Difference between revisions of "Part:BBa K4179020"

Revision as of 14:26, 8 October 2022

Luc_Blast_OraCell

The composite part comprises a Blasticidin S deaminase (BBa_K1943015), which is a Blasticidin resistance gene with the Kozak sequence, under the CMV constitutive promoter (BBa_I712004). Downstream to it an SV40PolyA terminator (BBa_K4235020) is found. Together in this composite, a cTNT promoter (BBa_K4179021) is cloned downstream to the 8XGTIIC motifs (BBa_K4179017), followed by the luciferase gene (BBa_I712019) and finally the 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. Decursin inhibits expression of luciferase gene by inhibiting the Hippo pathway. 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. To read more about the OraCell measurement tool, visit the team's wiki page.

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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 213
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 1090

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 <partinfo>BBa_K4179020 short</partinfo>
-The composite part comprises a Blasticidin S deaminase [https://parts.igem.org/Part:BBa_K1943015 (BBa_K1943015)], which is a Blasticidin resistance gene with the Kozak sequence. Downstream to this under the CMV [https://parts.igem.org/Part:BBa_I712004 (BBa_I712004)] which is a constitutive promoter, an SV40PolyA terminator [https://parts.igem.org/Part:BBa_K4235020 (BBa_K4235020)] is cloned. Together in this composite, a cTNT promoter [https://parts.igem.org/Part:BBa_K4179021 (BBa_K4179021)] is found, downstream to the 8XGTIIC motifs [https://parts.igem.org/Part:BBa_K4179017 (BBa_K4179017)] followed by the luciferase gene [https://parts.igem.org/Part:BBa_I712019 (BBa_I712019)] and the low most part SV40 Poly(A)[https://parts.igem.org/Part:BBa_K4235020 (BBa_K4235020)].
+The composite part comprises a Blasticidin S deaminase [https://parts.igem.org/Part:BBa_K1943015 (BBa_K1943015)], which is a Blasticidin resistance gene with the Kozak sequence, under the CMV constitutive promoter [https://parts.igem.org/Part:BBa_I712004 (BBa_I712004)]. Downstream to it an SV40PolyA terminator [https://parts.igem.org/Part:BBa_K4235020 (BBa_K4235020)] is found. Together in this composite, a cTNT promoter [https://parts.igem.org/Part:BBa_K4179021 (BBa_K4179021)] is cloned downstream to the 8XGTIIC motifs [https://parts.igem.org/Part:BBa_K4179017 (BBa_K4179017)], followed by the luciferase gene [https://parts.igem.org/Part:BBa_I712019 (BBa_I712019)] and finally the SV40 Poly(A) [https://parts.igem.org/Part:BBa_K4235020 (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.
+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. Decursin inhibits expression of luciferase gene by inhibiting the Hippo pathway. 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. To read more about the OraCell measurement tool, visit the team's wiki page.
 ==References==