ribosomal binding site switching an effective strategy for high-throughput cloning constructions核糖体结合位点转换的一种有效策略,高通量克隆结构.pdfVIP

Ribosomal Binding Site Switching: An Effective Strategy for High-Throughput Cloning Constructions 1. 1,2. 1,2 1 1 1 1 Yangbo Hu , Lipeng Feng , Yunlong Li , Yong Zhang , Pei Lu , Simon Rayner , Shiyun Chen * 1 Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China, 2 Graduate University of the Chinese Academy of Sciences, Beijing, China Abstract 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 eff