Difference between revisions of "Part:BBa K3081033"

Revision as of 19:12, 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

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3081033 short</partinfo>
-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 <i>E.coli</i> 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.
 https://2019.igem.org/wiki/images/4/49/T--Peking--R1%2B%2820bp%29_ssrA.gif