:Chiba/System_2"><span>Version 2</span></a></li>                   </ul>
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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>
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<br><br>
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[[Team:Chiba/System 2|<<Back]]<br><br>
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<font size=6>Version 2 :</font>
===<font size="5">Abstract</font>===
===<font size="5">Abstract</font>===
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BBa_R0061は-35および-10の間にlux box を含む配列である。継続的に下流遺伝子の転写を行う。しかしながら，LuxR-AHL複合体によって下流の遺伝子転写を抑制する。<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>
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私たちは，Plux invの下流にRBSおよびGFP遺伝子を挿入したプラスミド (Plux inv-GFP) を作製し，抑制機能を確認した。<br>
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Biobrick number R0061 contains lux box between -35 and -10 and fuctions LuxR-AHL inverter.
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We've prepare Plux inv-GFP and characterized about it.
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<br><br><br>
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===<font size="5">Experiments</font>===
===<font size="5">Experiments</font>===
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===1.Preparing Plux inv-GFP===
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===1.Construction of Plux inv-GFP===
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1．Plux inv－GFPの作製
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クローニングのprocessをFig. 1に示す。作製したPlux inv-GFPを形質転換した株は緑色蛍光を示した。また，sequenceも正しいことを確認した。
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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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Preparing Plux inv-GFP process is shown in '''Fig. 1'''.
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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===
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Plux inv-GFP およびLuxR generatorである Plac-LuxRをDH10B株に共形質転換した。コロニーをつついてLB液体培地(AHL0および1000 nM)で12 h培養し,
 
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スピンダウンしてペレットを観察した。
 
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[[Image:lux inverter function.png|frame|center|Fig. 2 assay protocol]]
 
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strain:DH10B<br>
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sample<br>
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1,Plux inv-GFP and Plac-LuxR(PSB1A3) AHL0 nM and AHL 1000 nM<br>
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2,Plux inv-GFP (Positive control)<br>
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3,Plux inv    (Negative control)<br>
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 +
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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.
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[[Image:Plux inv function.png|none|frame|Fig. 2 characterization of Plux inv-GFP]]
<br><br><br>
<br><br><br>
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===<font size="5">Results</font>===
===<font size="5">Results</font>===
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[[Image:Plux inv results.png|none|right|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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<br><br><br>
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[[Image:Plux inv results.png|thumb|left|Fig. 3 ]]
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===Conclusion===
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We're not able to confirm LuxR repression.<br>
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However,''iGEMTokyo_tech2010'' team also charcterized about '''Plux inv''' and succeeded in repressing transcription .<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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