METHOD OF DUAL-ADAPTER RECOMBINATION FOR EFFICIENT CONCATENATION OF MULTIPLE DNA FRAGMENTS IN SHUFFLED OR SPECIFIED ARRANGEMENTS

• US 20130298265A1
• Filed: 10/12/2011
• Published: 11/07/2013
• Est. Priority Date: 10/12/2010
• Status: Active Grant

• Current Assignee: Cornell University
• Original Assignee: Cornell University
• Inventors: Collmer, Alan, Cunnac, Sbastien
• Granted Patent: US 9,689,012 B2
• Time in Patent Office: Days
• US Class Current: 800/13
• 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 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1041 Ribosome/Polysome display, e.g. SPERT, ARM
• 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
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor

Abstract

The present invention relates to methods of assembling a plurality of genetic units to form synthetic genetic constructs. This method involves appending universal adapter oligonucleotides and flexible adapter oligonucleotides to the 5′ and 3′ ends of separate genetic units to be assembled to form separate dual extended genetic units. The dual extended genetic units are assembled together via homologous recombination between the flexible adapter oligonucleotide portions of the dual extended units to form synthetic genetic constructs. The present invention further relates to synthetic genetic constructs formed using the methods of the present invention, and vectors, cells, and organisms containing such synthetic genetic constructs.

Claims

1. A method of assembling synthetic genetic constructs comprising a plurality of genetic units, said method comprising:

providing a plurality of separate genetic units each having 5′ and

3′

ends;

appending universal adapter oligonucleotides to the 5′ and

3′

ends of each separate genetic unit to form separate extended genetic units each having 5′ and

3′

ends;

attaching flexible adapter oligonucleotides to the 5′ and

3′

ends of separate extended genetic units to form separate dual extended genetic units; and

assembling together the separate dual extended genetic units via homologous recombination between the flexible adapter oligonucleotides of the dual extended genetic units to form the synthetic genetic constructs.

2. The method of claim 1, wherein said appending the universal adapter oligonucleotides to the 5′

and 3′

ends of each genetic unit involves using one or more enzymes selected from the group consisting of ligases, polymerases, and restriction enzymes.

3. The method of claim 2, wherein said appending the universal adapter oligonucleotides to each genetic unit to form extended genetic units involves using a polymerase, said method further comprising:

providing one or more sets of universal adapter oligonucleotide primers, each set comprising (1) a first oligonucleotide primer comprising a genetic unit specific portion and a 5′

universal adapter specific portion and (2) a second oligonucleotide primer comprising a genetic unit specific portion and a 5′

universal adapter specific portion, wherein the universal adapter specific portions of the primers in a primer set comprise different nucleotide sequences;

providing a polymerase

blending the universal adapter oligonucleotide primer sets and the polymerase with the one or more separate genetic units to form a polymerase chain reaction mixture; and

subjecting the polymerase reaction mixture to one or more polymerase chain reaction cycles to append the universal adapter oligonucleotides to 5′ and

3′

ends of each separate genetic unit to form separate extended genetic units.

4. The method of claim 1, wherein each flexible adapter oligonucleotide comprise a universal adapter specific portion and a flexible adapter specific portion.

5. The method of claim 1, wherein said attaching the flexible adapter oligonucleotides to the separate extended genetic units involves using an enzyme selected from the group consisting of polymerases, ligases, and restriction enzymes.

6. The method of claim 5, wherein said attaching the flexible adapter oligonucleotides to the separate extended genetic units comprises using a polymerase, said method further comprising:

providing a plurality of flexible adapter oligonucleotide primer sets, each set comprising (1) a first oligonucleotide primer comprising a universal adapter specific portion and a 5′

flexible adapter specific portion and (2) a second oligonucleotide primer comprising a universal adapter specific portion and a 5′

flexible adapter specific portion;

providing a polymerase

blending the plurality of flexible adapter oligonucleotide primer sets and the polymerase with the separate extended genetic units to form a polymerase chain reaction mixture; and

subjecting the polymerase reaction mixture to two or more polymerase chain reaction cycles to attach the flexible adapter oligonucleotides to the 5′ and

3′

ends of the separate extended genetic units to form the separate dual extended genetic units.

7. The method of claim 1, wherein homologous recombination occurs between identical flexible adapter oligonucleotide portions of the separate dual extended genetic units.

8. The method of claim 1, wherein said assembling together the separate genetic units further comprises:

providing a linearized nucleic acid vector comprising flexible adapter specific portions at its terminal ends and

joining the separate dual extended genetic units with the nucleic acid vector to form a re-circularized vector containing the synthetic genetic construct.

9. The method of claim 8 further comprising:

isolating the re-circularized nucleic acid vector containing the synthetic genetic construct.

10. The method of claim 8, wherein the nucleic acid vector is selected from the group consisting of a plasmid vector, a shuttle vector, a viral vector, an expression vector, and a cloning vector.

11. The method of claim 1, wherein said assembling together the separate genetic units is carried out by in vitro homologous recombination, said method further comprising:

providing one or more recombinant proteins or cell extracts having the property of promoting homologous recombination and

blending the one or more recombinant proteins or cell extracts with the separate dual extended genetic units under conditions suitable for homologous recombination and assembly of the dual extended genetic units to form the synthetic genetic construct.

12. The method of claim 1, wherein said assembling together the separate genetic units is carried out by in vivo homologous recombination, said method further comprising:

transforming a competent host cell with the separate dual extended genetic units under conditions suitable for in vivo homologous recombination to occur between identical flexible adapter oligonucleotide portions of the dual extended genetic units thereby assembling the separate genetic units into a synthetic genetic construct.

