Part:BBa_K5441016
PB_Gluc_BAX
High-efficiency prostate cancer cells killing system with BAX apoptosis regulator
PB promoter with Gluc and BAX at its downstream.
Evaluation of killing ability theoretically
The wild-type probasin promoter exhibits prostate-specific activity, as shown by its differential expression in different cell types[1]. In previous research, the probasin promoter (ARR2PB) has also been used to express the BAX gene in androgen receptor-positive prostate cancer cells[2]. BAX is an apoptosis regulator, controlling mitochondrial dysfunction and cell killing, and this characteristic of BAX can be utilised to kill cancer cells. The sequence also includes the gene to encode Gaussia Luciferase for the detection of prostate cancer cells.
Evaluation of killing ability experimentally
Firstly, a mammalian plasmid containing the gene PB-Gluc-Bax is cloned. To verify whether the gene was ligated using mammalian plasmid, we utilized plasmid extraction, followed by restriction digestion. The diagram shown below is the plasmid map and the results of the linear plasmid under gel electrophoresis.
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Plasmid map for pENTR1A-PB-Gluc-Bax |
Gel electrophoresis result for linearised pENTR1A-PB-Gluc-Bax |
To test for the effect of killer gene BAX on prostate cancer cells, we seeded PSMA-positive prostate cancer cells (MLLB-2) into 36 wells, all containing the same cell concentration (30,000 cells/mm2), in a 96-well plate. The reactions between PB-Gluc-BAX and cancer cells can be monitored by the MTT Cell Viability Assay and can be measured by absorption using the wavelength 540 nm, which is positively correlated with the number of cells.
Absorption of the reaction between PB-Gluc-BAX and cancer cells.
The setup without pENTR1A-PB-Gluc-Bax demonstrates a faster growth rate on the second day than first (p < 0.001). This proves that our wells were suitable mediums for the cells to grow from days 0 to 2 of the experiment.
In low (0.00124 μg / μL) and high (0.00496 μg / μL) concentrations of pENTR1A-PB-Gluc-Bax, the rate of increase in cancer cells is significantly slower than the setups without BAX transfected from day 0 to day 2 (p < 0.05). A significant drop in the number of cancer cells was also observed for the setup with a medium (0.00248 μg / μL) concentration of plasmids transfected.
In future studies, we plan to quantify the amount of Gluc expressed with the Gaussia Luciferase Flash Assay, using a multimode plate reader to measure the luminescence given out by the reaction. The result is predicted to show similar correlations with the MTT Cell Assay.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1510
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
[1] Zhang, J., Gao, N., DeGraff, D. J., Yu, X., Sun, Q., Case, T. C., Kasper, S., & Matusik, R. J. (2010). Characterization of cis elements of the probasin promoter necessary for prostate‐specific gene expression. The Prostate, 70(9), 934–951. https://doi.org/10.1002/pros.21128 [2] Andriani, F., Nan, B., Yu, J., Li, X., Weigel, N. L., McPhaul, M. J., Kasper, S., Kagawa, S., Fang, B., Matusik, R. J., Denner, L., & Marcelli, M. (2001). Use of the Probasin promoter ARR2PB to express BAX in androgen Receptor-Positive Prostate cancer cells. JNCI Journal of the National Cancer Institute, 93(17), 1314–1324. https://doi.org/10.1093/jnci/93.17.1314
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