Difference between revisions of "Part:BBa K3365000"
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<partinfo>BBa_K3365000 short</partinfo>
 
<partinfo>BBa_K3365000 short</partinfo>
  
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An RNAP &#969; subunit, RpoZ is fused to dCas9 C-terminal.
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An RNA polymerase ω subunit is fused to the C-terminal of dCas9.
  
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===Usage and Biology===
 
===Usage and Biology===
 
The dCas9 is a Cas9 nuclease mutant. Mutations D10A and H840A in the RucC and HNH domains, respectively, abolish cleavage but do not impair DNA binding. The dCas9 provides a simple and robust technology for gene repression and activation, and can target almost any DNA sequence aided by the sgRNA. The ω subunit can activate transcription by recruiting the RNAP holoenzyme. The fusion between dCas9 and ω subunit can activate gene transcription downstream of the protospacer. The CRISPR interference inhibits transcription by sterically blocking the RNA polymerase(RNAP).
 
The dCas9 is a Cas9 nuclease mutant. Mutations D10A and H840A in the RucC and HNH domains, respectively, abolish cleavage but do not impair DNA binding. The dCas9 provides a simple and robust technology for gene repression and activation, and can target almost any DNA sequence aided by the sgRNA. The ω subunit can activate transcription by recruiting the RNAP holoenzyme. The fusion between dCas9 and ω subunit can activate gene transcription downstream of the protospacer. The CRISPR interference inhibits transcription by sterically blocking the RNA polymerase(RNAP).
  
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===Results===
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<center>[[File:DCas9_omega.png]]</center>
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<p>We got three kinds of transformants, each harboring (a)pResponse-RFP, (b)pResponse-RFP plus pActivator without sgRNA, (c)pResponse-RFP plus pActivator with sgRNA. Overnight cultures of different colonies were transferred and grow into early logarithmic stage. Then, we added tetracycline to culture (b) and (c) to a final concentration of 100ug/ml, and incubated them overnight at 30℃. Samples were prepared according to protocol and loaded to 96-well plate. Below is the data acquired from microplate reader, with excitation wavelength at 584nm and emission wavelength at 607nm. Florescence/OD600 indicates the intensity of mRFP.</p>  
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<center>{{#tag:html|<img style="max-width: 500%" src="https://static.igem.org/mediawiki/parts/3/32/T--SJTU-BioX-Shanghai--result_abc.png" alt="" />}}</center>  
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<center><b>Figure1.</b> Gene circuit</center>
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<center><b>Figure 3.</b> <i> Fluorescence intensity of three kinds transformants before and after tetracycline induction</i> </center>
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<center>[[File:Mechanism_of_CRISPR-mediated_transcriptional_activation.png]]</center>
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<center><b>Figure2.</b> Mechanism of CRISPR-mediated transcriptional activation</center>
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Latest revision as of 18:32, 27 October 2020


dCas9-ω

An RNA polymerase ω subunit is fused to the C-terminal of dCas9.

Usage and Biology

The dCas9 is a Cas9 nuclease mutant. Mutations D10A and H840A in the RucC and HNH domains, respectively, abolish cleavage but do not impair DNA binding. The dCas9 provides a simple and robust technology for gene repression and activation, and can target almost any DNA sequence aided by the sgRNA. The ω subunit can activate transcription by recruiting the RNAP holoenzyme. The fusion between dCas9 and ω subunit can activate gene transcription downstream of the protospacer. The CRISPR interference inhibits transcription by sterically blocking the RNA polymerase(RNAP).

DCas9 omega.png
Figure1. Gene circuit


Mechanism of CRISPR-mediated transcriptional activation.png


Figure2. Mechanism of CRISPR-mediated transcriptional activation


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1340
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1340
    Illegal NheI site found at 1099
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1340
    Illegal BamHI site found at 3378
    Illegal BamHI site found at 4212
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1340
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1340
  • 1000
    COMPATIBLE WITH RFC[1000]