# US 11,896,626 B2 · Multistrain population control systems and methods

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- Number: US 11,896,626 B2
- Document ID: US11896626B2
- Status: Granted
- Kind: B2
- Application number: 16/972,405
- Filed: 2019-06-07
- Published: 2021-06-10
- Granted: 2024-02-13
- Earliest claimed: 2018-06-08
- Assignees: The Regents of the University of California
- Applicants: The Regents of the University of California
- Inventors: Jeff Hasty; Michael Julius Liao; Muhammad Omar Din
- Web page: https://patentgrep.com/patent/US11896626B2

## CPC

- A61K 35/74: Bacteria (therapeutic use of a bacterial protein A61K38/00)
- C07K 14/245: Escherichia (G)
- C12N 1/20: Bacteria; Culture media therefor
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C12P 39/00: Processes involving microorganisms of different genera in the same process, simultaneously
- A61K 2035/11: {Medicinal preparations comprising living procariotic cells}
- A61K 2035/115: {Probiotics}
- C12R 2001/19: Escherichia coli
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS

## Abstract

Provided herein are multi-strain population control systems, methods, kits, and compositions. Also provided are methods, systems, kits, and compositions for culturing bacterial cells in multi-strain ecosystems, and temporally arranged multi-strain ecosystems or cultures using a synchronized lysis circuit in combination with multiple toxin/antitoxin systems to cycle continuously over a long period of time.

## Claims

### Claim 1

A method of maintaining a bacterial co-culture, the method comprising:
culturing a first bacterial strain for a first period of time in a growth environment;
adding a second bacterial strain to the growth environment and culturing the second bacterial strain for a second period of time;
adding a third bacterial strain to the growth environment and culturing the third bacterial strain for a third period of time,
wherein each of the first, second, and third bacterial strains comprises a toxin system;
wherein the toxin system of the first bacterial strain produces a first toxin/first antitoxin pair and a third antitoxin from a third toxin/third antitoxin pair wherein the first bacterial strain does not produce the third toxin;
wherein the toxin system of the second bacterial strain produces a second toxin/second antitoxin pair and the first antitoxin from the first toxin/first antitoxin pair wherein the second bacterial strain does not produce the first toxin; and
wherein the toxin system of the third bacterial strain produces a third toxin/third antitoxin pair and the second antitoxin from the second toxin/second antitoxin pair wherein the third bacterial strain does not produce the second toxin, and
wherein each of the first, second, and third bacterial strains comprises
a lysis plasmid having a lysis gene under the control of an activatable promoter; and
an activator plasmid having an activator gene, the expression of which promotes the accumulation of a quorum-sensing molecule,
wherein both the activatable promoter of the lysis gene and the expression of the activator gene is activated by the quorum-sensing molecule.

### Claim 2 (depends on 1)

The method of claim 1, wherein the quorum-sensing molecule is the same in each of the first, second, and third bacterial strains.

### Claim 3 (depends on 1)

The method of claim 1, wherein the toxin system of the first bacterial strain, the second bacterial strain, and the third bacterial strain is independently encoded in a genome of at least one of the first, second, and third bacterial strains, on a plasmid integrated into the genome of at least one of the first, second, and third bacterial strains, on multiple plasmids integrated into the genome of at least one of the first, second, and third bacterial strains, or a combination thereof.

### Claim 4 (depends on 1)

The method claim 1, wherein in each of the at least the first, second, and third bacterial strains the lysis plasmid comprises a lysis gene, an activatable promoter, and a reporter gene; and the activator plasmid comprises an activator gene, a degradation tag, and a reporter gene.

### Claim 5 (depends on 1)

The method claim 1, wherein the lysis gene in each of the at least the first, second, and third bacterial strains is E from a bacteriophage ΦX174.

### Claim 6 (depends on 1)

The method claim 1, wherein the activatable promoter in each of the at least the first, second, and third bacterial strains is a LuxR-AHL activatable luxI promoter and the activator gene is a LuxI.

### Claim 7 (depends on 1)

The method claim 1, wherein one or more of the first, second, and third bacterial strains encodes a heterologous nucleic acid and/or a heterologous protein operably linked to a promoter.

### Claim 8 (depends on 7)

The method of claim 7, wherein the heterologous nucleic acid and/or heterologous protein is a therapeutic agent.

### Claim 9 (depends on 8)

The method of claim 8, wherein the therapeutic agent is selected from the group consisting of: an inhibitory nucleic acid, a cytokine, an enzyme, a peptide hormone, a fusion protein, a clotting factor, a toxin, an antimicrobial peptide, and an antibody or antigen-binding fragment thereof.

### Claim 10

A method of maintaining a bacterial co-culture, the method comprising:
providing n bacterial strains comprising at least a first bacterial strain, a second bacterial strain, and an nth bacterial strain that are distinct from each other;
co-culturing each of the bacterial strains sequentially for one or more independent periods of time in a growth environment;
wherein n is at least 3;
wherein each of the n bacterial strains comprises a toxin system;
wherein the toxin system of the first bacterial strain produces a first toxin/first antitoxin pair and an nth antitoxin from an nth toxin/nth antitoxin pair, wherein the first toxin is effective against the nth bacterial strain;
wherein the toxin system of the second bacterial strain produces a second toxin/second antitoxin pair and the first antitoxin from the first toxin/first antitoxin pair, wherein the second toxin is effective against the first bacterial strain; and
wherein the toxin system of the first bacterial strain produces an nth toxin/nth antitoxin pair and an n−1th antitoxin from an n−1th toxin/n−1th antitoxin pair, wherein the nth toxin is effective against the n−1th bacterial strain.

### Claim 11 (depends on 10)

The method of claim 10, wherein the toxin system of the n bacterial strains is independently encoded in a genome of at least one of the first, second, and nth bacterial strains, on a plasmid integrated into the genome of at least one of the first, second, and nth bacterial strains, on multiple plasmids integrated into the genome of at least one of the first, second, and nth bacterial strains, or a combination thereof.

### Claim 12 (depends on 10)

The method claim 10, wherein one or more of the periods of time partially or completely overlap.

### Claim 13 (depends on 10)

The method claim 10, wherein one or more of the n bacterial strains encodes a heterologous nucleic acid and/or a heterologous protein operably linked to a promoter.

### Claim 14 (depends on 13)

The method of claim 13, wherein the heterologous nucleic acid and/or heterologous protein is a therapeutic agent.

### Claim 15 (depends on 14)

The method of claim 14, wherein the therapeutic agent is selected from the group consisting of: an inhibitory nucleic acid, a cytokine, an enzyme, a peptide hormone, a fusion protein, a clotting factor, a toxin, an antimicrobial peptide, and an antibody or antigen-binding fragment thereof.

### Claim 16 (depends on 10)

The method claim 10, wherein each of the second bacterial strain through the nth bacterial strain does not produce the second toxin of the toxin system of the n−1th strain, and the first bacterial strain does not produce the nth toxin of the toxin system of the nth bacterial strain.