Difference between revisions of "Part:BBa K1031441"
 
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<p>For detailed information concerning HcaR, please visit <a href="http://2013.igem.org/Team:Peking/Project/BioSensors/HcaR#Mileston1">2013 Peking iGEM Biosensor HcaR</a></p>
 
<p>For detailed information concerning HcaR, please visit <a href="http://2013.igem.org/Team:Peking/Project/BioSensors/HcaR#Mileston1">2013 Peking iGEM Biosensor HcaR</a></p>
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In order to fine-tune the performance of HcaR biosensor, we constructed a library of ''Pc'' constitutive promoters, collocating with RBS (Ribosome Binding Site) at different intensity. BBa_K1031441 is composed of three elements, the constitutive ''Pc'' promoter J23106[https://parts.igem.org/Part:BBa_J23106], coding sequence of HcaR and terminator B0015[https://parts.igem.org/Part:BBa_B0015]. ('''Fig 1'''). This circuit expressing HcaR is collocated with RBS B0034.
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In order to fine-tune the performance of HcaR biosensor, we constructed a library of ''Pc'' constitutive promoters, collocating with a library of RBS (Ribosome Binding Site) at different intensity. BBa_K1031441 is composed of three elements, the constitutive ''Pc'' promoter <html><a href="https://parts.igem.org/Part:BBa_J23106">J23106</a></html>, coding sequence of HcaR and terminator <html><a href="https://parts.igem.org/Part:BBa_B0015">B0015</a></html>. ('''Fig 1''').
  
 
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<img src="https://static.igem.org/mediawiki/igem.org/thumb/a/af/Peking2013_106-HcaR.png/800px-Peking2013_106-HcaR.png", width=600px; />
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<img src="https://static.igem.org/mediawiki/igem.org/thumb/a/af/Peking2013_106-HcaR.png/800px-Peking2013_106-HcaR.png" style="width:600px; margin-left:170px" />
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<p style="text-align:center"><b>Fig 1</b> Construction of biosensor circuit ''Ph''/J23106-HcaR. The orange arrowheads represent promoters, RBS is shown as a green oval, and the red circle stands for B0015 terminator.
 
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'''Fig 1''' Construction of biosensor circuit ''Ph''/J23106-HcaR. The orange arrowheads represent promoters, RBS is shown as a green oval, and the red circle stands for B0015 terminator.
 
  
  
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'''Tuning'''
 
'''Tuning'''
  
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Induction ratio of HcaR biosensor library test utilizes different'' Pc'' promoters, collocating with RBS (Ribosome Binding Site) at different intensity to control the expression of HcaR. The dashed box refers to data for ''Ph''/J23106-HcaR biosensor circuit ('''Fig 2''') in collocation with RBS B0034.
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Induction ratio of HcaR biosensor library test utilizes different'' Pc'' promoters, collocating with RBSs (Ribosome Binding Sites) at different intensity to optimize the performance of HcaR. The first three lines are data for HcaR biosensor circuit adopting Pc J23106 in collocation with RBS B0031, B0032 and B0034 respectively. The dashed box refers to data for ''Ph''/J23106-HcaR biosensor circuit ('''Fig 2''') in collocation with RBS B0032, which obtains the optimal performance among them.  
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This figure shows that when using B0034, the biosensor circuit adopting J23106 obtains the optimal performance.  
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<img src="https://static.igem.org/mediawiki/igem.org/thumb/d/da/Peking2013_part_Pc_library_HcaR_106.png/800px-Peking2013_part_Pc_library_HcaR_106.png", width=600px; />
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<img src="https://static.igem.org/mediawiki/igem.org/thumb/9/96/Peking2013_part_K1031441.png/800px-Peking2013_part_K1031441.png" style="width:600px; margin-left:170px" />
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<p style="text-align:center"><b>Fig 2</b> Induction ratio of HcaR biosensor library test. Horizontal axis stands for different HcaR biosensor with <i>Pc</i> promoters of different strength. The expression strength of these constitutive promoters, J23113, J23109, J23114, J23105, J23106 is 21, 106, 256, 623, and 1185, respectively, according to the Parts' registry. PPA was tested following <a href="http://2013.igem.org/Team:Peking/Team/Notebook/Protocols">Test Protocol 1</a> as inducer. Vertical axis represents the ON-OFF induction ratio. The dashed box refers to performance for ''Ph''/J23106-HcaR biosensor adopting RBS B0032.
 
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'''Fig 2''' Induction ratio of HcaR biosensor library test. Horizontal axis stands for different HcaR biosensor with ''Pc'' promoters of different strength. The expression strength of these constitutive promoters, J23113, J23109, J23114, J23105, J23106 is 21, 106, 256, 623, and 1185, respectively, according to the Partsregistry. Four kinds of aromatics shown with different color intensities, were tested following Test Protocol 1 [http://2013.igem.org/Team:Peking/Team/Notebook/Protocols]. Vertical axis represents the ON-OFF induction ratio. The dashed box refers to performance for ''Ph''/J23106-HcaR biosensor circuit.
 
