Difference between revisions of "Part:BBa K1031916"
 
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<p>For detailed information concerning XylS, please visit <a href="http://2013.igem.org/Team:Peking/Project/BioSensors/XylS">2013 Peking iGEM Biosensor XylS</a></p>
 
<p>For detailed information concerning XylS, please visit <a href="http://2013.igem.org/Team:Peking/Project/BioSensors/XylS">2013 Peking iGEM Biosensor XylS</a></p>
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In order to fine-tune the performance of XylS biosensor, we constructed a library of ''Pc'' constitutive promoters at different intensity. BBa_K1031916 is composed of three elements, the constitutive ''Pc'' promoter J23114[https://parts.igem.org/Part:BBa_J23114], coding sequence of XylS and terminator B0015[https://parts.igem.org/Part:BBa_B0015]. ('''Fig 1''')
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In order to fine-tune the performance of XylS biosensor, we constructed a library of ''Pc'' constitutive promoters at different intensity. BBa_K1031916 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 XylS 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/thumb/3/33/Peking2013_part_114-XylS.png/800px-Peking2013_part_114-XylS.png", width=600px; />
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<img src="https://static.igem.org/mediawiki/2013/thumb/3/33/Peking2013_part_114-XylS.png/800px-Peking2013_part_114-XylS.png" style="width:600px; margin-left:170px" />
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<p style="text-align:center"><b>Fig 1</b> Construction of biosensor circuit <i>Pm</i>/J23114-XylS. Orange arrowheads represent promoters. RBSs are shown as green ovals and squares in dark red stand for B0015 terminator.
 
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'''Fig 1''' Construction of biosensor circuit ''Pm''/J23114-XylS. Orange arrowheads represent promoters. RBSs are shown as green ovals and squares in dark red stand for B0015 terminator.
 
  
  
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== '''Data shown''' ==
 
== '''Data shown''' ==
  
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'''Construction and tuning'''
  
 
Induction ratio of XylS 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''')
 
Induction ratio of XylS 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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<img src="https://static.igem.org/mediawiki/2013/thumb/8/80/Peking2013_part_Pc_library_114.png/800px-Peking2013_part_Pc_library_114.png", width=600px; />
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<img src="https://static.igem.org/mediawiki/2013/thumb/8/80/Peking2013_part_Pc_library_114.png/800px-Peking2013_part_Pc_library_114.png" style="width:600px; margin-left:170px" />
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<p style="text-align:center"><b>Fig 2</b> Horizontal axis stands for different XylS 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. Four kinds of aromatics, namely BzO, 2-MxeBzO, 3-MeBzO and 4-MeBzO, 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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'''Fig 2''' Horizontal axis stands for different XylS 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. Four kinds of aromatics, namely BzO, 2-MxeBzO, 3-MeBzO and 4-MeBzO, 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 ''Pm''/J23114-XylS biosensor circuit.  
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'''ON/OFF test'''
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20 compounds showed apparent activation effect with the induction ratios over 20, they are listed as follows: BzO, 2-MeBzO, 3-MeBzO, 4-MeBzO, 2-FBzO, 4-FBzO, 2-ClBzO, 3-ClBzO, 4-ClBzO, 2-BrBzO, 4-BrBzO, 3-IBzO, 3-MeOBzO, SaA, 3-MeOSaA, 4-ClSaA, 5-ClSaA, 3-MeBAD, 3-ClTOL '''(Fig.3)'''
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<img src="https://static.igem.org/mediawiki/2013/f/f3/Peking2013_XylS_data.png" style="width:800px;margin-left:60px"  ></a>
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<b>Fig.3</b> (a) The induction ratios of all 78 typical aromatic compounds in the <a href="http://2013.igem.org/Team:Peking/Team/Notebook/Protocols#Content3">ON/OFF test</a> following <a href="http://2013.igem.org/Team:Peking/Team/Notebook/Protocols#Content1">Test Protocol 1</a>. XylS biosensor could respond to 24 out of 78 aromatics with the induction ratio higher than 20, mainly benzoate, salicylic and their derivatives. </br>(b) The aromatics-sensing profile of XylS biosensor is summarized from (a), highlighted in red in the aromatics spectrum. The structure formula of typical inducers are listed around the central spectrum, near their chemical formula.
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<b>Dose-response curve to benzoate, salicylic acid and derivatives</b>
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Pm/J23114-XylS biosensor circuit was subjected to induction experiments with concentration of inducer ranging at 10 µM, 30µM,100µM, 300µM and 1000µM according to protocol 1. (<b>Fig.4</b>).
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<img src="https://static.igem.org/mediawiki/2013/f/fd/Peking2013_XylS_Induce.png" style="width:800px;margin-left:60px"  ></a>
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<b>Fig.4</b>  (a) Dose-response curves of XylS biosensor induced by benzoate and its derivatives. X-axis stands for concentration gradient of inducers at 10µM, 30µM, 100µM, 300µM and 1000µM. Different colors represent different kinds of inducers. Y-axis shows induction ratios.  (b) Dose-response curves of XylS biosensor induced by salicylate and its derivatives. X-axis stands for concentration gradient of inducers at 10µM, 30µM, 100µM, 300µM and 1000µM. Y-axis denotes induction ratios. Different colors denote different kinds of inducers.
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Latest revision as of 23:36, 27 September 2013

