# US 9,434,974 B2 · Installation of genomes or partial genomes into cells or cell-like systems

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- Number: US 9,434,974 B2
- Document ID: US9434974B2
- Status: Granted
- Kind: B2
- Application number: 11/644,713
- Filed: 2006-12-22
- Published: 2007-11-22
- Granted: 2016-09-06
- Earliest claimed: 2005-12-23
- Assignees: Synthetic Genomics, Inc.
- Applicants: John I. Glass; Lei Young; Carole Lartigue; Nacyra Assad-Garcia; Hamilton O. Smith; Clyde A. Hutchison; J. Craig Venter
- Inventors: John I. Glass; Lei Young; Carole Lartigue; Nacyra Assad-Garcia; Hamilton O. Smith; Clyde A. Hutchison; J. Craig Venter
- Web page: https://patentgrep.com/patent/US9434974B2

## CPC

- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C12N 2510/00: Genetically modified cells
- 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)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A method is provided for introducing a genome into a cell or cell-like system. The introduced genome may occur in nature, be manmade with or without automation, or may be a hybrid of naturally occurring and manmade materials. The genome is obtained outside of a cell with minimal damage. Materials such as a proteins, RNAs, polycations, nucleoid condensation proteins, or gene translation systems may accompany the genome. The genome is installed into a naturally occurring cell or into a manmade cell-like system. A cell-like system or synthetic cell resulting from the practice of the provided method may be designed and used to yield gene-expression products, such as desired proteins. By enabling the synthesis of cells or cell-like systems comprising a wide variety of genomes, accompanying materials and membrane types, the provided method makes possible a broader field of experimentation and bioengineering than has been available using prior art methods.

## Claims

### Claim 1

A method for making a bacterial synthetic cell, the method comprising:
preparing a Mycoplasma capricolum recipient cell for installation of a bacterial genome;
isolating a genome from a Mycoplasma mycoides cell by suspending the isolated genome in an agarose suspension; and
introducing the isolated genome into the Mycoplasma capricolum recipient cell by contacting the recipient cell with the genome.
wherein the genome of the Mycoplasma capricolum recipient cell is removed or inactivated to produce the recipient cell prior to, or after, introduction of the isolated genome by means of a physical, genetic or selective method or a combination thereof;
wherein the introduced genome is self-replicating; the introduced genome does not recombine with a genome of the recipient cell; and the recipient cell takes on the phenotype programmed by the introduced genome thereby producing a synthetic cell.

### Claim 2 (depends on 1)

The method of claim 1, wherein the isolating step further comprises:
a) suspending the Mycoplasma mycoides cell in a melted agarose composition;
b) digesting the suspended Mycoplasma mycoides cell with a lysis mixture effective to lyse the cell, wherein the lysis mixture comprises a proteinase and a detergent;
c) washing the lysed Mycoplasma mycoides cell to form a suspension of the Mycoplasma mycoides genome in the melted agarose composition; and
d) optionally separating linear DNA molecules, RNA, and peptides from the Mycoplasma mycoides genome.

### Claim 3 (depends on 1)

The method of claim 1, wherein:
a) the isolating step further comprises optionally removing, digesting or melting the agarose in the genome suspension; and
b) the introducing step further comprises combining the genome with the Mycoplasma capricolum recipient cell in the presence of cationic liposomes and the genomic DNA is introduced via lipofection.

### Claim 4 (depends on 1)

The method of claim 1, wherein:
a) the isolating step further comprises optionally removing, digesting or melting the agarose in the genome suspension; and
b) the introducing step further comprises combining the genome with the Mycoplasma capricolum recipient cell in the presence of polyethylene glycol.

### Claim 5 (depends on 1)

The method of claim 1, wherein the Mycoplasma capricolum recipient cell comprises a ghost cell.

### Claim 6 (depends on 1)

The method of claim 1, wherein the genome of the Mycoplasma capricolum recipient cell is removed prior to introducing the isolated bacterial genome.

### Claim 7 (depends on 1)

The method of claim 1, wherein the genome of the Mycoplasma capricolum recipient cell is transiently present in the cell upon introduction of the isolated genome and is then removed by cell division to make a bacterial synthetic cell comprising the isolated genome.