Device

Part:BBa_K1932005

Designed by: Jin Liu   Group: iGEM16_Jilin_China   (2016-10-14)


This device is made up of BBa_K1932000;BBa_K1932001;BBa_K1932004

This device was improved from the part of BBa_K1166005(https://parts.igem.org/Part:BBa_K1166005) registered by TecMonterrey(http://2013.igem.org/Team:TecMonterrey) and was 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:

This device was improved from the part of BBa_K1166005(https://parts.igem.org/Part:BBa_K1166005) registered by TecMonterrey(http://2013.igem.org/Team:TecMonterrey), which encodes the fusion protein, TAT-apoptin. To use the biobrick in Bifidobacterium, BBa_K1932000 and BBa_K1932001 were added. Compared with the old part, the device could stably exist and replicate in Bifidobacterium and the expression of TAT-apoptin in Bifidobacterium was up-regulated.

This part 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).

T--Jilin_China--p5-1.png

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

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). However, we haven't succeeded in inserting the part into the vector, pSB1C3.


T--Jilin_China--p5-2.png

Fig.2. (1) control (only DH5α); (2) colony of DH5α that transformed with the BBa_K1932005 (TAT-Apoptin-device+pSB1C3 vector)

The detailed protocols of these experiments were shown in table 1 and table 2.

T--Jilin_China--T1.png

T--Jilin_China--T2.png

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. 3).

T--Jilin_China--p5-4.png

Fig. 3.Different temperature induced with precipitation

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


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 1059


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