Difference between revisions of "Part:BBa K3777028"

 
 
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<partinfo>BBa_K3777028 short</partinfo>
 
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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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===Usage and Biology===
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<b><font size="3">Usage and Biology</font></b>
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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>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.
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https://static.igem.org/mediawiki/parts/thumb/d/d3/LuxR-Plux-dCas9.PNG/800px-LuxR-Plux-dCas9.PNG
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<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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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>

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