Engineered
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.
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 the attBB′,
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; and
(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.
2. A method according to claim 1 , wherein the third nucleic acid sequence comprises an antibiotic resistance gene.
3. A method according to claim 1 , wherein step (d) comprises transfecting the selected bacteria with a plasmid encoding the recombinase.
4. A method according to claim 3 , wherein expression of the recombinase from the plasmid encoding the recombinase is temperature sensitive, and step (d) further comprises incubating the selected bacteria at the permissive temperature for expression of the recombinase.
5. A method according to claim 1 , wherein the heterologous nucleic acid sequence of interest comprises a sequence encoding an antigen from a cancer cell, tumor, or infectious agent.
6. A method according to claim 1 , wherein the pre-determined site of the genome is a virulence gene encoded by the genome, and wherein introduction of the heterologous nucleic acid sequence of interest disrupts expression of the virulence gene or disrupts a coding sequence of the virulence gene.
7. A method according to claim 1 , wherein the bacteria are Listeria bacteria.
8. A method according to claim 6 , wherein the bacteria are Listeria bacteria and the virulence gene is a PrfA-dependent gene.
9. A method according to claim 8 , wherein the PrfA-dependent gene is actA or inlB.