Difference between revisions of "Part:BBa K1445001"
 
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<partinfo>BBa_K1445001 short</partinfo>
 
<partinfo>BBa_K1445001 short</partinfo>
  
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The M13ori-pCas9 composite part consists of the M13 origin of replication (M13ori) and the pCas9 part (BBa_K1218011) containing a tracrRNA sequence, the cas9 gene, and a minimal CRISPR array.
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This composite part consists of parts BBa_K1445000 (M13ori) and BBa_K1218011 (cas9).
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The M13 origin of replication is a non-coding sequence that facilitates the uptake of a plasmid as a single-stranded phagemid into the M13 or Fd phage. It does not code for phage coat proteins; therefore cannot produce phage on its own.
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The cas9 construct from ''Streptococcus pyogenes'' consists of the native trcrRNA and promoter region upstream of the type II cas9 endonuclease. Downstream of the Cas9 protein is a minimal CRISPR array that includes two CRISPR repeats flanking a spacer region. It is this spacer region that determines what DNA sequence is targeted by the Cas9 endonuclease. The spacer sequence can be easily replaced through a digestion-ligation reaction using BsaI.
  
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M13ori (BBa_K1445000) is a 500bp noncoding sequence that is recognized by the gene II of the M13 phage, which results in the uptake of the plasmid containing the ori into an assembled phage coat. This part does not contain any complete phage genes so phage production requires a Helper Phagemid such as M13K07. There will be some packaging of Helper Phagemid but their packaging signal weakened so parts containing the M13ori are preferentially packaged.
 
  
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pCas9 contains the native trcrRNA and promoter region upstream of the Cas9 protein. The type II Cas9 protein is the native, active version from streptococcus pyogenes with the exceptrion of a SNP to erase an illegal EcoRI cleavage site. Downstream of the Cas9 protein is a minimal CRISPR array. This minimal array only includes two CRISPR repeats flanking a spacer region. It is this spacer region that determines which DNA sequence is targeted by the Cas9 endonuclease. Spacer sequence can be easily replaced with the protocol found here.
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===Usage and Biology===
  
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This composite part was packaged into M13 phage using the M13K07 helper phagemid. Conjugated BW23115 ''E. coli'' were infected with the progeny phage and plated onto LB agar plates containing chloramphenicol to select for infected cells.
  
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<!-- Add more about the biology of this part here -->
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[[File:Boulder_M13ori-pCas9_FIGURE_1.png|300px|thumb|none|Figure 1: Conjugated BW23115 ''E. coli''  infected with BBa_K1445001 on a pSB1C3 backbone, grown on LB agar with chloramphenicol at 170 ug/mL.]]
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===Usage and Biology===
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[[File:Boulder_M13ori-pCas9_FIGURE_1.png|300px|thumb|left|Boulder_M13ori-pCas9_FIGURE_1.png]]
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The addition of the M13 origin of replication allows for successful delivery of the Cas9 machinery to conjugated cells via recombinant M13 phage as demonstrated by the presence of colonies in Figure 1.
  
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K1445001 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1445001 SequenceAndFeatures</partinfo>

Latest revision as of 12:19, 17 October 2014

Endogenous Type II CRISPR-Cas9 phagemid

This composite part consists of parts BBa_K1445000 (M13ori) and BBa_K1218011 (cas9).

The M13 origin of replication is a non-coding sequence that facilitates the uptake of a plasmid as a single-stranded phagemid into the M13 or Fd phage. It does not code for phage coat proteins; therefore cannot produce phage on its own.

The cas9 construct from Streptococcus pyogenes consists of the native trcrRNA and promoter region upstream of the type II cas9 endonuclease. Downstream of the Cas9 protein is a minimal CRISPR array that includes two CRISPR repeats flanking a spacer region. It is this spacer region that determines what DNA sequence is targeted by the Cas9 endonuclease. The spacer sequence can be easily replaced through a digestion-ligation reaction using BsaI.


Usage and Biology

This composite part was packaged into M13 phage using the M13K07 helper phagemid. Conjugated BW23115 E. coli were infected with the progeny phage and plated onto LB agar plates containing chloramphenicol to select for infected cells.

Figure 1: Conjugated BW23115 E. coli infected with BBa_K1445001 on a pSB1C3 backbone, grown on LB agar with chloramphenicol at 170 ug/mL.

The addition of the M13 origin of replication allows for successful delivery of the Cas9 machinery to conjugated cells via recombinant M13 phage as demonstrated by the presence of colonies in Figure 1.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 2179
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 4458
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 5400
    Illegal BsaI.rc site found at 5377