Part:BBa_K3365061

Target transcription activating unit upstream of RFP

This part is an on-target detecting platform for dCas9-ω. We put this part in a plasmid and transfer it into E.coli, which was knocked out rpoZ. For convenience, we called it △rpoZ-MG1655. If dCas9-ω is binding to target sequence, the subnit Omega fused to dCas9 C-terminal can activate the expression of reporter by recruiting the RNAP holoenzyme and the cell would emit red fluorescence,which means dCas9-ωis on-target, else the weak promoter and the lack of rpoZ makes the reporter cannot express or just have a quite low expression level.

Fig.1: Schematic diagram of this part

We co-transformed plasmid contained this part and pWJ66 with dCas9-ωin △rpoZ-MG1655 to determine if the system can work well. The transformed △rpoZ-MG1655 without pWJ66 is used as control. We took isolated colonies to pre-inoculate in 5mL of Luria-Bertani (LB) , incubating overnight in a shaker at 37℃. After that we detect the fluorescence intensity of the bacterial solution by microplate reader every two hours.Ths result is shown as follow.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 66
  Illegal NheI site found at 89
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 812
  Illegal XhoI site found at 803
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 667
  Illegal AgeI site found at 779
  
• 1000
  COMPATIBLE WITH RFC[1000]