Analyzing the Function of Essential Genes by Plasmid Shuffling

Investigations on gene essentiality have important implications in several fields of basic and applied research. A variety of strategies have been developed over the years to identify essential genes. Here, we describe an implemented plasmid shuffling method useful to assess the essentiality of overlapped genes under very stringent conditions. A host strain harboring the chromosomal deletion of the genes of interest is complemented by a thermosensitive plasmid carrying the copy of gene 1, gene 2, and rpsL allele, conferring streptomycin sensitivity to an otherwise resistant strain. A compatible plasmid harboring a different selectable marker and the copy of gene 2 only is transformed into the host strain, resulting in the coexistence of two plasmids. These cells are grown at high temperatures in a medium containing streptomycin. Under such conditions, viable cells are expected to contain only the incoming plasmid and to carry suppressor mutation(s) that bypass the loss of the essential gene 1. The system may thus represent a valuable tool to identify interactions between essential proteins and cell pathways.