The TGV transgenic vectors for single-copy gene expression from the Escherichia coli chromosome

Plasmid-based cloning and expression of genes in Escherichia coli can have several problems: plasmid destabilization; toxicity of gene products; inabi lity to achieve complete repression of gene expression-, non-physio logical overexpression of the cloned gene; titration of regulatory proteins and th e requirement for antibiotic selection. We describe a simple system for clo ning and expression of genes in single copy in the E, coli chromosome, usin g a non-antibiotic selection for transgene insertion. The transgene is inse rted into a vector containing homology to the chromosomal region flanking t he attachment site for phage lambda. This vector is then linearized and int roduced into a recombination-proficient E. coli strain carrying a temperatu re-sensitive lambda prophage. Selection for replacement of the prophage wit h the transgene is performed at high temperature, Once in the chromosome, t ransgenes can be moved into other lysogenic E. coli strains using standard phage-mediated transduction techniques, selecting against a resident propha ge. Additional vector constructs provide an arabinose-inducible promoter (P BAD), PRAD plus a translation-initiation sequence, and optional chloramphen icol-, tetracycline-, or kanamycin-resistance cassettes. These Transgenic E . coli Vectors (TGV) allow drug-free, single-copy expression of genes from the E. coli chromosome, and are useful for genetic studies of gene function . (C) 2001 Elsevier Science B.V. All rights reserved.