Vectors for ligation-independent construction of lacZ gene fusions and cloning of PCR products using a nicking endonuclease

Several ligation-independent cloning methods have been developed that offer advantages for construction of recombinant plasmids at high efficiency while minimizing cloning artifacts. Here we report new plasmid vectors that use the nicking endonuclease Nt.BspQI to generate extended single stranded tails for direct cloning of PCR products. The vectors include pLacCOs1, a ColE1-derivative plasmid imparting resistance to ampicillin, which allows facile construction of lacZ translational fusions and pKanCOs1, a pSC101-derivative cloning vector that imparts resistance to kanamycin, for cloning of PCR amplicons from genomic DNA as well as from ampicillin-based plasmids. We have successfully used these plasmids to directionally clone and characterize bacterial promoters that exhibit temperature regulated expression, as well as for cloning a variety of PCR products. In all cases, constructs with the correct configurations were generated at high efficiency and with a minimal number of manipulations. The cloning vectors can also be easily modified to incorporate additional reporter genes or to express epitope-tagged gene products.

► Plasmids have been constructed for direct cloning of PCR products. ► Cloning uses a nicking endonuclease without the need for T4 DNA ligase. ► Vectors were designed to make lacZ gene fusions and to clone any PCR product. ► Gene fusions enabled the study gene regulation in Yersinia pestis.