101. Ribosomal binding site switching: an effective strategy for high-throughput cloning constructions
- Author
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Yong Zhang, Shiyun Chen, Yangbo Hu, Simon Rayner, Pei Lu, Lipeng Feng, and Yunlong Li
- Subjects
Applied Microbiology ,Genetic Vectors ,Molecular Sequence Data ,Gene Expression ,lcsh:Medicine ,Computational biology ,Biology ,Molecular cloning ,Polymerase Chain Reaction ,Biochemistry ,Microbiology ,Marker gene ,Molecular Genetics ,DNA amplification ,Plasmid ,Two-Hybrid System Techniques ,Molecular Cell Biology ,Escherichia coli ,Genetics ,Cloning, Molecular ,lcsh:Science ,Selectable marker ,Cloning ,Binding Sites ,Multidisciplinary ,Base Sequence ,Nucleotides ,lcsh:R ,Computational Biology ,DNA ,Molecular biology ,TA cloning ,Ribosomal binding site ,Nucleic acids ,Protein Biosynthesis ,Biological Assay ,lcsh:Q ,Transformation, Bacterial ,Genetic Engineering ,Ligation ,Ribosomes ,Plasmids ,Research Article ,Biotechnology - 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 efficiency, makes our strategy extremely useful in HTP cloning constructions.
- Published
- 2012