期刊
CURRENT MICROBIOLOGY
卷 66, 期 6, 页码 548-554出版社
SPRINGER
DOI: 10.1007/s00284-013-0312-y
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资金
- National Natural Science Foundation of China (NSFC) [3107 0091]
- Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP) [20100131110035]
- Hi-tech Research and Development Program of China [2011AA100902]
Three putative promoter regions were identified preceding the nisZ gene in Lactococcus lactis HSM-22. To investigate their function in the control of nisZ biosynthesis, green fluorescence protein (GFP) was adopted as probe to determine activities of the three promoters. The results showed that PnisZ-0 containing two sets of the -35 and -10 regions exhibited the same maximum activity as promoter PnisZ-2 containing the putative promoter region near the start codon. However, the GFP expression level directed by PnisZ-0 was twofold higher than that found with PnisZ-2 under low-dose nisin, indicating that promoter PnisZ-1 distant from the start codon could be important in response to the inducer nisin. Then, Pnis-2 was randomized to develop functional promoters through the degenerate oligonucleotide approach in L. lactis. 35 inducible promoters and 14 constitutive promoters were obtained, covering 3-5 logs of expression levels in small increments of activity. Sequence analysis revealed that base changes in both consensus sequence and spacing sequence resulted in remarkable decrease of promoter activity, while the sequence outside -35 and -10 regions would influence the promoter function radically. The functional promoters were evaluated for the efficiency and stability to control beta-galactosidase (Gal) expression in L. lactis. High correlation was obtained between the Gal activity and promoter strength, suggesting that promoters developed here have the potential for fine tuning gene expression in L. lactis.
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