Part:BBa_K4179020

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

OraCell cells showed a periodic behavior of decursin’s effect on luciferase expression via the Hippo pathway over time. Additionally, following a 22-hour incubation time with decursin, the luminescence was markedly decreased compared to the same incubation time without decursin. This result also showed that the OraCell cells showed a clear bioluminescent signal compared to the negative controls, the same cells without the addition of luciferin and CHO cells that do not express luciferase.(See figure.1-Decursin incubation time calibration)

Figure 1. Decursin incubation time calibration. OraCell cells were incubated with decursin with various time points ranging from 1-22 hours, lysed, luciferin was added and bioluminescence was measured. In addition, there were two types of negative control used, OraCell cells without luciferin added and CHO cells that do not express luciferase. The positive control was OraCell cells incubated without decursin.

After the incubation time calibration test, a decursin incubation time experiment was conducted (shown in figure.2-Decursin incubation time) reinforced the trend shown previously (Figure.1), that there’s a periodic oscillation of decursin’s impact on the Hippo pathway over time. It can also be seen that in the negative control, cell without decursin, following a 16-hour incubation there was a decrease in luminescence compared to the three previous time points.

Figure 2. Decursin incubation time. OraCell cells were incubated with and without decursin at various time points ranging from 1-16 hours, lysed, luciferin was added, and bioluminescence was measured. The cells without decursin served as the positive control, to see how much decursin impacted the cells’ bioluminescence.

Following a 3-hour incubation with decursin, there was no clear trend to be discerned from the calibration curve. However, following a 16-hour incubation time with decursin, there was a clear trend that with increased decursin there was increased luminescence(figure.3-Calibration Curve of decursin concentration to luminescence at 3 and 16 hours.) This result along with others (Figures 1 and 2), brought a potential new understanding of decursin’s impact on the OraCell system and the Hippo pathway as a whole. Their working theory is that decursin is capable of increasing or decreasing luciferase expression as explained on their wiki site.

Figure 3. Calibration Curve of decursin concentration to luminescence at 3 and 16 hours. OraCell cells were incubated with six concentrations of decursin ranging from 0-240 µM for two time points, 3 and 16 hours. The cells were then lysed, luciferin was added, and bioluminescence was measured. The luminescence was normalized to the zero concentration of decursin for each time point, respectively.

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