Difference between revisions of "Part:BBa K3081032"
 
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This composite part is the principal design of the inducible CRISPR-based DNA replication interference system, with the 18 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 18 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.
  
 +
Our experiments reveal that multi-input control is achievable. The variables include length of sgRNA. We observed corresponding changes in cell morphology (cell elongation in most cases) among the 18 bp, 19bp, and 20bp sgRNA targeting to the R1+ DnaA box on <i>E.coli</i> genome replication initiation region, OriC.
 
https://2019.igem.org/wiki/images/4/49/T--Peking--R1%2B%2820bp%29_ssrA.gif
 
https://2019.igem.org/wiki/images/4/49/T--Peking--R1%2B%2820bp%29_ssrA.gif
  
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R1+20bp
+
R1+ssrA 20bp
  
 
https://2019.igem.org/wiki/images/6/6b/T--Peking--R1%2B%2819bp%29_ssrA.gif
 
https://2019.igem.org/wiki/images/6/6b/T--Peking--R1%2B%2819bp%29_ssrA.gif
  
−
R1+19bp
+
R1+ssrA 19bp
  
 
https://2019.igem.org/wiki/images/0/05/T--Peking--R1%2B%2818bp%29_ssrA.gif
 
https://2019.igem.org/wiki/images/0/05/T--Peking--R1%2B%2818bp%29_ssrA.gif
  
−
R1+18bp
+
R1+ssrA 18bp
 +
 
 +
 
 +
（Considering the technical problem, if the reader is interested in our results, welcome to our presentation and poster session to watch the original movie.）
 +
 
 +
 
 +
Reference：
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 +
[1]Wolański M, Donczew R, Zawilakpawlik A, et al. oriC-encoded instructions for the initiation of bacterial chromosome replication.[J]. Frontiers in Microbiology, 2015, 5(735):735.
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Latest revision as of 19:42, 21 October 2019


pBAD-dCas9-ssrA-J23119-R1+(18bp)

This composite part is the principal design of the inducible CRISPR-based DNA replication interference system, with the 18 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.

Our experiments reveal that multi-input control is achievable. The variables include length of sgRNA. We observed corresponding changes in cell morphology (cell elongation in most cases) among the 18 bp, 19bp, and 20bp sgRNA targeting to the R1+ DnaA box on E.coli genome replication initiation region, OriC. 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


（Considering the technical problem, if the reader is interested in our results, welcome to our presentation and poster session to watch the original movie.）


Reference：

[1]Wolański M, Donczew R, Zawilakpawlik A, et al. oriC-encoded instructions for the initiation of bacterial chromosome replication.[J]. Frontiers in Microbiology, 2015, 5(735):735.


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