J23114-XylS-Terminator

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


Characterization

In order to fine-tune the performance of XylS biosensor, we constructed a library of Pc constitutive promoters at different intensity. BBa_K1031916 is composed of three elements, the constitutive Pc promoter J23114, coding sequence of XylS and terminator B0015. (Fig 1)

Fig 1 Construction of biosensor circuit Pm/J23114-XylS. Orange arrowheads represent promoters. RBSs 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 BglII site found at 93
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 815
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 278



Data shown

Construction and tuning

Induction ratio of XylS 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) This figure shows the biosensor circuit adopting J23114 obtains the optimal performance.

Fig 2 Horizontal axis stands for different XylS 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. Four kinds of aromatics, namely BzO, 2-MxeBzO, 3-MeBzO and 4-MeBzO, shown with different color intensities, were tested following Test Protocol 1. Vertical axis represents the ON/OFF induction ratio. The dashed box refers to performance for Pm/J23114-XylS biosensor circuit.


ON/OFF test

20 compounds showed apparent activation effect with the induction ratios over 20, they are listed as follows: BzO, 2-MeBzO, 3-MeBzO, 4-MeBzO, 2-FBzO, 4-FBzO, 2-ClBzO, 3-ClBzO, 4-ClBzO, 2-BrBzO, 4-BrBzO, 3-IBzO, 3-MeOBzO, SaA, 3-MeOSaA, 4-ClSaA, 5-ClSaA, 3-MeBAD, 3-ClTOL (Fig.3)


Fig.3 (a) The induction ratios of all 78 typical aromatic compounds in the ON/OFF test following Test Protocol 1. XylS biosensor could respond to 24 out of 78 aromatics with the induction ratio higher than 20, mainly benzoate, salicylic and their derivatives.
(b) The aromatics-sensing profile of XylS biosensor is summarized from (a), highlighted in red in the aromatics spectrum. The structure formula of typical inducers are listed around the central spectrum, near their chemical formula.



Dose-response curve to benzoate, salicylic acid and derivatives
Pm/J23114-XylS biosensor circuit was subjected to induction experiments with concentration of inducer ranging at 10 µM, 30µM,100µM, 300µM and 1000µM according to protocol 1. (Fig.4).

Fig.4 (a) Dose-response curves of XylS biosensor induced by benzoate and its derivatives. X-axis stands for concentration gradient of inducers at 10µM, 30µM, 100µM, 300µM and 1000µM. Different colors represent different kinds of inducers. Y-axis shows induction ratios. (b) Dose-response curves of XylS biosensor induced by salicylate and its derivatives. X-axis stands for concentration gradient of inducers at 10µM, 30µM, 100µM, 300µM and 1000µM. Y-axis denotes induction ratios. Different colors denote different kinds of inducers.