Difference between revisions of "Part:BBa K3081057"
 
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<partinfo>BBa_K3081057 short</partinfo>
 
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This composite part is the principal design of the inducible CRISPR-based DNA replication interference system, with the 20 bp sgRNA targeting to the bottom strand of initiation site on p15A origin. In this part, we add a degradation signal peptide "ssrA" to the dCas9 to alleviate its effect.  
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This composite part uses a CRISPR-based DNA replication interference system for the copy number control of the p15A plasmid. The dCas9 is expressed in an inducible manner with a ssrA degradation tag fused to its C-terminal to minimize the basal expression. The sgRNA has a 20-bp complementary sequence with the bottom strand (the strand that replication activator (RNA II) binds to) of initiation site (+1) on p15A origin. It can efficiently downregulate the plasmid copy number.
 
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https://2019.igem.org/wiki/images/3/3b/T--Peking--p15A_control_schematic.png
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https://2019.igem.org/wiki/images/c/ca/T--Peking--p15A_mechanism.png
 
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Mechanism of CRISPRri-based p15A plasmid copy number control
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<b>Design of the p15A plasmid copy number control system</b>
 
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<center>
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https://2019.igem.org/wiki/images/f/fa/T--Peking--p15A_result.png
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<b>The effect of 4 target sites on p15A origin</b>
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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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===Usage and Biology===
 
===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3081057 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3081057 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display  
 
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K3081057 parameters</partinfo>
 
<partinfo>BBa_K3081057 parameters</partinfo>
 
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Latest revision as of 19:18, 21 October 2019

CRISPR-based replication interference system for p15A plasmid copy number control, ini(+) site

This composite part uses a CRISPR-based DNA replication interference system for the copy number control of the p15A plasmid. The dCas9 is expressed in an inducible manner with a ssrA degradation tag fused to its C-terminal to minimize the basal expression. The sgRNA has a 20-bp complementary sequence with the bottom strand (the strand that replication activator (RNA II) binds to) of initiation site (+1) on p15A origin. It can efficiently downregulate the plasmid copy number.

T--Peking--p15A_mechanism.png

Design of the p15A plasmid copy number control system

T--Peking--p15A_result.png

The effect of 4 target sites on p15A origin


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 2321
    Illegal NheI site found at 5495
    Illegal NheI site found at 5518
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
    Illegal BamHI site found at 4600
  • 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