Part:BBa_K2457006

Cas9 device + Optimized sgRNA

This BioBrick is composed by BBa_K2457000 (AraC + pBAD), BBa_K2457001 (Cas9) and BBa_K2457002 (simple guide RNA). Which AraC + pBAD regules the Cas9 expression through your repressor and your inductor: Glucose and L-Arabinose, respectively. Cas9 is a protein that with the assistance of sgRNA cleaves a target-site in the bacterial genome. This construction was developed by Amazonas_Brazil team to be a fundamental part of the CRISPeasy toolbox, divided in CRISPReasy-out and CRISPReasy-in.

CRISPeasy-out is a standardized knock out approach that aims to turn the current method in an easier way to engineer the bacterial genome by Non-Homologous End Joining (NHEJ).

CRISPeasy-in is a standardized knock-in approach that aims to turn the current method in an easier way to insert a donor template into the bacterial genome by Homology-Directed Repair (HDR).

Figure 1: BBa K2457006 circuit

Sequencing

Figure 2:Sequencing electropherogram from BBa_K2457006.

Figure 3:Alignment of the designed sequence and our final construction from BBa_K2457006.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1207
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1042
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 1024