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Ribosomal Binding Site Switching: An Effective Strategy for High-Throughput Cloning Constructions
作者:Hu, YB (Hu, Yangbo)[ 1 ] ; Feng, LP (Feng, Lipeng)[ 1,2 ] ; Li, YL (Li, Yunlong)[ 1,2 ] ; Zhang, Y (Zhang, Yong)[ 1 ] ; Lu, P (Lu, Pei)[ 1 ] ; Rayner, S (Rayner, Simon)[ 1 ] ; Chen, SY (Chen, Shiyun)[ 1 ]
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PLOS ONE
卷: 7  期: 11
文献号: e50142
DOI: 10.1371/journal.pone.0050142
出版年: NOV 21 2012
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摘要
Direct cloning of PCR fragments by TA cloning or blunt end ligation are two simple methods which would greatly benefit high-throughput (HTP) cloning constructions if the efficiency can be improved. In this study, we have developed a ribosomal binding site (RBS) switching strategy for direct cloning of PCR fragments. RBS is an A/G rich region upstream of the translational start codon and is essential for gene expression. Change from A/G to T/C in the RBS blocks its activity and thereby abolishes gene expression. Based on this property, we introduced an inactive RBS upstream of a selectable marker gene, and designed a fragment insertion site within this inactive RBS. Forward and reverse insertions of specifically tailed fragments will respectively form an active and inactive RBS, thus all background from vector self-ligation and fragment reverse insertions will be eliminated due to the non-expression of the marker gene. The effectiveness of our strategy for TA cloning and blunt end ligation are confirmed. Application of this strategy to gene over-expression, a bacterial two-hybrid system, a bacterial one-hybrid system, and promoter bank construction are also verified. The advantages of this simple procedure, together with its low cost and high efficiency, makes our strategy extremely useful in HTP cloning constructions.
关键词
KeyWords PlusOSITIVE-SELECTION VECTORS; PCR PRODUCTS; ESCHERICHIA-COLI; BACILLUS-SUBTILIS; IN-VIVO; GENE; RECOMBINATION; SEQUENCE
作者信息
通讯作者地址: Chen, SY (通讯作者)
显示增强组织信息的名称        Chinese Acad Sci, Wuhan Inst Virol, Key Lab Agr & Environm Microbiol, Wuhan, Peoples R China.
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显示增强组织信息的名称        [ 1 ] Chinese Acad Sci, Wuhan Inst Virol, Key Lab Agr & Environm Microbiol, Wuhan, Peoples R China
显示增强组织信息的名称        [ 2 ] Chinese Acad Sci, Grad Univ, Beijing, Peoples R China
电子邮件地址:sychen@wh.iov.cn
基金资助致谢
基金资助机构        授权号
National Natural Science Foundation of China        
31170133
National Transgenic Program of China        
2011ZX08009-003-001-003
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出版商
PUBLIC LIBRARY SCIENCE, 1160 BATTERY STREET, STE 100, SAN FRANCISCO, CA 94111 USA
类别 / 分类
研究方向:Science & Technology - Other Topics
Web of Science 类别:Multidisciplinary Sciences
文献信息
文献类型:Article
语种:English
入藏号: WOS:000311821000189
PubMed ID: 23185557
ISSN: 1932-6203
期刊信息
Impact Factor (影响因子): Journal Citation Reports®
其他信息
IDS 号: 047ER
Web of Science 核心合集中的 "引用的参考文献": 30
Web of Science 核心合集中的 "被引频次": 0，