期刊
JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY
卷 24, 期 4, 页码 431-439出版社
KOREAN SOC MICROBIOLOGY & BIOTECHNOLOGY
DOI: 10.4014/jmb.1311.11009
关键词
Bacillus subtilis; Bacillus pumilus; green fluorescent protein; mannanase; P43 promoter; secretory expression
资金
- Fundamental Research Funds for the Central Universities of China [WF1214047]
- National High Technology Research and Development Program of China [2013AA102109]
- National Major Science and Technology Projects of China [2012ZX09304009]
We report the construction of two Bacillus subtilis expression vectors, pBNS1/pBNS2. Both vectors are based on the strong promoter P43 and the ampicillin resistance gene expression cassette. Additionally, a fragment with the Shine-Dalgarno sequence and a multiple cloning site (BamHI, SalI, SacI, XhoI, PstI, SphI) were inserted. The coding region for the amyQ (encoding an amylase) signal peptide was fused to the promoter P43 of pBNS1 to construct the secreted expression vector pBNS2. The applicability of vectors was tested by first generating the expression vectors pBNS1-GFP/pBNS2-GFP and then detecting for green fluorescent protein gene expression. Next, the mannanase gene from B. pumilus Nsic-2 was fused to vector pBNS2 and we measured the mannanase activity in the supernatant. The mannanase total enzyme activity was 8.65 U/ml, which was 6 times higher than that of the parent strain. Our work provides a feasible way to achieve an effective transformation system for gene expression in B. subtilis and is the first report to achieve B. pumilus mannanase secretory expression in B. subtilis.
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