Antibiotic-free plasmid
View Patent ↗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. An engineered gram-negative bacterium comprising:
a drugless plasmid encoding a cI repressor protein comprising the sequence as set forth in SEQ ID NO:2; and
a heterologous polynucleotide in a nonessential region of the bacterial chromosome that encodes a product which is toxic to the bacterium,
wherein expression of the cI repressor protein regulates expression of the heterologous polynucleotide in the bacterium.
2. The bacterium of claim 1 , wherein the drugless plasmid further encodes a heterologous immunogen or protein.
3. The bacterium of claim 1 , wherein the heterologous polynucleotide comprises a sacB gene which encodes a sacB protein comprising the sequence as set forth in SEQ ID NO:4.
4. The bacterium of claim 3 , wherein the sacB gene is operably linked to a promoter comprising the sequence as set forth in SEQ ID NO:5.
5. The bacterium of claim 1 , wherein the heterologous polynucleotide is operably linked to a promoter.
6. The bacterium of claim 5 , wherein the promoter is a promoter from λ phage.
7. The bacterium of claim 1 , wherein the nonessential region comprises at least one of a deA gene and a purN gene.
8. The bacterium of claim 1 , wherein the bacterium is selected from Avibacterium, Brucella, Escherichia coli, Haemophilus, Salmonella, Shigella, Pasteurella , and Rimeirella.
9. The bacterium of claim 3 , wherein the sacB gene comprises the sequence of SEQ ID NO:3.
10. A method comprising:
inserting in a nonessential region of a bacterial chromosome of a gram negative bacterium, by allelic replacement, a heterologous polynucleotide that encodes a product which is toxic to the bacteriurn;
constructing a DNA plasmid encoding a cI repressor protein comprising the sequence as set forth in SEQ ID NO:2;
transforming the bacterium with the DNA plasmid, wherein expression of the cI repressor protein in the bacterium regulates expression of the heterologous polynucleotide in the bacterium; and
growing the transformed bacterium in the presence of sucrose at a temperature ranging from 30° C. to 42° C.
11. The method of claim 10 , wherein the method further comprises recovering the DNA plasmid after growing the transformed bacterium.
12. The method of claim 10 , wherein the DNA plasmid further encodes a heterologous immunogen or protein, and wherein the method further comprises recovering the heterologous immunogen or protein after growing the transformed bacterium.
13. An engineered gram-negative bacterium comprising:
a drugless plasmid encoding a cI repressor protein comprising the sequence as set forth in SEQ ID NO:2; and
a heterologous polynucleotide in a nonessential region of the bacterial chromosome,
wherein the heterologous polynucleotide comprises a gene encoding a product toxic to the bacterium operably linked to a promoter,
wherein the heterologous polynucleotide comprises the sequence as set forth in SEQ ID NO:75, and
wherein expression of the cI repressor protein regulates expression of the gene encoding the product toxic to the bacterium.
14. The bacterium of claim 13 , wherein the heterologous polynucleotide is inserted in each of the deA gene and the purN gene of the bacterial chromosome.