Synthetic genomes

• US 20070264688A1
• Filed: 12/06/2006
• Published: 11/15/2007
• Est. Priority Date: 12/06/2005
• Status: Active Grant

• Current Assignee: Telesis Bio, Inc.
• Original Assignee: Synthetic Genomics, Inc.
• Inventors: Smith, Hamilton, Venter, J., Hutchison, Clyde
• Granted Patent: US 10,041,060 B2
• Time in Patent Office: Days
• US Class Current: 435/69.100
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

Abstract

Methods are provided for constructing a synthetic genome, comprising generating and assembling nucleic acid cassettes comprising portions of the genome, wherein at least one of the nucleic acid cassettes is constructed from nucleic acid components that have been chemically synthesized, or from copies of the chemically synthesized nucleic acid components. In one embodiment, the entire synthetic genome is constructed from nucleic acid components that have been chemically synthesized, or from copies of the chemically synthesized nucleic acid components. Rational methods may be used to design the synthetic genome (e.g., to establish a minimal genome and/or to optimize the function of genes within a genome, such as by mutating or rearranging the order of the genes). Synthetic genomes of the invention may be introduced into vesicles (e.g., bacterial cells from which part or all of the resident genome has been removed, or synthetic vesicles) to generate synthetic cells. Synthetic genomes or synthetic cells may be used for a variety of purposes, including the generation of synthetic fuels, such as hydrogen or ethanol.

Claims

1. A method for constructing a synthetic genome comprising:

assembling nucleic acid cassettes that comprise portions of the synthetic genome, wherein at least one of the nucleic acid cassettes is constructed from nucleic acid components that have been chemically synthesized, or from copies of chemically synthesized nucleic acid components.

2. The method of claim 1, wherein one or more of the nucleic acid cassettes are prepared by assembling chemically synthesized, overlapping oligonucleotides of about 50 nucleotides.

3. The method of claim 1, wherein the cassettes are about 4 kilobases to about 7 kilobases in length.

4. The method of claim 1, wherein the cassettes are about 4.5 kilobases to about 6.5 kilobases in length.

5. The method of claim 1, wherein the cassettes are about 5 kilobases in length.

6. The method of claim 1, wherein the cassettes overlap adjacent cassettes by at least about 200 nucleotides.

7. The method of claim 1, wherein the synthetic genome is a eukaryotic cellular organelle.

8. The method of claim 1, wherein the synthetic genome is a bacterial genome.

9. The method of claim 1, wherein the synthetic genome is a minimal genome.

10. The method of claim 1, wherein the synthetic genome is a minimal replicating genome.

11. The method of claim 1, wherein the synthetic genome is substantially identical to a naturally occurring genome.

12. The method of claim 1, wherein the synthetic genome is a non-naturally occurring genome.

13. The method of claim 1, wherein one or more of the cassettes can be readily removed and replaced in the synthetic genome.

14. The method of claim 1, wherein an entire synthetic genome is constructed from nucleic acid components that have been chemically synthesized, or from copies of the chemically synthesized nucleic acid components.

15. The method of claim 1, wherein the synthetic genome is a synthetic cellular genome.

16. The method of claim 15, wherein an entire synthetic cellular genome is constructed from nucleic acid components that have been chemically synthesized or from copies of the chemically synthesized nucleic acid components.

17. The method of claim 1, wherein the synthetic genome further comprises sequences that allow production of a product of interest.

18. The method of claim 17, wherein the product of interest is an energy source.

19. The method of claim 14, wherein the entire synthetic genome further comprises sequences that allow production of a product of interest.

21. The method of claim 15, wherein the synthetic cellular genome further comprises sequences that allow production of a product of interest.

22. The method of claim 21, wherein the product of interest is an energy source.

23. The method of claim 16, wherein the entire synthetic cellular genome further comprises sequences that allow production of a product of interest.

24. The method of claim 23, wherein the product of interest is an energy source.

25. The method of claim 1, further comprising rationally designing components of the synthetic genome.

26. The method of claim 1, further comprising constructing a synthetic cell, by introducing the synthetic genome into a vesicle.

27. The method of claim 15, further comprising constructing a self-replicating synthetic cell, by introducing the synthetic cellular genome into a vesicle, optionally under conditions effective for replication of the synthetic cell.

28. The method of claim 26, further comprising producing a product of interest by culturing the synthetic cell under conditions effective to produce the product.

29. The method of claim 27, further comprising producing a product of interest by culturing the self-replicating synthetic cell under conditions effective to replicate the synthetic cell and to produce the product.

30. The method of claim 1, further comprising automating the method.

31. The method of claim 1, further comprising the synthetic genome.

33. The method of claim 26, further comprising the synthetic cell.

20. The method of paragraph 19, wherein the product of interest is an energy source.

32. A synthetic genome.

34. A synthetic cell comprising a synthetic genome.

35. A method for making a synthetic cell, comprising:

removing part or all of a resident genome from a microorganism; and

replacing the resident genome with a synthetic genome that exhibits at least one property that is different from the resident genome.

36. The method of claim 35, further comprising the synthetic cell.

37. A synthetic cell comprising:

a microorganism of one species from which part or all of the resident genome has been removed; and

a synthetic genome which exhibits at least one property that is different from the resident genome.

38. A method comprising:

designing a synthetic genome;

constructing the synthetic genome;

introducing the synthetic genome into a biological system; and

expressing the synthetic genome.