%0 Journal Article
%T Fast, easy and efficient: site-specific insertion of transgenes into Enterobacterial chromosomes using Tn7 without need for selection of the insertion event
%A Gregory J McKenzie
%A Nancy L Craig
%J BMC Microbiology
%D 2006
%I BioMed Central
%R 10.1186/1471-2180-6-39
%X We present a simple, rapid and highly efficient method for transgene insertion into the chromosome of Escherichia coli, Salmonella or Shigella at a benign chromosomal site using the site-specific recombination machinery of the transposon Tn7. This method requires very few manipulations. The transgene is cloned into a temperature-sensitive delivery plasmid and transformed into bacterial cells. Growth at the permissive temperature with induction of the recombination machinery leads to transgene insertion, and subsequent growth at the nonpermissive temperature cures the delivery plasmid. Transgene insertion is highly site-specific, generating insertions solely at the Tn7 attachment site and so efficient that it is not necessary to select for the insertion.This method is more efficient and straightforward than other techniques for transgene insertion available for E. coli and related bacteria, making moving transgenes from plasmids to a chromosomal location a simple matter. The non-requirement for selection is particularly well suited for use in development of unmarked strains for environmental release, such as live-vector vaccine strains, and also for promoter-fusion studies, and experiments in which every bacterial cell must express a transgene construct.Expression of transgenes in bacteria is routinely accomplished by expressing the gene from an extrachromosomal plasmid. However, chromosomal expression, with its single-copy nature and inherent stability, is preferable in many situations. For example, in experiments where physiological levels of protein are desired, including genetic complementation experiments, even low copy number plasmids can yield non-physiological levels of expression making single-copy expression a better choice [1]. In addition, interpretation of experiments involving plasmids can often be confounded by the heterogeneity of the population arising from plasmid loss due to the imperfect segregation of most plasmids [2]. Recent experiments in Salm
%U http://www.biomedcentral.com/1471-2180/6/39