Difference between revisions of "Part:BBa K3081033"

 
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This composite part is the principal design of the inducible CRISPR-based DNA replication interference system, with the 19 bp sgRNA targeting to the R1+ DnaA box on E.coli genome replication initiation region, OriC. In natural situations, R1+ is a high affinity box for DnaA binding. By blocking the binding of DnaA protein to R1+ box using a 18bp sgRNA, alleviation of severe arrest and inhibition to the genome replication initiation is achieved. This part is an improvement of <partinfo>BBa_K3081009</partinfo>, which we add a degradation signal peptide ssrA to the dCas9. This  largely accelerates the degradation rate of dCas9 and weakens its overinhibtion on genome replication initiation targeted to the R1 box.
 
This composite part is the principal design of the inducible CRISPR-based DNA replication interference system, with the 19 bp sgRNA targeting to the R1+ DnaA box on E.coli genome replication initiation region, OriC. In natural situations, R1+ is a high affinity box for DnaA binding. By blocking the binding of DnaA protein to R1+ box using a 18bp sgRNA, alleviation of severe arrest and inhibition to the genome replication initiation is achieved. This part is an improvement of <partinfo>BBa_K3081009</partinfo>, which we add a degradation signal peptide ssrA to the dCas9. This  largely accelerates the degradation rate of dCas9 and weakens its overinhibtion on genome replication initiation targeted to the R1 box.
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https://2019.igem.org/wiki/images/4/49/T--Peking--R1%2B%2820bp%29_ssrA.gif
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R1+ssrA 20bp
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https://2019.igem.org/wiki/images/6/6b/T--Peking--R1%2B%2819bp%29_ssrA.gif
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R1+ssrA 19bp
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https://2019.igem.org/wiki/images/0/05/T--Peking--R1%2B%2818bp%29_ssrA.gif
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R1+ssrA 18bp
  
  

Revision as of 18:52, 21 October 2019


pBAD-dCas9-ssrA-J23119-R1+(19bp)

This composite part is the principal design of the inducible CRISPR-based DNA replication interference system, with the 19 bp sgRNA targeting to the R1+ DnaA box on E.coli genome replication initiation region, OriC. In natural situations, R1+ is a high affinity box for DnaA binding. By blocking the binding of DnaA protein to R1+ box using a 18bp sgRNA, alleviation of severe arrest and inhibition to the genome replication initiation is achieved. This part is an improvement of BBa_K3081009, which we add a degradation signal peptide ssrA to the dCas9. This largely accelerates the degradation rate of dCas9 and weakens its overinhibtion on genome replication initiation targeted to the R1 box.

T--Peking--R1%2B%2820bp%29_ssrA.gif

R1+ssrA 20bp

T--Peking--R1%2B%2819bp%29_ssrA.gif

R1+ssrA 19bp

T--Peking--R1%2B%2818bp%29_ssrA.gif

R1+ssrA 18bp


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