Difference between revisions of "Part:BBa K3081007"
Line 3: Line 3:
 
<partinfo>BBa_K3081007 short</partinfo>
 
<partinfo>BBa_K3081007 short</partinfo>
  
−
This composite part is the principal design of the inducible CRISPR-based DNA replication interference system, with the 20 bp sgRNA targeting to the M+(top strand) DnaA box on E.coli genome replication initiation region, OriC. In natural situations, M+ is a low affinity box for DnaA binding. By blocking the binding of DnaA protein to M+ box, moderate arrest and inhibition of genome replication initiation is achieved.
+
This composite part is the principal design of the inducible CRISPR-based DNA replication interference system, with the 20 bp sgRNA targeting to the M+(top strand) DnaA box on E.coli genome replication initiation region, OriC. In natural situations, M+ is a low affinity box for DnaA binding. By blocking the binding of DnaA protein to M+ box, moderate arrest and inhibition of genome replication initiation is achieved. For more detailed information, see <partinfo>BBa_K3081058</partinfo>
  
−
For more detailed information, see <partinfo>BBa_K3081058</partinfo>
+
<h1>Design</h1>
 +
Based on CRISPR-interference method for transcription inhibition, we develop a novel approach for prokaryotic genome replication interference (CRISPRri). Hence, a 20-bp sgRNA is designed to be complementary to OriC, the genome replication origin (Figure 1). Instead of site-directed mutations one by one, CRISPRri allows for 20-bp scan each time. Although CRISPRri requires a PAM ("NGG") sequence to execute its function, we found a high occurrence frequency of PAM in the region of replication origin and all available sgRNAs can cover 76.2% (221 out of 290) of OriC.
 +
Seven different targeting sites for dCas9 is designed to test the effect on cell growth. We characterized M+ box in detail.
  
 
https://2019.igem.org/wiki/images/9/98/T--Peking--M%2Bgif.gif
 
https://2019.igem.org/wiki/images/9/98/T--Peking--M%2Bgif.gif

Revision as of 12:23, 21 October 2019


pBAD-dCas9-J23119-M+

This composite part is the principal design of the inducible CRISPR-based DNA replication interference system, with the 20 bp sgRNA targeting to the M+(top strand) DnaA box on E.coli genome replication initiation region, OriC. In natural situations, M+ is a low affinity box for DnaA binding. By blocking the binding of DnaA protein to M+ box, moderate arrest and inhibition of genome replication initiation is achieved. For more detailed information, see BBa_K3081058

Design

Based on CRISPR-interference method for transcription inhibition, we develop a novel approach for prokaryotic genome replication interference (CRISPRri). Hence, a 20-bp sgRNA is designed to be complementary to OriC, the genome replication origin (Figure 1). Instead of site-directed mutations one by one, CRISPRri allows for 20-bp scan each time. Although CRISPRri requires a PAM ("NGG") sequence to execute its function, we found a high occurrence frequency of PAM in the region of replication origin and all available sgRNAs can cover 76.2% (221 out of 290) of OriC. Seven different targeting sites for dCas9 is designed to test the effect on cell growth. We characterized M+ box in detail.

T--Peking--M%2Bgif.gif

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
    Illegal NheI site found at 5459
    Illegal NheI site found at 5482
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1470
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961