Part:BBa_K3777014
TetR-tetM-T7(tetO)-3WJdB
A biosensor device for tetracycline detection.It is worh mentioning that we added a antibiotic resistance gene into our circuits to ensure its robust performance.
Usage and Biology
The genetic circuit was composed of coding sequences of tetracycline repressor genes and tetM genes which was inserted into an expression vectors with a consitive promoter(BBa_J23114) as well as 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)
Tetracyclines (including tetracycline and chlortetracycline) inhibited protein synthesis by preventing aminoacyl-tRNA from binding to the ribosomal acceptor site. However, tetM gene confer antibiotic resistance by producing ribosomal protection proteins. So we hope this circuit could possess higher sensitivity and robust.
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.
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 2986 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2598
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2024
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