Difference between revisions of "Part:BBa K1031311"
 
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In order to fine-tune the performance of HbpR biosensor, we constructed a library of ''Pc'' constitutive promoters at different intensity. BBa_K1031311 is composed of three elements, the constitutive ''Pc'' promoter <html><a href="https://parts.igem.org/Part:BBa_J23114">J23114</a></html>, coding sequence of HbpR and terminator <html><a href="https://parts.igem.org/Part:BBa_B0015">B0015</a></html>. ('''Fig 1''')
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In order to fine-tune the performance of HbpR biosensor, we constructed a library of constitutive promoters at different intensity. BBa_K1031311 is composed of three elements, the constitutive promoter <html><a href="https://parts.igem.org/Part:BBa_J23114">J23114</a></html>, coding sequence of HbpR 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/2013/d/db/Peking2013_part_HbpR_construction_.png"  
 
<img src="https://static.igem.org/mediawiki/2013/d/db/Peking2013_part_HbpR_construction_.png"  
 
style="width:400px; margin-left:250px" />
 
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<p style="text-align:center"><b>Fig 1</b> Construction of biosensor circuit <i>Pm</i>/J23114-HbpR. Orange arrowheads represent promoters. RBSs are shown as green ovals and squares in dark red stand for B0015 terminator.
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<p style="text-align:center"><b>Fig 1</b> Construction of biosensor circuit <i>Pc</i>/J23114-HbpR. Orange arrowheads represent promoters. RBSs(Ribosome Binding Sites) are shown as green ovals and squares in dark red stand for B0015 terminator.
 
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'''Construction and tuning'''  
 
'''Construction and tuning'''  
  
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Induction ratio of HbpR biosensor library from ON/OFF test that utilizes different'' Pc'' promoters to control the expression of XylS. The dashed box refers to data for ''Pm''/J23114-XylS biosensor circuit ('''Fig 2''')
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Induction ratio of HbpR biosensor library from ON/OFF test that utilizes different constitutive promoters to control the expression of HbpR. The dashed box refers to data for ''Pc''/J23114-HbpR biosensor circuit ('''Fig 2''')
 
This figure shows the biosensor circuit adopting J23114 obtains the optimal performance.  
 
This figure shows the biosensor circuit adopting J23114 obtains the optimal performance.  
  
 
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<img src="" style="width:600px; margin-left:170px" />
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<img src="https://static.igem.org/mediawiki/2013/e/e9/Peking2013_part_HbpR_Pc_library.png"  
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<p style="text-align:center"><b>Fig 2</b> Horizontal axis stands for different HbpR biosensor with <i>Pc</i> promoters of different strength. The expression strength of these constitutive promoters, J23113, J23109, J23117, J23114 is 21, 106, 623 and 256, respectively, according to the Parts' registry. Two kinds of aromatics, namely 2-HBP and 2-ABP, shown with different color intensities, were tested following <a href="http://2013.igem.org/Team:Peking/Team/Notebook/Protocols">Test Protocol 1</a>. Vertical axis represents the ON/OFF induction ratio. The dashed box refers to performance for <i>Pm</i>/J23114-XylS biosensor circuit.  
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style="width:500px; margin-left:200px" />
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<p style="text-align:center"><b>Fig 2</b> Horizontal axis stands for different HbpR biosensor with constitutive promoters of different strength. The expression strength of these constitutive promoters, J23113, J23109, J23117, J23114 is 21, 106, 623 and 256, respectively, according to the Parts' registry. Two kinds of aromatics, namely 2-HBP and 2-ABP, shown with different color intensities, were tested following <a href="http://2013.igem.org/Team:Peking/Team/Notebook/Protocols">Test Protocol 1</a>. Vertical axis represents the ON/OFF induction ratio calculated from fluorescence intensity. The dashed box refers to performance for <i>Pc</i>/J23114-HbpR biosensor circuit.  
 
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'''ON/OFF test'''
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Induction ratio of HbpR ON/OFF test when exposed to 78 aromatic compounds utilizing J23114 as constitutive promoter. It is shown that HbpR biosensor responds specific to 2-HBP and 2-ABP.
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<img src="https://static.igem.org/mediawiki/igem.org/a/a0/HbpR_Figure6_2013Peking_WH.png"
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style="width:800px; margin-left:75px" />
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<p style="text-align:center"><b>Fig 3</b> Horizontal axis stands for different aromatic compounds utilized to induce HbpR biosensor. Vertical axis represents ON/OFF induction ratio of HbpR biosensor calculated from fluorescence intensity when exposed to corresponding aromatics. It is tested following <a href="http://2013.igem.org/Team:Peking/Team/Notebook/Protocols">Test Protocol 1</a>. 
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'''Dose-response curve plotting'''
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Dose-response curve of HbpR biosensor adopting J23114 were plotted when exposed to 2-HBP and 2-ABP to further describe characterization of HbpR.
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<img src="https://static.igem.org/mediawiki/igem.org/e/e6/HbpR_Figure7_2013Peking_WH.png"
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style="width:500px; margin-left:200px" />
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<p style="text-align:center"><b>Fig 4</b> Two inducers are shown in different color intensity. Horizontal axis stands for inducer at different concentration. Vertical axis represents induction ratio calculated from fluorescence intensity. It is tested following <a href="http://2013.igem.org/Team:Peking/Team/Notebook/Protocols">Test Protocol 1</a>. 
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 16:09, 28 October 2013

J23114-HbpR-Terminator

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


Characterization

In order to fine-tune the performance of HbpR biosensor, we constructed a library of constitutive promoters at different intensity. BBa_K1031311 is composed of three elements, the constitutive promoter J23114, coding sequence of HbpR and terminator B0015. (Fig 1)

Fig 1 Construction of biosensor circuit Pc/J23114-HbpR. Orange arrowheads represent promoters. RBSs(Ribosome Binding Sites) are shown as green ovals and squares in dark red stand 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
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1735
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 449



Data shown

Construction and tuning

Induction ratio of HbpR biosensor library from ON/OFF test that utilizes different constitutive promoters to control the expression of HbpR. The dashed box refers to data for Pc/J23114-HbpR biosensor circuit (Fig 2) This figure shows the biosensor circuit adopting J23114 obtains the optimal performance.

Fig 2 Horizontal axis stands for different HbpR biosensor with constitutive promoters of different strength. The expression strength of these constitutive promoters, J23113, J23109, J23117, J23114 is 21, 106, 623 and 256, respectively, according to the Parts' registry. Two kinds of aromatics, namely 2-HBP and 2-ABP, shown with different color intensities, were tested following Test Protocol 1. Vertical axis represents the ON/OFF induction ratio calculated from fluorescence intensity. The dashed box refers to performance for Pc/J23114-HbpR biosensor circuit.


ON/OFF test

Induction ratio of HbpR ON/OFF test when exposed to 78 aromatic compounds utilizing J23114 as constitutive promoter. It is shown that HbpR biosensor responds specific to 2-HBP and 2-ABP.

Fig 3 Horizontal axis stands for different aromatic compounds utilized to induce HbpR biosensor. Vertical axis represents ON/OFF induction ratio of HbpR biosensor calculated from fluorescence intensity when exposed to corresponding aromatics. It is tested following Test Protocol 1.


Dose-response curve plotting

Dose-response curve of HbpR biosensor adopting J23114 were plotted when exposed to 2-HBP and 2-ABP to further describe characterization of HbpR.

Fig 4 Two inducers are shown in different color intensity. Horizontal axis stands for inducer at different concentration. Vertical axis represents induction ratio calculated from fluorescence intensity. It is tested following Test Protocol 1.