Engineered listeria and methods of use thereof

Abstract

The invention provides a bacterium containing a polynucleotide comprising a nucleic acid encoding a heterologous antigen, as well as fusion protein partners. Also provided are vectors for mediating site-specific recombination and vectors comprising removable antibiotic resistance genes.

What is claimed is: 
     
         1 . A method of introducing a heterologous nucleic acid sequence of interest into a bacterial genome, comprising:
 (a) recombinantly introducing a bacterial attachment site (attBB′) which is not present in the bacterial genome into a pre-determined site of the genome of a population of bacteria;   (b) contacting the population of bacteria with a plasmid comprising
 (i) a first nucleic acid sequence encoding a first recombinase binding site heterologous to the bacterial genome and a second nucleic acid sequence encoding a second recombinase binding site heterologous to the bacterial genome, wherein the first and second recombinase binding sites are two Lox sites or two Frt sites, 
 (ii) a third nucleic acid sequence encoding a selection marker, wherein the first and second recombinase binding sites flank the third nucleic acid sequence encoding a selection marker, 
 (iii) a fourth nucleic acid sequence encoding a phage integrase selected to mediate integration at the first bacterial attachment site, wherein the first and second recombinase binding sites flank the fourth nucleic acid sequence, 
 (iv) a fifth nucleic acid sequence not flanked by the recombinase binding sites, the fifth nucleic acid sequence encoding the heterologous nucleic acid sequence of interest, and 
 (v) a sixth nucleic acid sequence not flanked by the recombinase binding sites, the sixth nucleic acid sequence encoding a phage attachment site (attPP′) site compatible with first bacterial attachment site, 
   under conditions selected to cause integration of the plasmid into the pre-determined site of the genome mediated by the phage integrase, wherein the plasmid lacks an origin of replication that is active in the bacteria;   (c) selecting bacteria from the population of bacteria which have integrated the plasmid into the pre-determined site of the genome using expression of the selection marker;   (d) transiently expressing a recombinase within the selected bacteria, wherein if the first and second recombinase binding sites are two Lox sites the recombinase is Cre recombinase, and wherein if the first and second recombinase binding sites are two Lox sites the recombinase is FLP recombinase, wherein the recombinase mediates excision of the third and fourth nucleic acid sequences from the pre-determined site of the genome.