  
  
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'''ON/OFF test'''
 
'''ON/OFF test'''
  
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The best performed biosensor circuit ''Ph''/J23106-HcaR is then subjected to the On-Off test about 78 aromatics according to protocol 1[1]. Results showed that HcaR worked as a specific sensor to PPA ('''Fig 4''').
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The best performed biosensor ''Ph''/J23106-HcaR with RBS B0032 is then subjected to the On-Off test about 78 aromatics according to <html><a href="http://2013.igem.org/Team:Peking/Team/Notebook/Protocols">Text protocol 1</a></html>. Results showed that HcaR worked as a specific sensor to PPA ('''Fig 4''').
  
 
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<img src="https://static.igem.org/mediawiki/igem.org/thumb/9/92/Peking2013_part_HcaR_On-Off.jpg/800px-Peking2013_part_HcaR_On-Off.jpg", width=600px; />
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<img src="https://static.igem.org/mediawiki/igem.org/0/09/Peking2013_HcaR_OF_J23106-B0032.jpg", width=820px; />
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<p style="text-align:center"><b>Fig 3</b> Results of On-Off test about biosensor HcaR. HcaR specifically responds to PPA (1000 μM) with the induction ration over 2.
 
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'''Fig 3''' Results of On-Off test about biosensor HcaR. HcaR specifically responds to PPA (1000 μM) with the induction ration over 2.
 
  
  
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'''Dose-response curve'''
 
'''Dose-response curve'''
  
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PPA was used to test dose-response curve of ''Ph''/J23106-HcaR at the concentration at 1µM, 5µM, 10µM, 50µM, 100µM, 500µM and 1000µM  ('''Fig 3''')
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PPA was used to test dose-response curve of ''Ph''/J23106-HcaR. Two biosensor circuits adopting RBS B0032 and B0034 are tested respectively.('''Fig 3''')
  
 
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<p style="text-align:center"><b>Fig 4</b> Dose response curve of biosensor circuit <i>Ph</i>/J23106-HcaR induced by PPA. X-axis stands for concentration gradient of inducers at 1µM, 5µM, 10µM, 50µM, 100µM, 500µM and 1000µM. Y-axis shows induction ratio reflected via fluorescence intensity.Two biosensor circuits adopting B0032 and B0034 are shown in different color intensity.
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'''Fig 3''' Dose response curve of biosensor circuit ''Ph''/J23106-HcaR induced by PPA. X-axis stands for concentration gradient of inducers at 1µM, 5µM, 10µM, 50µM, 100µM, 500µM and 1000µM. Y-axis shows induction ratio reflected via fluorescence intensity.
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Latest revision as of 21:46, 27 September 2013

J23106-HcaR

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


Characterization

In order to fine-tune the performance of HcaR biosensor, we constructed a library of Pc constitutive promoters, collocating with a library of RBS (Ribosome Binding Site) at different intensity. BBa_K1031441 is composed of three elements, the constitutive Pc promoter J23106, coding sequence of HcaR and terminator B0015. (Fig 1).

Fig 1 Construction of biosensor circuit ''Ph''/J23106-HcaR. The orange arrowheads represent promoters, RBS is shown as a green oval, and the red circle stands for B0015 terminator.


Sequence and Features

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 892
    Illegal AgeI site found at 565
  • 1000
    COMPATIBLE WITH RFC[1000]


Data shown

Tuning

Induction ratio of HcaR biosensor library test utilizes different Pc promoters, collocating with RBSs (Ribosome Binding Sites) at different intensity to optimize the performance of HcaR. The first three lines are data for HcaR biosensor circuit adopting Pc J23106 in collocation with RBS B0031, B0032 and B0034 respectively. The dashed box refers to data for Ph/J23106-HcaR biosensor circuit (Fig 2) in collocation with RBS B0032, which obtains the optimal performance among them.

Fig 2 Induction ratio of HcaR biosensor library test. Horizontal axis stands for different HcaR biosensor with Pc promoters of different strength. The expression strength of these constitutive promoters, J23113, J23109, J23114, J23105, J23106 is 21, 106, 256, 623, and 1185, respectively, according to the Parts' registry. PPA was tested following Test Protocol 1 as inducer. Vertical axis represents the ON-OFF induction ratio. The dashed box refers to performance for ''Ph''/J23106-HcaR biosensor adopting RBS B0032.


ON/OFF test

The best performed biosensor Ph/J23106-HcaR with RBS B0032 is then subjected to the On-Off test about 78 aromatics according to Text protocol 1. Results showed that HcaR worked as a specific sensor to PPA (Fig 4).

Fig 3 Results of On-Off test about biosensor HcaR. HcaR specifically responds to PPA (1000 μM) with the induction ration over 2.


Dose-response curve

PPA was used to test dose-response curve of Ph/J23106-HcaR. Two biosensor circuits adopting RBS B0032 and B0034 are tested respectively.(Fig 3)

Fig 4 Dose response curve of biosensor circuit Ph/J23106-HcaR induced by PPA. X-axis stands for concentration gradient of inducers at 1µM, 5µM, 10µM, 50µM, 100µM, 500µM and 1000µM. Y-axis shows induction ratio reflected via fluorescence intensity.Two biosensor circuits adopting B0032 and B0034 are shown in different color intensity.