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Ribosomal binding site switching: an effective strategy for high-throughput cloning constructions. PLoS One. 2012 谁帮忙在web of science 上帮忙查询下 |
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zhouqi0903
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3楼2016-02-21 11:48:27
liouzhan654
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guangyi: 金币+5, 谢谢 2016-02-21 11:14:47
感谢参与，应助指数 +1
guangyi: 金币+5, 谢谢 2016-02-21 11:14:47
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目前被引用次数为0次 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 ] 查看 ResearcherID 和 ORCID PLOS ONE 卷: 7 期: 11 文献号: e50142 DOI: 10.1371/journal.pone.0050142 出版年: NOV 21 2012 查看期刊信息 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 Plus  OSITIVE-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. 地址: 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 查看基金资助信息 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 |
2楼2016-02-21 11:11:37
liouzhan654
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4楼2016-02-21 14:03:10