Difference between revisions of "Part:BBa K3081046"
 
(One intermediate revision by one other user not shown)
Line 2: Line 2:
 
<partinfo>BBa_K3081046 short</partinfo>
 
<partinfo>BBa_K3081046 short</partinfo>
  
−
Unlike traditional CRISPRi system, our CRISPRri system<partinfo>BBa_K3081060</partinfo> is driven by an arabinose-inducible promoter with low leakage and great switch property. The expression of sgRNA is driven by a novel inducible T7 system. But the sgRNA is a relatively meaningless sequence. And two BsaI sites flanking the meaningless sequence allow for easy assembly of intended sgRNA sequence by Golden Gate method. After Golden Gate assembly, the ‘sgRNA’ sequence will be replaced by our carefully selected sequences (like M+, <partinfo>BBa_K3081107</partinfo>; R1+,<partinfo>BBa_K3081108</partinfo>; etc.). These newly generated pBAD-dCAS9-T7-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.
+
Unlike traditional CRISPRi system, our CRISPRri system<partinfo>BBa_K3081060</partinfo> is driven by an arabinose-inducible promoter with low leakage and great switch property. The expression of sgRNA is driven by a novel inducible T7 system. But the sgRNA is a relatively meaningless sequence. And two BsaI sites flanking the meaningless sequence allow for easy assembly of intended sgRNA sequence by Golden Gate method. After Golden Gate assembly, the ‘sgRNA’ sequence will be replaced by our carefully selected sequences (like M+, <partinfo>BBa_K3081107</partinfo>; R1+,<partinfo>BBa_K3081108</partinfo>; etc.). These newly generated pBAD-dCAS9-T7-M+/R1/… parts each contains a meaningful sgRNA which guides the dCas9 protein to different boxes on <i>E.coli</i> genome replication initiation region, OriC, thus blocking the binding of relevant proteins essential for replication initiation to their boxes.
  
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3081046 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3081046 SequenceAndFeatures</partinfo>
  
 +
 +
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.
 +
 +
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 19:21, 21 October 2019

pBAD-dCas9-T7-sgRNA

Unlike traditional CRISPRi system, our CRISPRri systemBBa_K3081060 is driven by an arabinose-inducible promoter with low leakage and great switch property. The expression of sgRNA is driven by a novel inducible T7 system. But the sgRNA is a relatively meaningless sequence. And two BsaI sites flanking the meaningless sequence allow for easy assembly of intended sgRNA sequence by Golden Gate method. 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-T7-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.

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 2321
    Illegal NheI site found at 6708
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
    Illegal BamHI site found at 4600
    Illegal XhoI site found at 6437
  • 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 6627
    Illegal BsaI.rc site found at 6223
    Illegal SapI site found at 961
    Illegal SapI.rc site found at 5369


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.