Difference between revisions of "Part:BBa K4179020"
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===Characterization===
 
===Characterization===
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<b>Stable Integration</b>
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To establish a stable clonal cell line, cells were transfected with the BlastR-modified luciferase plasmid and subjected to a high concentration of blasticidin. Then, we used FACS (Bigfoot Spectral Cell Sorter) to perform single-sorting cell into individual wells on a plate and allowed cells to proliferate before testing their luciferase expression with luciferin.
 +
 +
<b>Luciferase assay</b>
 +
 +
To measure the transfected cell reporter gene, luciferase, we performed a luminescence assay. This was done both to validate the transfection itself, and to study how decursin affects the gene expression.
 +
We used a Biotium’s Firefly Luciferase Assay Kit 2.0, cells were lysed, and luciferin was added to the lysate. This resulted in an oxidation reaction that emitted a measurable signal. The signal was measured using the bioluminescent mode of a plate reader. Our positive control for the experiment was E. coli β-10 cells transfected with a p.lux plasmid that contains the genes encoding for both luciferase and luciferin.
 +
 +
<b>Decursin assay</b>
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Revision as of 15:19, 8 October 2022


Luc_Blast_OraCell

Figure 1: The Hippo pathway [2]: red X showing decursin's effect

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 (figure 1). 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.


Characterization

Stable Integration

To establish a stable clonal cell line, cells were transfected with the BlastR-modified luciferase plasmid and subjected to a high concentration of blasticidin. Then, we used FACS (Bigfoot Spectral Cell Sorter) to perform single-sorting cell into individual wells on a plate and allowed cells to proliferate before testing their luciferase expression with luciferin.

Luciferase assay

To measure the transfected cell reporter gene, luciferase, we performed a luminescence assay. This was done both to validate the transfection itself, and to study how decursin affects the gene expression. We used a Biotium’s Firefly Luciferase Assay Kit 2.0, cells were lysed, and luciferin was added to the lysate. This resulted in an oxidation reaction that emitted a measurable signal. The signal was measured using the bioluminescent mode of a plate reader. Our positive control for the experiment was E. coli β-10 cells transfected with a p.lux plasmid that contains the genes encoding for both luciferase and luciferin.

Decursin assay



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

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 2. Liu, Q., Liu, X., & Song, G. (2021). The Hippo Pathway: A Master Regulatory Network Important in Cancer. Cells, 10(6), 1416. https://doi.org/10.3390/cells10061416