Coding

Part:BBa_K1932004

Designed by: Jin Liu   Group: iGEM16_Jilin_China   (2016-10-14)
Revision as of 21:33, 19 October 2016 by Mavis1 (Talk | contribs)


TAT+apoptin

TAT-Apoptin is a fusion protein with the transduction domain of HIV-1 and apoptin. Since apoptin only functions inside the cells, the peptide TAT is added to direct the transfer of the apoptin into the cell. Once transferred into the tumor cells, apoptin can induce the apoptosis of them potently.

Characterization:

The part of BBa_K1932004 was synthesized and cloned in a pGH vector by Generay Biotechnology. The plasmid was cut by the restriction enzymes, EcoRⅠ and PstⅠ, and separated by 1% agarose gel(Fig.1).

T--Jilin_China--p4-1.png

Fig.1. (1) Marker ; (2) pGH+TAT-Apoptindigested with EcoRⅠ and PstⅠ

The sequence of TAT-Apoptin was ligated into the vector pSB1C3 by T4 ligase at 16℃ overnight,and the ligated construct was transformed into the E.coli(Fig.2).

T--Jilin_China--p4-2.png

Fig.2. (1) control (only DH5α);(2) DH5α transformed with BBa_K1932004 (the TAT-Apoptin+pSB1C3 vector)

To ensure the insertion of the right-size sequence, the sequence of TAT-Apoptin was cut again and tested by agarose gel electrophoresis (Fig.3).

T--Jilin_China--p4-3.png

Fig.3.(1)Marker;(2) pSB1C3+TAT-Apoptin (BBa_K1932004) digested with EcoRⅠ and PstⅠ

Once the size of this sequence was confirmed, the bacteria containing the construct were sent to the Comate Bioscience Company for DNA sequencing for further verification. The detailed protocols of these experiments were shown in table 1 and table 2.

T--Jilin_China--T1.png

T--Jilin_China--T2.png

Since this sequence encodes the protein that undertakes the function to kill cancer cells in vivo, special assays were done to validate its function.

The construct we cloned first to confirm the function of apoptin was transfected into many kinds of cells to compare the toxicity in normal cells and cancer cells. The result of the transfection assay was shown in Figure 4 and it display robustly that this protein could kill cancer cells and do not affect normal cells significantly. MTT assay was done to validate the function of apoptin in inhibiting the cancer cells’ capacity of replication in vitro (Fig.5).

T--Jilin_China--p4-4.png

Fig.4.The Western Blot indicated that MCF-7 cells can express apoptin after transfection, and the expression was more distinctafter MCF-7 cells were transfected with 5μl plasmid (5μl) than with 2μl plasmid (2μl). But the expression of apoptin could not be detected in MCF-7 cells without tranfection(control).

T--Jilin_China--p4-5%EF%BC%88A%EF%BC%89.png

T--Jilin_China--p4-5%EF%BC%88B%EF%BC%89.png

Fig.5.Apoptin can inhibit cell proliferation of MCF-7 cells but leaving MCF-10A cells unharmed.

To investigate the characterization of TAT-apoptin further, wound scratch-healing assays were performed in MCF-7 cells and the results showed clearly that the ability of migration was also suppressed by the expression of apoptin in cancer cells (Fig.6).

T--Jilin_China--p4-6.png 

            A                       B

Fig.6.The cell migration ability of MCF-7 cells was weakened obviously after we transfected pGH-apoptin into it (B) compared to the MCF-7 cells without the construct (A).

All the assays demonstrated that the fused protein, TAT-apoptin, was an ideal drug to inhibit the growth, replication and migration of cancer cells in vitro, and the subsequent experiments were needed to examined the function of the TAT-apoptin protein in vivo.

References:

【1】Danen-Van Oorschot, A. A. A. M., Fischer, D. F., Grimbergen, J. E., Klein, B., Zhuang, S. M., Falkenburg, J. H. F., ... &Noteborn, M. H. M. (1997).Apoptin induces apoptosis in human transformed and malignant cells but not in normal cells. Proceedings of the National Academy of Sciences, 94(11), 5843-5847.

【2】Li, J., Wang, H., Ma, Z., Fan, W., Li, Y., Han, B., ... & Wang, J. (2012). TAT-Apoptin induces apoptosis in the human bladder cancer EJ cell line and regulates Bax, Bcl-2, caspase-3 and survivin expression. ExpTher Med, 3(6), 1033-8.

【3】Ma, J. L., Han, S. X., Zhao, J., Zhang, D., Wang, L., Li, Y. D., & Zhu, Q. (2012). Systemic delivery of lentivirus-mediated secretable TAT-apoptin eradicates hepatocellular carcinoma xenografts in nude mice. International journal of oncology, 41(3), 1013-1020.

【4】Zhou, S., Zhang, M., & Wang, J. (2011).Tumor-targeted delivery of TAT-Apoptin fusion gene using Escherichia coli Nissle 1917 to colorectal cancer.Medical hypotheses, 76(4), 533-534.

【5】Guelen, L., Paterson, H., Gäken, J., Meyers, M., Farzaneh, F., &Tavassoli, M. (2004). TAT-apoptin is efficiently delivered and induces apoptosis in cancer cells. Oncogene, 23(5), 1153-1165.

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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
None