Revision as of 21:04, 19 October 2016

orf1 and orf2 from Bifidobacterium

Part BBa_K1932001was originally cloned from the plasmid of Bifidobacterium that contains two orf sequences.（The plasmid had a G + C content of 62.0%, and contained two open reading frames, orf1 and orf2）.This sequence is essential for the shuttling of the plasmid from E.coli to Bifidobacterium.

Characterization:

The part of BBa_K1932001 was synthesized and cloned into a pGH vector by Generay Biotechnology. This plasmid was cut with the restriction enzyme, EcoRⅠand PstⅠ, and separated by 1% agarose gel (Fig.1).

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Fig.1. (1): pGH+PMB1; (2) marker;(3)pGH+PMB1 digested with EcoRⅠ and PstⅠ

The sequence of PMB1 was ligated into the vector pSB1C3 by ligase in 16℃ overnight, and the ligated products were transformed into the E.coli(Fig.2). However, we haven't succeeded in inserting the part into the vector, pSB1C3.

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Fig.2. (1) control only DH5α; (2) DH5α transformed with BBa_K1932001 (the PMB1+pSB1C3 vector)

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

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The sequence of PMB1 differs from the sequence of pMB1 from the vector, pSB1C3,which was shown with the tool of BLAST online(Fig.3).

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Fig.3. The sequence of PMB1 is different from the sequence of pMB1 in pSB1C3

References:

【1】Rossi, M., Brigidi, P., y Rodriguez, A. G. V., &Matteuzzi, D. (1996).Characterization of the plasmid pMB1 from Bifidobacterium longum and its use for shuttle vector construction.Research in microbiology, 147(3), 133-143.

【2】Matteuzzi, D.,Brigidi, P., Rossi, M., & Di, D. (1990).Characterization and molecular cloning of Bifidobacterium longum cryptic plasmid pMB1.Letters in applied microbiology, 11(4), 220-223.

【3】Xu, Y. F., Zhu, L. P., Hu, B., Fu, G. F., Zhang, H. Y., Wang, J. J., &Xu, G. X. (2007). A new expression plasmid in Bifidobacterium longum as a delivery system of endostatin for cancer gene therapy.Cancer gene therapy, 14(2), 151-157.

【4】Corneau, N., Émond, É., &LaPointe, G. (2004).Molecular characterization of three plasmids from Bifidobacterium longum.Plasmid, 51(2), 87-100.

【5】Hu, B., Kou, L., Li, C., Zhu, L. P., Fan, Y. R., Wu, Z. W., ... &Xu, G. X. (2009). Bifidobacterium longum as a delivery system of TRAIL and endostatin cooperates with chemotherapeutic drugs to inhibit hypoxic tumor growth.Cancer gene therapy, 16(8), 655-663.

Sequence and Features