Difference between revisions of "Part:BBa K1031211"
 
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== '''Characterization''' ==
 
== '''Characterization''' ==
  
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Prof.V.Shringler provided 2013 Peking iGEM Team with pVI401 composed of Pr-DmpR on vector pVI398, from which K1031211 is derived. K1031211 is composed of the natural promoter of DmpR, Pr and modified DmpR coding sequence with two Pst1 sites removed. To characterize DmpR, we constructed biosensor circuit composed of Pr-DmpR and Po-sfGFP.  
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Professor V.Shringler provided 2013 Peking iGEM Team with pVI401 composed of <html><i>Pr</i></html>-DmpR on vector pVI398, from which K1031211 is derived. K1031211 is composed of the natural promoter of DmpR, <html><i>Pr</i></html> and modified coding sequence with two Pst1 sites removed. To characterize DmpR, we constructed a biosensor circuit composed of <html><i>Pr</i></html>-DmpR and <html><i>Po</i></html>-sfGFP.  
 
('''Fig 1''')
 
('''Fig 1''')
  
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<img src="https://static.igem.org/mediawiki/2013/thumb/7/73/Peking2013_part_Construction_Pr-DmpR.png/800px-Peking2013_part_Construction_Pr-DmpR.png"  
 
<img src="https://static.igem.org/mediawiki/2013/thumb/7/73/Peking2013_part_Construction_Pr-DmpR.png/800px-Peking2013_part_Construction_Pr-DmpR.png"  
 
style="width:600px; margin-left:170px" />  
 
style="width:600px; margin-left:170px" />  
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<p style="text-align:center"><b>Fig 1</b> Construction of biosensor circuit <i>Po</i>/Pr-DmpR. Orange arrowheads represent promoters. RBSs are shown as green ovals.
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<p style="text-align:center"><b>Fig 1</b> Construction of biosensor circuit <i>Po</i>/DmpR. Orange arrowheads represent promoters. RBSs(Ribosome Binding Sites) are shown as green ovals.
 
</html>
 
</html>
  
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'''ON/OFF test'''
 
'''ON/OFF test'''
  
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We evaluated the performance of DmpR using our own protocols and almost every protocol mentioned in the previous studies. Results showed that the <html><a href="http://2013.igem.org/Team:Peking/Team/Notebook/Protocols#Content2">Test Protocol 3</a></html> works the best, with which we tested the ON-OFF ratios of all the 78 aromatic compounds. 7 compounds out of the 78 showed significant induction ratios (>2):Phl, 2-MePhl, 2-ClPhl, 3-ClPhl, Cat, 4-NtPhl and 2-APhl ('''Fig 2''')
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We evaluated the performance of DmpR using our own protocols and almost every protocol mentioned in the previous studies. Results showed that the <html><a href="http://2013.igem.org/Team:Peking/Team/Notebook/Protocols#Content2">Test Protocol 3</a></html> works the best, with which we tested the ON/OFF ratios of all the 78 aromatic compounds. 7 compounds out of the 78 showed significant induction ratios (>2),they are Phl, 2-MePhl, 2-ClPhl, 3-ClPhl, Cat, 4-NtPhl and 2-APhl ('''Fig 2''')
  
 
<html>
 
<html>
 
<img src="https://static.igem.org/mediawiki/igem.org/7/7b/Peking2013_DmpR_Figure8.png"  
 
<img src="https://static.igem.org/mediawiki/igem.org/7/7b/Peking2013_DmpR_Figure8.png"  
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style="width:800px; margin-left:120px" />
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style="width:800px; margin-left:70px" />
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<p style="text-align:center"><b>Fig 2</b>  <b>Left</b> Horizontal axis stands for different aromatic compounds.Vertical axis represents the ON/OFF induction ratio calculated from fluorescence intensity. <b>Right</b> Radar graph shows the aromatic sensing profile. The efficient inducers are shown as their chemical structure.
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<p style="text-align:center"><b>Fig 2</b>  <b>Left</b> Horizontal axis stands for different aromatic compounds.Vertical axis represents the ON/OFF induction ratio calculated from fluorescence intensity. <b>Right</b> Radar graph shows the aromatics-sensing profile of DmpR. The efficient and typical inducers are shown as their chemical structure.
 
</html>
 
</html>
  
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'''Dose-Response curve'''
 
'''Dose-Response curve'''
  
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7 aromatics at different concentration was induced to plot dose-response curves for detailed information concerning DmpR.   
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7 efficient aromatics at different concentration was induced to DmpR biosensor to plot dose-response curves for detailed information concerning DmpR.   
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<html>
 
<html>
 
<img src="https://static.igem.org/mediawiki/igem.org/0/0d/Peking2013_DmpR_Figure9.png"
 
<img src="https://static.igem.org/mediawiki/igem.org/0/0d/Peking2013_DmpR_Figure9.png"
 
style="width:600px; margin-left:170px" />
 
style="width:600px; margin-left:170px" />
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<p style="text-align:center"><b>Fig 3</b> Dose-response curves of DmpR biosensor induced by 7 strong inducers (phenol, its homologs and derivatives). The induction ratio was calculated by dividing the fluorescence intensity of biosensor exposed to inducers by the basal fluorescence intensity of the biosensor.
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<p style="text-align:center"><b>Fig 3</b> Dose-response curves of DmpR biosensor induced by 7 strong inducers (phenol, as well as its homologs and derivatives). The induction ratio was calculated by dividing the fluorescence intensity of biosensor exposed to inducers by the basal fluorescence intensity of the biosensor.
 
</html>
 
</html>
  

Latest revision as of 16:01, 28 October 2013

Pr-DmpR

For detailed information concerning DmpR, please visit 2013 Peking iGEM Biosensor DmpR


Characterization

Professor V.Shringler provided 2013 Peking iGEM Team with pVI401 composed of Pr-DmpR on vector pVI398, from which K1031211 is derived. K1031211 is composed of the natural promoter of DmpR, Pr and modified coding sequence with two Pst1 sites removed. To characterize DmpR, we constructed a biosensor circuit composed of Pr-DmpR and Po-sfGFP. (Fig 1)

Fig 1 Construction of biosensor circuit Po/DmpR. Orange arrowheads represent promoters. RBSs(Ribosome Binding Sites) are shown as green ovals.


Sequence and Features

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 160
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 638
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 323
    Illegal BsaI.rc site found at 864
    Illegal SapI.rc site found at 1521



Data shown

ON/OFF test

We evaluated the performance of DmpR using our own protocols and almost every protocol mentioned in the previous studies. Results showed that the Test Protocol 3 works the best, with which we tested the ON/OFF ratios of all the 78 aromatic compounds. 7 compounds out of the 78 showed significant induction ratios (>2),they are Phl, 2-MePhl, 2-ClPhl, 3-ClPhl, Cat, 4-NtPhl and 2-APhl (Fig 2)

Fig 2 Left Horizontal axis stands for different aromatic compounds.Vertical axis represents the ON/OFF induction ratio calculated from fluorescence intensity. Right Radar graph shows the aromatics-sensing profile of DmpR. The efficient and typical inducers are shown as their chemical structure.


Dose-Response curve

7 efficient aromatics at different concentration was induced to DmpR biosensor to plot dose-response curves for detailed information concerning DmpR.

Fig 3 Dose-response curves of DmpR biosensor induced by 7 strong inducers (phenol, as well as its homologs and derivatives). The induction ratio was calculated by dividing the fluorescence intensity of biosensor exposed to inducers by the basal fluorescence intensity of the biosensor.