Bifunctional lacZα-ccdB Genes for Selective Cloning of PCR Products

Abstract

The use of PCR-amplified DNA fragments is a classical approach to generate recombinant DNA. To facilitate the cloning of PCR products, we have constructed two new pKIL vectors that allow selection of recombinants. The multiple cloning sites (MCS) of these plasmids contain two adjacent AspeI sites and a unique HindII site. Cleavage of these vectors with AspeI produce linearized molecules with a single thymidine nucleotide at the 3′ends allowing TA cloning of Taqamplified fragments. On the other hand, cleavage with HindII can be used for the cloning of blunt-ended PCR products generated by other DNA polymerases. The LacZα-Ccdb fusion protein produced by these plasmids has retained both the CcdB killer activity and the ability to α-complement the truncated LacZ ΔM15. This bifunctionality allowed us to show that small PCR products (<1000 bp) that do not disrupt lacZα efficiently do inactivate CcdB, which demonstrates that the CcdB-based selection is well adapted for cloning of PCR products, especially for small size fragments.