Part:BBa_K1487022

Promoter + Guide RNA (gRNA) target for acfB

This part contains guide RNA (gRNA) that can target accessory colonization factor B (acfB) gene under strong constitutive promoter J23119. This part contains a 42bp binding handle, 20bp targeting region, and a 40bp terminator. This part is ready to use and can form a complex with Cas9 protein to direct a double strand break at a complementary sequence.

This part was tested in Escherichia coli using Reporter part BBa_K1487064, which contains a complementary sequence that is able to be targeted by this gRNA. The reporter is designed with a constitutive promoter so that it continuously produces eYFP (eYFP sequence was cloned from BBa_E0030). Once targeted and cut by the Cas9/gRNA complex, the cells stop producing eYFP and relative fluorescence will decrease. This reporter also confers chloramphenicol resistance, so if it is cleaved in the presence of chloramphenicol-containing media, it will result in cell death. This allowed us to measure the effectiveness of the gRNA/Cas9 targeting by measuring fluorescence as well as cell viability.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]