Part:BBa_K3777024
KB2-tetR-T7(tetO)-3WJdB-Ptet-sgRNA
A biosensor device for better tetracycline detection.
Usage and Biology
Compared to our another part TetR-tetM-T7(tetO)-3WJdB(BBa_K3777014),it adds a binding site which can be regonized by dcas9 proteins and sgRNA.(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. But BS2 will be regonized and cut by dca9 proteins and
sgRNA leading to lower fluorescence expression.
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.
The figure shows that our cuircuit can recognize different concentrations of tetracycline. What is more, CRISPRi can really inhibit our fluorescence expression
Reference：Alam Khalid K,Tawiah Kwaku D,Lichte Matthew F,Porciani David,Burke Donald H. A Fluorescent Split Aptamer for Visualizing RNA-RNA Assembly In Vivo.[J]. ACS synthetic biology,2017,6(9)
Sequence and Features
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 |