Generating a custom TA-cloning expression plasmid for Lactococcus lactis.

The growing popularity of the lactic acid bacterium Lactococcus lactis has increased demand for novel high-throughput cloning methods. Here we describe a general TA-cloning methodology and demonstrate its feasibility using the plasmid pNZ8148. PCR products were directly ligated into a linear, PCR-amplified and XcmI-digested pNZ8148 derivative that was termed pNZ-T. Cloning using pNZ-T yielded a high proportion of insert-containing plasmids on transformation. Although demonstrated with L. lactis, the technique presented here is organism-independent and can be implemented in other plasmids.