Fast, easy and efficient: site-specific insertion of transgenes into enterobacterial chromosomes using Tn7 without need for selection of the insertion event.
BACKGROUND
Inserting transgenes into bacterial chromosomes is generally quite involved, requiring a selection for cells carrying the insertion, usually for drug-resistance, or multiple cumbersome manipulations, or both. Several approaches use phage lambda red recombination, which allows for the possibility of mutagenesis of the transgene during a PCR step.


RESULTS
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.


CONCLUSIONS
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.
McKenzie
Gregory J
GJ
Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. gmckenz1@jhmi.edu
Craig
Nancy L
NL
D010582:Bacteriophage lambda / Q000235:genetics; D002876:Chromosomes, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004251:DNA Transposable Elements / Q000235:genetics*; D004755:Enterobacteriaceae / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D016297:Mutagenesis, Site-Directed / Q000379:methods* / Q000592:standards; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic / Q000235:genetics; D014170:Transformation, Genetic; D019076:Transgenes / Q000235:genetics*
D017931:DNA Primers; D004251:DNA Transposable Elements