Part:BBa_K2845046:Design
TetR fused the C-terminus of split EPIC luciferase
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 482
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 186
Design Notes
TetR is a transcription factor repressing the expression of tetA, a membrane protein responsible for resistance against the antibiotic tetracycline. TetR constitutively binds to the operator site in the promoter of tetA and suppresses its expression. Only when sensing the antibiotic or its homologues, it is released from the operator site and transcription is activated. We use the constitutively bound state of TetR to attach the C-terminus of split EPIC luciferase to DNA. This biobrick is designed to be used in a system together with a fusion protein consisting of the N-terminus of the split EPIC luciferase fused to OmpR and the binding sites of OmpR and TetR placed next to each other on a high copy plasmid. Since TetR is constitutively binding to its binding site, the two luciferase halves are only brought into proximity when OmpR binds to its binding site next to the TetR-split EPIC luciferase fusion protein. This allows the visualization of transcription factor binding by circumventing the time-consuming step of transcription and translation.