Part:BBa_K4179020
Luc_Blast_OraCell
The composite part comprises a Blasticidin S deaminase (BBa_K1943015), which is a Blasticidin resistance gene with the Kozak sequence. Downstream to this under the CMV (BBa_I712004) which is a constitutive promoter, an SV40PolyA terminator (BBa_K4235020) is cloned. Together in this composite, a cTNT promoter (BBa_K4179021) is found, downstream to the 8XGTIIC motifs (BBa_K4179017) followed by the luciferase gene (BBa_I712019) and the low most part SV40 Poly(A)(BBa_K4235020).
Usage and Biology
The team of Technion 2022 used this composite as a part of their cell line measurement system called Oracell. This biological system quantifies their manufactured metabolite-decursin by utilizing its effect on the Hippo pathway which is a signaling pathway that regulated proliferation and apoptosis in mammalian cells [1]. TEAD transcriptional factors bind to the 8XGTIIC motifs and regulate the expression of the luciferase gene which is cloned downstream to these motifs. Luciferase is used as a reporter gene that emits luminescence. Thus, the quantity of this metabolite is measured according to the luminescence values, by using a calibration curve. This composite is transfected to CHO (Chinese Hamster Ovarian) cells by stable integration. The role of the Blasticidin S deaminase here is to accomplish a successful selection for positive cells after transfection.
References
1.Yu, F. X., & Guan, K. L. (2013). The Hippo pathway: Regulators and regulations. In Geneand Development (Vol. 27, Issue 4, pp. 355–371). doi: 10.1101/gad.210773.112 Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 213
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1090
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