Difference between revisions of "Part:BBa K3777025"
 
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<partinfo>BBa_K3777013 short</partinfo>
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<partinfo>BBa_K3777025 short</partinfo>
  
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Basic biosensor device for tetracycline detection.
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Basic biosensor device for Chlortetracycline detection.
  
 
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<b><font size="3">Usage and Biology</font></b>
 
<b><font size="3">Usage and Biology</font></b>
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<br>The genetic circuit was composed of a coding sequence of  tetracycline repressor  which was inserted into an expression vectors with a consitive promoter(BBa_J23114) and RBS(BBa_K3777030), as well as 3WJdB(BBa_K3777000) under the control of T7 promoter (BBa_K3777006).  The terminator we used were BBa_B0010 and BBa_M36305.(Fig 1)
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<br>Compared to our another part ctcS-tetM-T7(ctcO)-3WJdB(BBa_K3777018),it adds a binding site which can be regonized by dcas9 proteins and sgRNA.(Fig 1)  
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<br>When tet was absent, TetR would bind to the inducible promoter（PI）and prevent RNA polymerase from initiating transcription, thus repressing the expression of reporter gene. If tet was present, TetR would no longer able to bind to the promoter, resulting in the expression of reporter gene.
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<br>When CTR was absent, ctcs would bind to the inducible promoter（PI）and prevent RNA polymerase from initiating transcription, thus repressing the expression of reporter gene. If tet was present, TetR would no longer able to bind to the promoter, resulting in the expression of reporter gene. But KB2 will bind to 3WJDB leading to lower fluorescent .
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<br>We expressed this circuit in the <i>E. coli </i> BL21(DE3) cells for tetracycline detection. Thus we could roughly deduce the concentration of the antibiotics in the sample according to the fluorescence intensity.  
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https://static.igem.org/mediawiki/parts/thumb/d/d9/KB2-CtcS-T7%28ctcO%29-3WJdB-PctcS-sgRNA.PNG/798px-KB2-CtcS-T7%28ctcO%29-3WJdB-PctcS-sgRNA.PNG
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https://static.igem.org/mediawiki/parts/thumb/3/3d/Tetr-tetO-3WJdB.PNG/799px-Tetr-tetO-3WJdB.PNG  
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<br>                          Fig.1  Schematic overview of the genetic circuit.
 
<br>                          Fig.1  Schematic overview of the genetic circuit.
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<br><b><font size="3">Results</font></b>
 
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<br>To verify the functionality of the biosensor, we performed a plate-reader experiment and measured optical density and fluorescence intensity every hour. We observed a correlation between concentration of antibiotics in the sample and intensity of fluorescent signal.
 
  
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<br>Reference：Alam Khalid K,Tawiah Kwaku D,Lichte Matthew F,Porciani David,Burke Donald H. A Fluorescent Split Aptamer for Visualizing RNA-RNA Assembly In Vivo.[J]. ACS synthetic biology,2017,6(9)
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3777013 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K3777025 SequenceAndFeatures</partinfo>
  
  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K3777013 parameters</partinfo>
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<partinfo>BBa_K3777025 parameters</partinfo>
 
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Latest revision as of 02:56, 22 October 2021


KB2-CtcS-T7(ctcO)-3WJdB-PctcS-sgRNA

Basic biosensor device for Chlortetracycline detection.

Usage and Biology
Compared to our another part ctcS-tetM-T7(ctcO)-3WJdB(BBa_K3777018),it adds a binding site which can be regonized by dcas9 proteins and sgRNA.(Fig 1)
When CTR was absent, ctcs would bind to the inducible promoter（PI）and prevent RNA polymerase from initiating transcription, thus repressing the expression of reporter gene. If tet was present, TetR would no longer able to bind to the promoter, resulting in the expression of reporter gene. But KB2 will bind to 3WJDB leading to lower fluorescent . 798px-KB2-CtcS-T7%28ctcO%29-3WJdB-PctcS-sgRNA.PNG
Fig.1 Schematic overview of the genetic circuit.


Reference：Alam Khalid K,Tawiah Kwaku D,Lichte Matthew F,Porciani David,Burke Donald H. A Fluorescent Split Aptamer for Visualizing RNA-RNA Assembly In Vivo.[J]. ACS synthetic biology,2017,6(9)
Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 260
    Illegal NheI site found at 283
    Illegal NheI site found at 1164
    Illegal NotI site found at 832
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 121
    Illegal XhoI site found at 841
    Illegal XhoI site found at 1305
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 576