Selective expression of transduced or cloned DNA in minicells containing plasmid pKB280

Abstract

DERIVATIVES of coliphage λ, carrying inserts of essentially any prokaryotic or eukaryotic DNA, can now be constructed using novel λ cloning vectors. Often, the reason for constructing these derivatives is to determine the polypeptides encoded by the inserted DNA. Phage expression can be analysed by infection of anucleate Escherichia coli minicells in which all host syntheses are absent¹. However, analysis of the expression of the non-λ DNA insert in this system is complicated by the concurrent expression of λ DNA. Attempts to inhibit the expression of the λ genes by use of minicells from a λ lysogenic minicell-producing strain were unsuccessful, indicating that insufficient λ repressor was segregated into the minicells (Shaw, Epp, Pearson and J.N.R., in preparation). Recently a plasmid, pKB280, was constructed which carries the gene (cI) for the λ repressor protein and in which approximately 1% of the soluble protein of E. coli strains carrying this plasmid is λ repressor². Minicells produced by a derivative of E. coli DS410 carrying plasmid pKB280 have been infected by transducing and cloning phages. We report here that only the non-λ DNA is expressed in these minicells.