A New Method for High-efficiency Integration of Transgenes into Bacterial Chromosomes without a Drug Marker

This invention allows the insertion of foreign DNA into an innocuous site in the bacterial chromosome using the site-specific recombination system of Tn7. Drug selection is not required since this integration event occurs in up to 97% of cells treated. The plasmid that carries this system is curable, once the process is complete all transposition machinery is lost, and only Tn7 and the transgene remain. Finally, this method is very easy to use and is suitable for use in environmental isolates of bacteria, and in common lab strains. Transgene expression in bacteria is routinely accomplished using an extra-chromosomal plasmid. The use of plasmids raises various technical difficulties depending on the proposed use of the engineered organism. Such difficulties include requirement for antibiotic selection during growth, imperfect plasmid segregation among daughter cells, and expression of non-physiological protein levels. Chromosomal insertion of transgenes may alleviate each of these technical problems. Most systems for transgene insertion require the use of a drug marker to select for a relatively rare integration event. Those that do not involve drug selection rely on other selection strategies that involve multiple steps. Description (Set) Proposed Use (Set) Generation of genetically modified bacteria by stable integration of new genes into an innocuous site in the bacterial chromosome. Such an approach may be useful for a variety of purposes including, but not limited to, in vivo protein expression and expression of specific gene products in vaccine strains of Salmonella spp.

Inventor(s): Craig, Nancy

Type of Offer: Licensing

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