Difference between revisions of "Part:BBa K3081033"

Latest revision as of 19:43, 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 19bp 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

@@ 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 19bp 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
@@ Line 16: / Line 18: @@
 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.