Antibiotic-free plasmids
The present invention provides a method of maintaining a gram negative bacterium plasmid without the use of antibiotic selection pressure. Further, the invention relates to the drugless plasmids produced including drugless plasmids containing a heterologous gene. The invention also provides formulations and/or compositions comprising the drugless plasmids comprising a heterologous gene, formulations and/or compositions comprising a protein or an immunogen expressed using the drugless plasmids, and methods of administering such formulations and/or compositions to a host. The invention relates to gram negative bacteria containing the drugless plasmids.
1. A drugless plasmid comprising a polynucleotide encoding a cI repressor protein comprising the sequence of SEQ ID NO:2, wherein the expression of the cI repressor protein regulates the expression of a heterologous toxic gene product in a bacterial host.
2. The drugless plasmid of claim 1 , wherein the polynucleotide is operably linked to a promoter.
3. The drugless plasmid of claim 2 , wherein the promoter is a P1 promoter or a native cI promoter.
4. The drugless plasmid of claim 3 , wherein the P1 promoter comprises the sequence of SEQ ID NO: 29 or SEQ ID NO:83.
5. The drugless plasmid of claim 3 , wherein the native cI promoter comprises the sequence of SEQ ID NO:30.
6. The drugless plasmid of claim 1 , wherein the polynucleotide comprises the sequence of SEQ ID NO:1.
7. The drugless plasmid of claim 6 , wherein the polynucleotide is operably linked to a promoter.
8. The drugless plasmid of claim 7 , wherein the promoter is a P1 promoter or a native cI promoter.
9. The drugless plasmid of claim 8 , wherein the P1 promoter comprises the sequence of SEQ ID NO: 29 or SEQ ID NO:83.
10. The drugless plasmid of claim 8 , wherein the native cI promoter comprises the sequence of SEQ ID NO:30.
11. The drugless plasmid of claim 1 further comprising a heterologous polynucleotide encoding an immunogen or a protein.
12. The drugless plasmid of claim 11 , wherein the heterologous polynucleotide is operably linked to a promoter functional in eukaryotic or prokaryotic cells.
13. The drugless plasmid of claim 12 , wherein the promoter is a CMV promoter or a promoter isolated from a gram-negative bacterium.
14. An engineered gram-negative bacterium host strain comprising the drugless plasmid of claim 1 , wherein the bacterium further comprises one or more heterologous polynucleotides in a nonessential region of the bacterial chromosome, and wherein comprises one or more heterologous polynucleotides encodes a product that is toxic to the bacterium host.
15. The bacterium strain of claim 14 , wherein the one or more heterologous polynucleotides encoding a product that is toxic to the bacterium host comprises a sacB gene.
16. The bacterium strain of claim 14 , wherein the one or more heterologous polynucleotides encoding a product that is toxic to the bacterium host is operably linked to a promoter.
17. The bacterium strain of claim 16 , wherein the promoter is a promoter from λ phage.
18. The bacterium strain of claim 14 , wherein the nonessential region comprises a deA gene or a purN gene.
19. The engineered gram-negative bacterium of claim 14 , wherein the gram-negative bacterium is selected from the group consisting of Avibacterium, Brucella, Escherichia coli, Haemophilus, Salmonella, Shigella, Pasteurella , and Rimeirella.
20. The engineered gram-negative bacterium of claim 14 , wherein the polynucleotide encoding the cI repressor protein is operably linked to a P1 promoter comprising the sequence of SEQ ID NO:29 or SEQ ID NO:83.
21. The engineered gram-negative bacterium of claim 14 , wherein the polynucleotide encoding the cI repressor protein is operably linked to a native cI promoter comprising the sequence of SEQ ID NO:30.
22. A composition comprising the drugless plasmid of claim 1 .
23. The composition of claim 22 further comprising a pharmaceutically acceptable carrier.