Part:BBa_K3777024
KB2-tetR-T7(tetO)-3WJdB-Ptet-sgRNA
Basic biosensor device for tetracycline detection.
Usage and Biology
The genetic circuit was composed of a coding sequence of tetracycline repressor which was inserted into an expression vectors with a consitive promoter(BBa_J23114) and RBS(BBa_K3777030), as well as 3WJdB(BBa_K3777000) under the control of T7 promoter (BBa_K3777006). The terminator we used were BBa_B0010 and BBa_M36305.(Fig 1)
When tet was absent, TetR would bind to the inducible promoter（PI）and prevent RNA polymerase from initiating transcription, thus repressing the expression of reporter gene. If tet was present, TetR would no longer able to bind to the promoter, resulting in the expression of reporter gene.
We expressed this circuit in the E. coli BL21(DE3) cells for tetracycline detection. Thus we could roughly deduce the concentration of the antibiotics in the sample according to the fluorescence intensity.
Fig.1 Schematic overview of the genetic circuit.
Results
To verify the functionality of the biosensor, we performed a plate-reader experiment and measured optical density and fluorescence intensity every hour. We observed a correlation between concentration of antibiotics in the sample and intensity of fluorescent signal.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 260
Illegal NheI site found at 283
Illegal NheI site found at 1262 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 121
Illegal XhoI site found at 1388 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |