Revision as of 19:55, 19 October 2016

This device is made up of BBa_K1932000;BBa_K1932001;BBa_K1932004

The device is designed for stable expression of the TAT-Apoptin. Among the subparts, BBa_K1932000 is included to regulate the expression of exogenous protein in Bifidobacterium with a strong promoter. BBa_K1932001 is included to increase the stability of the device in Bifidobacterium. BBa_K1932004 encodes TAT-Apoptin, which acts as the effector protein to kill the cancer cells.

Characterization:

The part of BBa_K1932005 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(Figure 1).

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

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

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.

The device was amplified in the competence bacterium DH5α, and was extracted and purified with the Plasmid Minipreparation Kit from BioTeke. To test the usage of our device in different laboratories, the expression of the protein was examined by the BIT-China with the method of SDS-PAGE (Fig. 4).

References:

【1】Missich, R., Sgorbati, B., & LeBlanc, D. J. (1994). Transformation of Bifidobacterium longum with pRM2, a constructed Escherichia coli-B.</i>longum</i> shuttle vector. Plasmid, 32(2), 208-211.

【2】Nakamura, T., Sasaki, T., Fujimori, M., Yazawa, K., Kano, Y., Amano, J., & Taniguchi, S. I. (2002). Cloned cytosine deaminase gene expression of Bifidobacterium longum and application to enzyme/pro-drug therapy of hypoxic solid tumors. Bioscience, biotechnology, and biochemistry, 66(11), 2362-2366.

【3】Matsumura, H., Takeuchi, A., & Kano, Y. (1997). Construction of Escherichia coli–Bifidobacterium longumshuttle vector transforming B.longum 105-A and 108-A. Bioscience, biotechnology, and biochemistry, 61(7), 1211-1212.

【4】Shkoporov, A. N., Efimov, B. A., Khokhlova, E. V., Steele, J. L., Kafarskaia, L. I., &Smeianov, V. V. (2008). Characterization of plasmids from human infant Bifidobacterium strains: sequence analysis and construction of E. coli–Bifidobacterium shuttle vectors. Plasmid, 60(2), 136-148.

【5】Hou, X., & Liu, J. E. (2006).Construction of Escherichia coli-Bifidobacterium longum shuttle vector and expression of tumor suppressor gene PTEN in B.longum. Actamicrobiologica Sinica, 46(3), 347-352.

Sequence and Features