13. The method of claim 12 further comprising:

isolating the synthetic genetic construct from the competent host cell.

14. The method of claim 12, wherein the competent host cell is selected from the group consisting of an animal cell, plant cell, archaebacterial cell, eubacterial cell, fungal cell, protist cell, and synthetic cell.

15. The method of claim 12 further comprising:

transforming the competent host cell with a linearized nucleic acid vector comprising flexible adapter specific portions at its terminal ends, wherein homologous recombination occurs between identical flexible adapter specific portions of the separate dual extended genetic units and the linearized vector to form a re-circularized nucleic acid vector comprising the synthetic genetic construct.

16. The method of claim 15, wherein the linearized vector is selected from the group consisting of plasmid vectors, shuttle vectors, cloning vectors, and expression vectors.

17. The method of claim 1, wherein the separate genetic units are randomly assembled.

18. The method of claim 1, wherein the separate genetic units are semi-randomly assembled.

19. The method of claim 1, wherein the separate genetic units are non-randomly assembled.

20. The method of claim 1, wherein the separate genetic units comprise artificial nucleotide sequences.

21. The method of claim 1, wherein the separate genetic units comprise nucleotide sequences derived from a genomic sequence of an animal, plant, archaebacteria, eubacteria, fungus, protist, virus, and combinations thereof.

22. The method of claim 1, wherein each separate genetic unit comprises one or more functional domains and/or modules of a single gene.

23. The method of claim 1, wherein each separate genetic unit comprises one or more functional genes.

24. The method of claim 1, wherein the synthetic genetic construct encodes one or more partial or complete biological pathways.

25. The method of claim 1, wherein the synthetic gene construct encodes a partial or complete genome.

26. The method of claim 1 further comprising:

isolating the synthetic genetic construct; and

transforming a host cell with the isolated synthetic genetic construct under conditions suitable to express the synthetic genetic construct.

27. The method of claim 26, wherein the host cell is selected from the group consisting of an animal cell, plant cell, archaebacterial cell, eubacterial cell, fungal cell, protist cell, and synthetic cell.

28. A synthetic genetic construct comprising a plurality of assembled separate genetic units, wherein each separate genetic unit comprises:

a gene specific portion;

a pair of universal adapter oligonucleotides appended to the 5′ and

3′

ends of the gene specific portion; and

a pair of flexible adapter oligonucleotides attached to the 5′ and

3′

ends of the universal adapter oligonucleotides appended to the gene specific portion.

29. The method of claim 28, wherein the separate genetic units comprise synthetic nucleotide sequences.

30. The method of claim 28, wherein the separate genetic units comprise artificial nucleotide sequences.

31. The method of claim 28, wherein the separate genetic units comprise nucleotide sequences derived from a genomic sequence of an animal, plant, archaebacteria, eubacteria, fungus, protist, virus, and combinations thereof.

32. The method of claim 28, wherein the separate genetic units comprises one or more functional domains and/or modules of a single gene.

33. The synthetic genetic construct of claim 28, wherein the separate genetic units comprise one or more functional genes.

34. The synthetic genetic construct of claim 28, wherein the synthetic genetic construct encodes one or more partial or complete biological pathways.

35. The synthetic genetic construct of claim 28, wherein the synthetic genetic construct encodes a partial or complete genome.

36. A nucleic acid vector comprising the synthetic genetic construct of claim 28.

37. A bacteriophage comprising the synthetic genetic construct of claim 28.

38. A host cell transformed with the synthetic genetic construct of claim 28.

39. The host cell of claim 38, wherein the host cell is selected from the group consisting of an animal cell, plant cell, archaebacterial cell, eubacterial cell, fungal cell, protist cell, and synthetic cell.

40. The host cell of claim 38, wherein expression of the synthetic genetic construct by the host cell provides a gain-of-function to the host cell.

41. The host cell of claim 38, wherein the gain-of-function provided to the host cell comprises enhanced growth, immunity, pathogen resistance, protein production, enhanced or new metabolic pathways for detoxification or transformation of endogenous or exogenous biological molecules, and combinations thereof.

42. The host cell of claim 38, wherein expression of the synthetic genetic construct by the host cell produces a biological product that is not endogenous to the host cell.

43. The host cell of claim 42, wherein the biological product is selected from the group consisting of an exogenous protein, a pharmaceutical, an agricultural chemical, biological energy source, an organic compound, a polyketide, a non-ribosomal peptide, and combinations thereof.

44. The host cell of claim 38, wherein expression of the synthetic genetic construct by the host cell provides a loss-of-function to the host cell.

45. The host cell of claim 38, wherein the synthetic genetic construct encodes one or more inhibitory nucleic acid molecules suitable for silencing expression of one or more genes of the host cell or one or more genes of a pathogen.

46. A plant comprising the transformed host cell of claim 38.

47. An animal comprising the transformed host cell of claim 38.

48. A kit for assembling a plurality of genetic units comprising:

one or more sets of universal adapter oligonucleotides;

a collection of flexible adapter oligonucleotides; and

reagents suitable for carrying out a homologous recombination reaction.

49. The kit of claim 48, wherein the collection of flexible adapter oligonucleotides comprise the nucleotide sequences of SEQ ID NOs:

1-20.

50. The kit of claim 48 further comprising:

a computer readable storage medium having stored thereof instructions and design parameters for assembling genetic units comprising flexible adapter oligonucleotides.

51. The kit of claim 48 further comprising

components for appending the universal adapter oligonucleotides to genetic units and the flexible adapter oligonucleotides to the universal adaptor oligonucleotides.