Regulatory

Part:BBa_K1932000

Designed by: Jin Liu   Group: iGEM16_Jilin_China   (2016-10-14)
Revision as of 21:14, 19 October 2016 by JinLiu (Talk | contribs)

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

BBa_K1932000 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).

T--Jilin_China--p0-1.png

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).

T--Jilin_China--p0-2.png

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).

T--Jilin_China--p0-3.png

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.

T--Jilin_China--T1.png

T--Jilin_China--T2.png

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).

T--Jilin_China--p0-4.png

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


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
    COMPATIBLE WITH RFC[1000]


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