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.
Highlights
The development of the polymerase chain reaction (PCR) represented a major technological breakthrough in the field of molecular biology and genetic engineering
A/G Content in the ribosomal binding site (RBS) Determines the Gene Expression To confirm the importance of A/G content for gene expression in the RBS sequence, we amplified five cat fragments with different RBS sequences, which were inserted into the pMD19T vector in the same orientation with the lac-O promoter (Figure 1A)
We proposed a strategy for clone construction in which a PCR fragment is inserted into an inactive RBS sequence with suitable A/G content upstream of the cat gene
Summary
The development of the polymerase chain reaction (PCR) represented a major technological breakthrough in the field of molecular biology and genetic engineering Many techniques such as restriction enzyme digestion and ligation [1], ligation-independent cloning (LIC) [2,3,4], in vivo ligation [5,6], and site-specific recombination systems [7,8,9] have been developed for the cloning of PCR products. For high flexibility DNA polymerase which contains 39 to 59 exonuclease activity, the PCR-generated fragments are blunt ended Direct ligation of these PCR fragments with blunt end vectors will greatly facilitate the cloning construction [10]. An efficient strategy to eliminate the background from vector self-ligation and reverse insertion of fragments is highly desirable for the application of direct cloning in HTP cloning construction
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