Difference between revisions of "Part:BBa K3777028"
 
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<partinfo>BBa_K3777013 short</partinfo>
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<partinfo>BBa_K3777028 short</partinfo>
  
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Basic biosensor device for tetracycline detection.
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A circuit that can produce dcas9 protein which can bind to our another part BS2(BBa_K3777005) leading to a better result.
  
 
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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 a coding sequence of  dcas9 protein which was inserted into an expression vectors with a Inducible promoter(BBa_R0062) and RBS(BBa_K3777030).The terminator we used was BBa_B0010 .(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>dcas9 proteins can bind to our another part BS2(BBa_K3777005),then sgRNA also binds to the BS2 (binding site of KB2), inhibiting fluorescence expression.
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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.  
 
<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/3/3d/Tetr-tetO-3WJdB.PNG/799px-Tetr-tetO-3WJdB.PNG  
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https://static.igem.org/mediawiki/parts/thumb/d/d3/LuxR-Plux-dCas9.PNG/800px-LuxR-Plux-dCas9.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>
 
<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/thumb/0/0b/CRISPR_i_2.png/800px-CRISPR_i_2.png
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<br>The figure shows that CRISPRi can really inhibit our fluorescence expression
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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_K3777028 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_K3777028 parameters</partinfo>
 
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Latest revision as of 22:47, 21 October 2021


luxR-Plux-dCas9

A circuit that can produce dcas9 protein which can bind to our another part BS2(BBa_K3777005) leading to a better result.

Usage and Biology
The genetic circuit was composed of a coding sequence of dcas9 protein which was inserted into an expression vectors with a Inducible promoter(BBa_R0062) and RBS(BBa_K3777030).The terminator we used was BBa_B0010 .(Fig 1)


dcas9 proteins can bind to our another part BS2(BBa_K3777005),then sgRNA also binds to the BS2 (binding site of KB2), inhibiting fluorescence expression.


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. 800px-LuxR-Plux-dCas9.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. 800px-CRISPR_i_2.png
The figure shows that CRISPRi can really inhibit our fluorescence expression
Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 2204
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 4483
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1022