12 The Minicell System as a Method for Studying Expression from Plasmid DNA

The major difficulty encountered when analyzing plasmid or phage encoded mRNA and polypeptides in whole (normal) bacterial cells is that the majority of these products are masked by those encoded by the host cell's chromosome. This difficulty often remains even when the genes of interest have been amplified by cloning into multicopy vectors. In relatively few cases does a very high rate of expression or the availability of a specific assay (e.g. zymogram staining or specific antisera) allow the experimenter to distinguish between the plasmid and chromosome-encoded products. It is usually necessary to label specifically the plasmid-encoded products in the absence of significant expression from the host cell's chromosome. In recent years, a number of techniques have been developed to achieve this. These include the use of bacterial minicells, the maxicell system, cell-free (in vitro) synthesis, selective expression from ColEl-type plasm ids after prolonged chloramphenicol treatemetn. These techniques have been designed to maximize expression from plasmid DNA while minimizing the level of background expression from the host chromosome or, in the case of the in vitro system, from mis-transcription or mis-translation.