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.
Verifying the strand replacement reaction. Promoter J23104 is much stronger than J23110. TetR-T7(tetO)-3WJdB has no KB2, so it is set as a negative control.
The result of this experiment proved that it’s valid to repress the fluorescence in vivo, which strongly support the feasibility of the ‘improved circuit’.
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]
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