Part:BBa_K3777028
luxR-Plux-dCas9
A circuit that can produce dcas9 protein which can bind to our another part BS2(BBa_K3777005) leading to a better result.
Usage and Biology
The genetic circuit was composed of a coding sequence of dcas9 protein which was inserted into an expression vectors with a Inducible promoter(BBa_R0062) and RBS(BBa_K3777030).The terminator we used was BBa_B0010 .(Fig 1)
dcas9 proteins can bind to our another part BS2(BBa_K3777005),leading to dcas9 proteins and sgRNA bind to the upstream binding site of KB2, inhibit 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
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2204
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 4483
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1022
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