Revision as of 19:47, 19 October 2016

A promoter and an RBS of the B.longum hup gene.

BBa_1932000 contains a part of the hup gene. This gene was cloned from Bifidobacterium to express the HU family protein, HB1, and it consisted a promoter, an RBS, a structural gene and a terminator. To use it conveniently and up-regulate the expression of downstream protein, we standardized this part to contain the sequence of this promoter and this RBS.

Characterization:

The part of BBa_K1932000 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).

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Fig.1.(1) Marker; (2) pGH+HU digested with EcoRⅠ and PstⅠ

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

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Fig.2. (1) control (only DH5α); (2) DH5α transformed by BBa_K1932000 (the HU+pSB1C3 vector)

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

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Fig.3.(1) Marker; (2) BBa_K1932000 (pSB1C3+HU) digested with EcoRⅠ and PSTⅠ;(3)BBa_K1932000(pSB1C3+HU)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.

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In addition to cloning and verifying of the HU sequence, we used rfp gene to check the function of this part to regulate the expression of rfp protein in B.longum. The cloning of HU sequence with rfp gene was assisted by the UESTC_China team ,following the molecular cloning protocol (Fig.4).

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Fig.4.The plasmid constructed by UESTC-China.

References:

【1】Takeuchi, A., Matsumura, H., & Kano, Y. (2002). Cloning and expression in Escherichia coli of a gene, hup, encoding the histone-like protein HU of Bifidobacterium longum.Bioscience, biotechnology, and biochemistry, 66(3), 598-603.

【2】Nakamura, T., Sasaki, T., Fujimori, M., Yazawa, K., Kano, Y., Ama no, 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】Klijn, A., Moine, D., Delley, M., Mercenier, A., Arigoni, F., &Pridmore, R. D. (2006).Construction of a reporter vector for the analysis of Bifidobacterium longum promoters.Applied and environmental microbiology, 72(11), 7401-7405.

【4】Hamaji, Y., Fujimori, M., Sasaki, T., Matsuhashi, H., Matsui-Seki, K., Shimatani-Shibata, Y., ... & TANIGUCHI, S. I. (2007).Strong enhancement of recombinant cytosine deaminase activity in Bifidobacterium longum for tumor-targeting enzyme/prodrug therapy.</i>Bioscience, biotechnology, and biochemistry, 71(4)</i>, 874-883.

Sequence and Features