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学科主题: 物理化学
题名: Rapid construction of a Bacterial Artificial Chromosomal (BAC) expression vector using designer DNA fragments
作者: Chen, Chao1;  Zhao, Xinqing1;  Jin, Yingyu2;  Zhao, Zongbao (Kent)3;  Suh, Joo-Won2
通讯作者: 赵心清
关键词: Heterologous expression ;  Bacterial Artificial Chromosomal (BAC) vector ;  In-Fusion cloning ;  lambda-RED-mediated homologous recombination ;  Biosynthetic gene cluster ;  Natural product
刊名: PLASMID
发表日期: 2014-11-01
DOI: 10.1016/j.plasmid.2014.10.002
卷: 76, 期:1, 页:79-86
收录类别: SCI
文章类型: Article
WOS标题词: Science & Technology ;  Life Sciences & Biomedicine
类目[WOS]: Genetics & Heredity
研究领域[WOS]: Genetics & Heredity
英文摘要: Bacterial artificial chromosomal (BAC) vectors are increasingly being used in cloning large DNA fragments containing complex biosynthetic pathways to facilitate heterologous production of microbial metabolites for drug development. To express inserted genes using Streptomyces species as the production hosts, an integration expression cassette is required to be inserted into the BAC vector, which includes genetic elements encoding a phage-specific attachment site, an integrase, an origin of transfer, a selection marker and a promoter. Due to the large sizes of DNA inserted into the BAC vectors, it is normally inefficient and time-consuming to assemble these fragments by routine PCR amplifications and restriction-ligations. Here we present a rapid method to insert fragments to construct BAC-based expression vectors. A DNA fragment of about 130 bp was designed, which contains upstream and downstream homologous sequences of both BAC vector and pIB139 plasmid carrying the whole integration expression cassette. In-Fusion cloning was performed using the designer DNA fragment to modify pIB139, followed by lambda-RED-mediated recombination to obtain the BAC-based expression vector. We demonstrated the effectiveness of this method by rapid construction of a BAC-based expression vector with an insert of about 120 kb that contains the entire gene cluster for biosynthesis of immunosuppressant FK506. The empty BAC-based expression vector constructed in this study can be conveniently used for construction of BAC libraries using either microbial pure culture or environmental DNA, and the selected BAC clones can be directly used for heterologous expression. Alternatively, if a BAC library has already been constructed using a commercial BAC vector, the selected BAC vectors can be manipulated using the method described here to get the BAC-based expression vectors with desired gene clusters for heterologous expression. The rapid construction of a BAC-based expression vector facilitates heterologous expression of large gene clusters for drug discovery. (C) 2014 Elsevier Inc. All rights reserved.
关键词[WOS]: BIOSYNTHETIC GENE-CLUSTER ;  STREPTOMYCES-COELICOLOR A3(2) ;  HETEROLOGOUS EXPRESSION ;  ESCHERICHIA-COLI ;  RECOMBINATION SYSTEM ;  CLONING ;  IDENTIFICATION ;  INACTIVATION ;  DISCOVERY ;  PATHWAYS
语种: 英语
WOS记录号: WOS:000345609000011
Citation statistics: 
内容类型: 期刊论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/144060
Appears in Collections:中国科学院大连化学物理研究所_期刊论文

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作者单位: 1.Dalian Univ Technol, Sch Life Sci & Biotechnol, Dalian 116024, Peoples R China
2.Myongji Univ, Div Biosci & Bioinformat, Yongin 449728, South Korea
3.Chinese Acad Sci, Dalian Inst Chem Phys, Dept Biotechnol, Dalian 116023, Peoples R China

Recommended Citation:
Chen, Chao,Zhao, Xinqing,Jin, Yingyu,et al. Rapid construction of a Bacterial Artificial Chromosomal (BAC) expression vector using designer DNA fragments[J]. PLASMID,2014,76(1):79-86.
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