Granted Patent B2

US 12,351,805 · App. 17/320,866 · Granted Jul 8, 2025

Inventors: Neel Satish Joshi (Somerville, MA); Peter Quoc Nguyen (Garden Grove, CA); Noemie-Manuelle Dorval Courchesne (Montreal, CA); Zahra Abdali (Montreal, CA)

Assignees: Northeastern University; The Royal Institution for the Advancement of Learning/McGill University

C12N15/70C07K1/22C07K1/30C07K1/34C07K14/245C12N1/20C07K2319/02C07K2319/21C12R2001/19

Abstract

Claims (25)

1. A method of producing a genetically modified Escherichia coli ( E. coli ) bacterium, comprising

genetically altering the bacterium to include a nucleic acid sequence encoding a recombinant bacterial collagen protein comprising a Sec domain for periplasmic localization and an N22 domain for directing the recombinant bacterial collagen protein to the outer membrane for secretion;

wherein the bacterial collagen protein comprises a trypsin-sensitive globular variable domain comprising the amino acid sequence set forth in SEQ ID NO: 2075, and at least one collagen domain comprising the amino acid sequence set forth in SEQ ID NO: 2076;

wherein the nucleic acid is under operation of a promoter to express the recombinant bacterial collagen protein.

2. The method of claim 1 , wherein the nucleic acid sequence further encodes a translational enhancing element (TEE).

3. The method of claim 1 , wherein the nucleic acid sequence further encodes each of the curli-specific accessory proteins selected from csgC, csgE, csgF, and csgG.

4. A genetically-modified Escherichia coli ( E. coli ) bacterium, comprising a nucleic acid sequence encoding recombinant bacterial collagen polypeptide comprising a Sec domain for periplasmic localization, an N22 domain for directing the recombinant protein to the outer membrane for secretion;

wherein the nucleic acid sequence is under the control of a promoter to express the recombinant polypeptide; and

wherein the bacterial collagen polypeptide comprises a trypsin-sensitive globular variable domain comprising the amino acid sequence set forth in SEQ ID NO: 2075, and at least one collagen domain comprising the amino acid sequence set forth in SEQ ID NO: 2076.

5. The genetically-modified bacterium of claim 4 , wherein the nucleic acid sequence further encodes a translational enhancing element (TEE).

6. The genetically-modified bacterium of claim 4 , wherein the nucleic acid sequence further encodes each of the curli-specific accessory proteins selected from csgC, csgE, csgF, and csgG.

7. The method of claim 1 , wherein the nucleic acid sequence further encodes at least curli-specific accessory proteins csgE, and csgG.

8. The method of claim 1 , wherein the bacterial collagen protein encoded by the nucleic acid sequence comprises a detectable protein tag selected from the group consisting of a poly-histidine tag, a myc tag, a FLAG tag, a hemagglutinin (HA) tag, and a V5 tag.

9. The method of claim 1 , wherein the bacterial collagen polypeptide is capable of stable triple-helix formation.

10. The method of claim 9 , wherein the bacterial collagen polypeptide triple-helix is capable of indefinite elongation.

11. The method of claim 1 , wherein the E. coli bacterium is non-pathogenic.

12. The method of claim 1 , wherein the E. coli bacterium lacks a functional endogenous curli operon.

13. The method of claim 1 , wherein the E. coli is mutant strain PQN4.

14. The genetically-modified bacterium of claim 4 , wherein the nucleic acid sequence further encodes at least curli-specific accessory proteins csgE, and csgG.

15. The genetically-modified bacterium of claim 4 , wherein the bacterial collagen protein encoded by the nucleic acid sequence comprises a detectable protein tag selected from the group consisting of a poly-histidine tag, a myc tag, a FLAG tag, a hemagglutinin (HA) tag, and a V5 tag.

16. The genetically-modified bacterium of claim 4 , wherein the bacterial collagen polypeptide is capable of stable triple-helix formation.

17. The genetically-modified bacterium of claim 16 , wherein the bacterial collagen polypeptide triple-helix is capable of indefinite elongation.

18. The genetically-modified bacterium of claim 4 , wherein said bacterium is non-pathogenic.

19. The genetically-modified bacterium of claim 4 , wherein said bacterium lacks a functional endogenous curli operon.

20. The genetically-modified bacterium of claim 4 , wherein said bacterium is mutant strain PQN4.

Assignments (2)

Continuity (3)

References Cited (15)