Difference between revisions of "Part:BBa K3081058"
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In the CRISPR replication inteference system, a 20-bp sgRNA is designed to be complementary to OriC, the genome replication origin. We use arabinose promoter to drive the expression of dCas9, while sgRNA is under the control of a J23119 promoter.
 
In the CRISPR replication inteference system, a 20-bp sgRNA is designed to be complementary to OriC, the genome replication origin. We use arabinose promoter to drive the expression of dCas9, while sgRNA is under the control of a J23119 promoter.
 
Seven different targeting sites for dCas9 is designed to test the effect on cell growth. Binding box nomenclature used here is the conventional name of binding box (e.g. "M") followed by a "+" or "-" which stands for the direction of sgRNA from 5' to 3'. For instance, M box with its bottom strand bound by dCas9 is thus called M+ box.
 
Seven different targeting sites for dCas9 is designed to test the effect on cell growth. Binding box nomenclature used here is the conventional name of binding box (e.g. "M") followed by a "+" or "-" which stands for the direction of sgRNA from 5' to 3'. For instance, M box with its bottom strand bound by dCas9 is thus called M+ box.
 +
 +
<center>https://2019.igem.org/wiki/images/4/4d/T--Peking--different_DnaA_boxes_v4.png </center> 
 +
<center> Figure 1: different targeting DnaA boxes on <i>E.coli</i> genome </center>
 +
  
 
This composite part is the principal design of the inducible CRISPR-based DNA replication interference system, but the intended sgRNAs are not assembled into the backbone, so this part contains a relatively meaningless sequence. Two BsaI sites flanking the meaningless sequence allow for easy assembly of intended sgRNA sequence by Golden Gate method. Length (412bp) of this meaningless sequence is far greater than that of normal sgRNAs (<20bp), facilitating easy detection of successfully assembled Golden Gate product when the colony PCR fragments are loaded onto gel.  
 
This composite part is the principal design of the inducible CRISPR-based DNA replication interference system, but the intended sgRNAs are not assembled into the backbone, so this part contains a relatively meaningless sequence. Two BsaI sites flanking the meaningless sequence allow for easy assembly of intended sgRNA sequence by Golden Gate method. Length (412bp) of this meaningless sequence is far greater than that of normal sgRNAs (<20bp), facilitating easy detection of successfully assembled Golden Gate product when the colony PCR fragments are loaded onto gel.  
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After Golden Gate assembly, the ‘sgRNA’ sequence will be replaced by our carefully selected sequences (like M+, BBa_K3081107; R1+, BBa_K3081108; etc.). These newly generated pBAD-dCAS9-J23119-M+/R1/… parts each contains a meaningful sgRNA which guides the dCas9 protein to different boxes on E.coli genome replication initiation region, OriC, thus blocking the binding of relevant proteins essential for replication initiation to their boxes. Arrest and inhibition of genome replication initiation is achieved in this way.
 
After Golden Gate assembly, the ‘sgRNA’ sequence will be replaced by our carefully selected sequences (like M+, BBa_K3081107; R1+, BBa_K3081108; etc.). These newly generated pBAD-dCAS9-J23119-M+/R1/… parts each contains a meaningful sgRNA which guides the dCas9 protein to different boxes on E.coli genome replication initiation region, OriC, thus blocking the binding of relevant proteins essential for replication initiation to their boxes. Arrest and inhibition of genome replication initiation is achieved in this way.
 
   
 
   
−
<center>https://2019.igem.org/wiki/images/4/4d/T--Peking--different_DnaA_boxes_v4.png </center> 
+
 
−
<center> Figure 1: different targeting DnaA boxes on <i>E.coli</i> genome </center>
+
  
 
<center>https://2019.igem.org/wiki/images/d/d0/T--Peking--sgRNA.png</center>  
 
<center>https://2019.igem.org/wiki/images/d/d0/T--Peking--sgRNA.png</center>  

Revision as of 15:27, 19 October 2019

pBAD-dCas9-J23119-sgRNA

In the CRISPR replication inteference system, a 20-bp sgRNA is designed to be complementary to OriC, the genome replication origin. We use arabinose promoter to drive the expression of dCas9, while sgRNA is under the control of a J23119 promoter. Seven different targeting sites for dCas9 is designed to test the effect on cell growth. Binding box nomenclature used here is the conventional name of binding box (e.g. "M") followed by a "+" or "-" which stands for the direction of sgRNA from 5' to 3'. For instance, M box with its bottom strand bound by dCas9 is thus called M+ box.

T--Peking--different_DnaA_boxes_v4.png
Figure 1: different targeting DnaA boxes on E.coli genome


This composite part is the principal design of the inducible CRISPR-based DNA replication interference system, but the intended sgRNAs are not assembled into the backbone, so this part contains a relatively meaningless sequence. Two BsaI sites flanking the meaningless sequence allow for easy assembly of intended sgRNA sequence by Golden Gate method. Length (412bp) of this meaningless sequence is far greater than that of normal sgRNAs (<20bp), facilitating easy detection of successfully assembled Golden Gate product when the colony PCR fragments are loaded onto gel.   After Golden Gate assembly, the ‘sgRNA’ sequence will be replaced by our carefully selected sequences (like M+, BBa_K3081107; R1+, BBa_K3081108; etc.). These newly generated pBAD-dCAS9-J23119-M+/R1/… parts each contains a meaningful sgRNA which guides the dCas9 protein to different boxes on E.coli genome replication initiation region, OriC, thus blocking the binding of relevant proteins essential for replication initiation to their boxes. Arrest and inhibition of genome replication initiation is achieved in this way.


T--Peking--sgRNA.png
Figure 2: Using Golden Gate Assembly to generate different sgRNAs

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
    Illegal XhoI site found at 5703
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 5893
    Illegal BsaI.rc site found at 5489
    Illegal SapI site found at 961