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学科主题物理化学
Rapid construction of a Bacterial Artificial Chromosomal (BAC) expression vector using designer DNA fragments
Chen, Chao1; Zhao, Xinqing1; Jin, Yingyu2; Zhao, Zongbao (Kent)3; Suh, Joo-Won2; Zhao XQ(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
2014-11-01
DOI10.1016/j.plasmid.2014.10.002
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
关键词[WOS]BIOSYNTHETIC GENE-CLUSTER ; STREPTOMYCES-COELICOLOR A3(2) ; HETEROLOGOUS EXPRESSION ; ESCHERICHIA-COLI ; RECOMBINATION SYSTEM ; CLONING ; IDENTIFICATION ; INACTIVATION ; DISCOVERY ; PATHWAYS
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.
语种英语
WOS记录号WOS:000345609000011
引用统计
被引频次:2[WOS]   [WOS记录]     [WOS相关记录]
文献类型期刊论文
条目标识符http://cas-ir.dicp.ac.cn/handle/321008/144060
专题中国科学院大连化学物理研究所
通讯作者Zhao XQ(赵心清)
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
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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.
APA Chen, Chao,Zhao, Xinqing,Jin, Yingyu,Zhao, Zongbao ,Suh, Joo-Won,&赵心清.(2014).Rapid construction of a Bacterial Artificial Chromosomal (BAC) expression vector using designer DNA fragments.PLASMID,76(1),79-86.
MLA Chen, Chao,et al."Rapid construction of a Bacterial Artificial Chromosomal (BAC) expression vector using designer DNA fragments".PLASMID 76.1(2014):79-86.
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