Difference between revisions of "Part:BBa K1932002"

Revision as of 16:17, 19 October 2016

SEC2 signal peptides

Sec2 contains a classical SP and a single transmembrane domain. The complete protein appears to be a homologue of a hypothetical secreted protein from B. longum NCC2705 (accession no. NP_695256) and is also encoded by the genomes of B. longum DJO10A (ZP_00121565). A probable acid phosphatase domain (COG0671) was identified in Sec2, which indicates involvement in lipid metabolism. Characterization: The prediction of the signal peptide was done with SingalP 4.1 Server (Fig.1), TMpred program (Fig.2) and TMHMM (Fig.3), and the results showed that it could direct the process of transmembrane by cutting the site between amino acid 34 and 35. Since we wanted to fuse it as a domain into our protein, we did the homology modeling and molecular dynamic simulation with the Phyre2 web portal for protein modeling and Hyperchem 8.0 (Fig.4), which showed that the two domains were separate and wouldn’t affect the function of each other. The part of BBa_K1932002 was synthesized and cloned in a pGH vector by Generay Biotechnology. The plasmid was cut by the restriction enzymes, EcoRⅠand PstⅠ, and separated by 1% agarose gel(Fig.5). The sequence of Sec2 was ligated into the vector pSB1C3 by T4 ligase at 16℃ overnight, and the ligated construct was transformed into the E.coli(Fig.6). To ensure the insertion of the right-size sequence, the sequence of Sec2 was cut again and tested by agarose gel electrophoresis (Fig.7). 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. References：【1】MacConaill, L. E., Fitzgerald, G. F., & van Sinderen, D. (2003). Investigation of protein export in Bifidobacteriumbreve UCC2003.Applied and environmental microbiology, 69(12), 6994-7001. 【2】Poquet, I., Ehrlich, S. D., &Gruss, A. (1998). An export-specific reporter designed for gram-positive bacteria: application to Lactococcuslactis. Journal of bacteriology, 180(7), 1904-1912. 【3】Schell, M. A., Karmirantzou, M., Snel, B., Vilanova, D., Berger, B., Pessi, G., ... &Pridmore, R. D. (2002). The genome sequence of Bifidobacteriumlongum reflects its adaptation to the human gastrointestinal tract. Proceedings of the National Academy of Sciences, 99(22), 14422-14427.

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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-Sec2 is predicted to contain a classical SP with a single transmembrane region. The portion of this protein fused to the nuclease comprises the first 77 amino acids of a putative 602-amino-acid protein. This protein is significantly similar to the permease component of an ABC-type transport system, and clear homologues of the gene are found in B. longum NCC2705 (accession no. NP_695398) and DJO10A (ZP_00121339)
+Sec2 contains a classical SP and a single transmembrane domain. The complete protein appears to be a homologue of a hypothetical secreted protein from B. longum NCC2705 (accession no. NP_695256) and is also encoded by the genomes of B. longum DJO10A (ZP_00121565). A probable acid phosphatase domain (COG0671) was identified in Sec2, which indicates involvement in lipid metabolism.
+Characterization:
+The prediction of the signal peptide was done with SingalP 4.1 Server (Fig.1), TMpred program (Fig.2) and TMHMM (Fig.3), and the results showed that it could direct the process of transmembrane by cutting the site between amino acid 34 and 35. Since we wanted to fuse it as a domain into our protein, we did the homology modeling and molecular dynamic simulation with the Phyre2 web portal for protein modeling and Hyperchem 8.0 (Fig.4), which showed that the two domains were separate and wouldn’t affect the function of each other.
+The part of BBa_K1932002 was synthesized and cloned in a pGH vector by Generay Biotechnology. The plasmid was cut by the restriction enzymes, EcoRⅠand PstⅠ, and separated by 1% agarose gel(Fig.5). The sequence of Sec2 was ligated into the vector pSB1C3 by T4 ligase at 16℃ overnight, and the ligated construct was transformed into the E.coli(Fig.6). To ensure the insertion of the right-size sequence, the sequence of Sec2 was cut again and tested by agarose gel electrophoresis (Fig.7). 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.
+References：
+【1】MacConaill, L. E., Fitzgerald, G. F., & van Sinderen, D. (2003). Investigation of protein export in Bifidobacteriumbreve UCC2003.Applied and environmental microbiology, 69(12), 6994-7001.
+【2】Poquet, I., Ehrlich, S. D., &Gruss, A. (1998). An export-specific reporter designed for gram-positive bacteria: application to Lactococcuslactis. Journal of bacteriology, 180(7), 1904-1912.
+【3】Schell, M. A., Karmirantzou, M., Snel, B., Vilanova, D., Berger, B., Pessi, G., ... &Pridmore, R. D. (2002). The genome sequence of Bifidobacteriumlongum reflects its adaptation to the human gastrointestinal tract. Proceedings of the National Academy of Sciences, 99(22), 14422-14427.
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