Difference between revisions of "Part:BBa K3777013"
 
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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 is composed of a coding sequence of     which was inserted into an expression vectors with     and RBS(BBa_K880005), as well as (BBa_K3386003) under the control of pMphR promoter (BBa_K3386002).  The terminator we used was BBa_B0029.
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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>When antibiotics are absent, aTF will bind to the inducible promoter（PI）and prevent RNA polymerase from initiating transcription, thus repressing the expression of reporter gene. If antibiotics are present, aTFs will bind with them and change their conformation. Therefore, they will no longer able to bind to the promoter, resulting in the expression of reporter gene. In our case, fluorescent signal increases only with the presence of antibiotics
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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>We expressed the circuit in the <i>E. coli </i> BL21(DE3) cells for     detection. Thus we could roughly deduce the concentration of the antibiotics in the sample according to the fluorescence intensity.  
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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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<img src="https://static.igem.org/mediawiki/parts/3/3d/Tetr-tetO-3WJdB.PNG" width="50%" >
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https://static.igem.org/mediawiki/parts/thumb/0/08/Tetr-bc.PNG/799px-Tetr-bc.PNG
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<br>                           Fig.1  Schematic overview of the genetic circuit.
 
<br><b><font size="3">Results</font></b>
 
<br><b><font size="3">Results</font></b>
 
<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.
 
<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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https://static.igem.org/mediawiki/parts/1/12/106-tetr%E2%91%A1.PNG
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<br>The figure shows that our cuircuit can recognize different concentrations of tetracycline, and the fluorescence value was positively correlated with  tetracycline concentration which proves that our part can Detect the concentration of tetracycline,and our reporter gene 3WJDB is surprisingly sensitive.
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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>

Latest revision as of 16:33, 21 October 2021


TetR-T7(tetO)-3WJdB

Basic biosensor device for tetracycline detection.

Usage and Biology
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)
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.
We expressed this circuit in the E. coli BL21(DE3) cells for tetracycline detection. Thus we could roughly deduce the concentration of the antibiotics in the sample according to the fluorescence intensity. 799px-Tetr-bc.PNG
Fig.1 Schematic overview of the genetic circuit.
Results
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. 106-tetr%E2%91%A1.PNG
The figure shows that our cuircuit can recognize different concentrations of tetracycline, and the fluorescence value was positively correlated with tetracycline concentration which proves that our part can Detect the concentration of tetracycline,and our reporter gene 3WJDB is surprisingly sensitive.

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 7
    Illegal NheI site found at 30
    Illegal NheI site found at 1009
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]