Difference between revisions of "Part:BBa K3777014"
 
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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>The genetic circuit was composed of coding sequences of  tetracycline repressor  genes and tetM genes which was inserted into an expression vectors with a consitive promoter(BBa_J23114) as well as 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 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>Tetracyclines (including tetracycline and chlortetracycline) inhibited protein synthesis by preventing aminoacyl-tRNA from binding to the ribosomal acceptor site. However, tetM gene confer antibiotic resistance by producing ribosomal protection proteins. So we hope this circuit could possess higher sensitivity and robust.
 
<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.  
 
<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.  
 
https://static.igem.org/mediawiki/parts/thumb/4/47/TetR-tetM-B.C..PNG/799px-TetR-tetM-B.C..PNG
 
https://static.igem.org/mediawiki/parts/thumb/4/47/TetR-tetM-B.C..PNG/799px-TetR-tetM-B.C..PNG
 
<br>                          Fig.1  Schematic overview of the genetic circuit.
 
<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>
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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>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.
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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 16:04, 21 October 2021


TetR-tetM-T7(tetO)-3WJdB

A biosensor device for tetracycline detection.It is worh mentioning that we added a antibiotic resistance gene into our circuits to ensure its robust performance.

Usage and Biology
The genetic circuit was composed of coding sequences of tetracycline repressor genes and tetM genes which was inserted into an expression vectors with a consitive promoter(BBa_J23114) as well as 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)
Tetracyclines (including tetracycline and chlortetracycline) inhibited protein synthesis by preventing aminoacyl-tRNA from binding to the ribosomal acceptor site. However, tetM gene confer antibiotic resistance by producing ribosomal protection proteins. So we hope this circuit could possess higher sensitivity and robust.
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-tetM-B.C..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.
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 2986
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 2598
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2024