Latest revision as of 21:14, 19 October 2016

orf1 and orf2 from Bifidobacterium

Part BBa_K1932001 was 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).

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.

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.

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).

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