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. (please view:[1])
![[1]](https://parts.igem.org/File:The_result_of_microplate_reader.jpeg){kind=link}
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 66
Illegal NheI site found at 89 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 812
Illegal XhoI site found at 803 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 667
Illegal AgeI site found at 779 - 1000COMPATIBLE WITH RFC[1000]
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