Part:BBa_K1445001

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