Composite

Part:BBa_K3365061

Designed by: Zou Aixuan   Group: iGEM20_SJTU-BioX-Shanghai   (2020-10-23)
Revision as of 13:11, 27 October 2020 by Gyaxxxxxx (Talk | contribs)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)


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.

Target binding site for dCas9 and subnit Omega.png

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. (please view:[1])

The result of microplate reader.jpeg

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]


[edit]
Categories
Parameters
None