Difference between revisions of "Part:BBa K3365000"
Line 7: Line 7:
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===
−
An RNAP ω subunit, RpoZ is fused to dCas9 C-terminal.
+
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).
 +
 
 +
===Results===
 +
<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>
 +
<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>
 +
<center><b>Figure 3.</b> <i> Fluorescence intensity of three kinds transformants before and after tetracycline induction</i> </center>
  
 
<!-- -->
 
<!-- -->

Revision as of 03:53, 24 October 2020


dCas9-ω

An RNAP ω subunit, RpoZ is fused to dCas9 C-terminal.

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]