Directed Molecular Evolution of Nitrite Oxido-reductase by DNA-shuffling

JUN-WEN LI; JIN-LAI ZHENG; XIN-WEI WANG; MIN JIN; FU-HUAN CHAO

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Volume 20 Issue 2

Apr. 2007

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Article Navigation > Biomedical and Environmental Sciences > 2007 > 20(2): 113-118

JUN-WEN LI, JIN-LAI ZHENG, XIN-WEI WANG, MIN JIN, FU-HUAN CHAO. Directed Molecular Evolution of Nitrite Oxido-reductase by DNA-shuffling[J]. Biomedical and Environmental Sciences, 2007, 20(2): 113-118.

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JUN-WEN LI, JIN-LAI ZHENG, XIN-WEI WANG, MIN JIN, FU-HUAN CHAO. Directed Molecular Evolution of Nitrite Oxido-reductase by DNA-shuffling[J]. Biomedical and Environmental Sciences, 2007, 20(2): 113-118.

Directed Molecular Evolution of Nitrite Oxido-reductase by DNA-shuffling

Institute of Environment and Health, 1 Dali Road, Tianjin 300050, China

Funds: 国家高技术研究发展计划(863计划)(2001AA214191)

Abstract

Objective To develtop directly molecular evolution of nitrite oxido-reductase using DNA-shuffling technique because nitrobacteria grow extremely slow and are unable to nitrify effectively inorganic nitrogen in wastewater treatment. Methods The norB gene coding the nitrite oxido-reductase in nitrobacteria was cloned and sequenced. Then, directed molecular evolution of nitrite oxido-reductase was developed by DNA-shuffling of 15 norB genes from different nitrobacteria. Results After DNA-shuffling with sexual PCR and staggered extension process PCR, the sequence was different from its parental DNA fragments and the homology ranged from 98% to 99%. The maximum nitrification rate of the modified bacterium of X16 by modified bacterium had the same characteristics of its parental bacteria of E. coli and could grow rapidly in normal cultures.Conclusion DNA-shuffling was successfully used to engineer E. coli, which had norB gene and could degrade inorganic nitrogen effectively.
- Directed evolution,
- DNA-shuffling,
- Nitrite oxido-reductase,
- norB gene,
- Nitrobacteria
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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

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Directed Molecular Evolution of Nitrite Oxido-reductase by DNA-shuffling

Institute of Environment and Health, 1 Dali Road, Tianjin 300050, China

Funds: 国家高技术研究发展计划(863计划)(2001AA214191)

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Abstract: Objective To develtop directly molecular evolution of nitrite oxido-reductase using DNA-shuffling technique because nitrobacteria grow extremely slow and are unable to nitrify effectively inorganic nitrogen in wastewater treatment. Methods The norB gene coding the nitrite oxido-reductase in nitrobacteria was cloned and sequenced. Then, directed molecular evolution of nitrite oxido-reductase was developed by DNA-shuffling of 15 norB genes from different nitrobacteria. Results After DNA-shuffling with sexual PCR and staggered extension process PCR, the sequence was different from its parental DNA fragments and the homology ranged from 98% to 99%. The maximum nitrification rate of the modified bacterium of X16 by modified bacterium had the same characteristics of its parental bacteria of E. coli and could grow rapidly in normal cultures.Conclusion DNA-shuffling was successfully used to engineer E. coli, which had norB gene and could degrade inorganic nitrogen effectively.

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