Iterative nucleic acid assembly using activation of vector-encoded traits

• US 8,053,191 B2
• Filed: 08/31/2007
• Issued: 11/08/2011
• Est. Priority Date: 08/31/2006
• Status: Active Grant

• Current Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Original Assignee: Westend Asset Clearinghouse Company, LLC
• Inventors: Blake, William J.
• Primary Examiner(s): CHUNDURU, SURYAPRABHA
• Application Number:
  US11/897,939
• Publication Number:
  US 20090155858A1
• Time in Patent Office: 1,530 Days
• Field of Search: 435/6, 435/91.2, 536/22.1
• US Class Current: 435/6.12
• 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 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• 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: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions

Abstract

Certain aspects of the present invention provide methods for assembling nucleic acid molecules using iterative activation of one or more vector-encoded traits to progressively assemble a longer nucleic acid insert. Aspects of the invention also provide kits, compositions, devices, and systems for assembling synthetic nucleic acids using iterative activation of one or more vector-encoded traits.

Claims

1. A method for assembling nucleic acid segments, the method comprising

providing a first and second population of nucleic acids, each population having at least a first, second, third and fourth restriction sites, wherein each population comprises at its 5′

end a 5′

activation sequence located between the first and second restriction sites and at its 3′

end a 3′

activation sequence located between the third and fourth restriction sites;

generating a first population of nucleic acid segments having the 5′

activation sequence but lacking the 3′

activation sequence by digesting the first population of nucleic acids using a first set of restriction enzymes that cleave the nucleic acids at the first and third restriction sites;

generating a second population of nucleic acid segments having the 3′

activation sequence but lacking the 5′

activation sequence by digesting the second population of nucleic acids using a second set of restriction enzymes that cleave the nucleic acids at the second and fourth restriction sites;

combining the first and second populations of nucleic acid segments with a first linearized nucleic acid vector that is digested with one or more restriction enzymes that produce restriction site overhangs that are complementary to the overhangs generated by the first and fourth restriction enzymes on the first and second populations of nucleic acid segments, wherein the first linearized nucleic acid vector comprises (i) at its 3′

end a coding sequence of a first marker gene 5′

of the first restriction site, and (ii) at its 5′

end a coding sequence of a second marker gene 3′

of the fourth restriction site, and

isolating ligated first nucleic acid vectors that express the first and the second marker genes, wherein expression of the first and the second marker genes is indicative of correct assembly of the first and second populations of nucleic acid segments and wherein expression of the first marker gene is regulated by the first activation sequence and expression of the second marker gene is regulated by the second activation sequence.

2. The method of claim 1, further comprising

digesting a third population of nucleic acids having at least first, second, third and fourth restriction sites, using the first set of restriction enzymes that cleave the nucleic acids at the first and third restriction sites,

digesting a fourth population of nucleic acids having at least first, second, third and fourth restriction sites, using the second set of restriction enzymes that cleave the nucleic acids at the second and fourth sites, wherein the third and fourth populations of nucleic acids comprise a 5′

first activation sequence located between the first and second restriction sites and a 3′

second activation sequence located between the third and fourth restriction sites, and digestion of the third population results in a third population of nucleic acid segments that comprises the first activation sequence but lacks the second activation sequence, and digestion of the fourth population results in a fourth population of nucleic acid segments that lacks a first activation sequence and comprises a second activation sequence,

combining in the presence of a ligase the third and fourth populations of nucleic acid segments with a second nucleic acid vector that is digested with one or more restriction enzymes that produce restriction site overhangs that are complementary to the overhangs generated by the first and fourth restriction enzymes on the third and fourth populations of nucleic acid segments, wherein the second nucleic acid vector comprises a coding sequence of a first marker gene 5′

of the first restriction site and a coding sequence of a second marker gene 3′

of the fourth restriction site,

selecting for ligated second nucleic acid vectors that express the first and the second marker genes, wherein expression of the first and the second marker genes is indicative of correct assembly of the third and fourth populations of nucleic acid segments and wherein expression of the first marker gene is regulated by the first activation sequence and expression of the second marker gene is regulated by the second activation sequence,

digesting the ligated second nucleic acid vector with restriction enzymes that cleave at the second and fourth restriction sites to release a fifth population of nucleic acid segments lacking a first activation sequence and comprising a second activation sequence,

digesting the ligated first nucleic acid vector with restriction enzymes that cleave at the first and third restriction sites to release a sixth population of nucleic acid segments comprising a first activation sequence and lacking a second activation sequence, and

combining the fifth and sixth populations of nucleic acid segments with a third nucleic acid vector digested with one or more restriction enzymes that produce restriction site overhangs that are complementary to the overhangs generated by the first and fourth restriction enzymes on the fifth and sixth populations of nucleic acid segments, wherein the third nucleic acid vector comprises a coding sequence of a third marker gene 5′

of the first restriction site and a coding sequence of a fourth marker gene 3′

of the fourth restriction site, and

selecting for ligated third nucleic acid vectors that express the third and fourth marker genes, wherein expression of the third and fourth marker genes is indicative of correct assembly of the fifth and sixth populations of nucleic acid segments and wherein expression of the first marker gene is regulated by the first activation sequence and expression of the second marker gene is regulated by the second activation sequence.

4. The method of claim 2, further comprising

digesting a third population of nucleic acids having at least first, second, third and fourth restriction sites, using a first set of restriction enzymes that cleave the nucleic acids at the first and third sites,

digesting a fourth population of nucleic acids having at least first, second, third and fourth restriction sites, using a second set of restriction enzymes that cleave the nucleic acids at the second and fourth sites, wherein the third and fourth populations of nucleic acids comprise a 5′

promoter sequence located between the first and second restriction sites and a 3′

promoter sequence between the third and fourth restriction sites, and digestion of the third population results in a third population of nucleic acid segments that comprises the 5′

promoter sequence but lacks the 3′

promoter sequence, and digestion of the fourth population results in a fourth population of nucleic acid segments that lacks a 5′

promoter sequence and comprises a 3′

promoter sequence,

combining in the presence of a ligase the third and fourth populations of nucleic acid segments with a second nucleic acid vector that is digested with restriction enzymes that cleave at the first and fourth restriction sites, wherein the second nucleic acid vector comprises a coding sequence of a first marker gene 5′

of the first restriction site, wherein the first marker gene expression is regulated by the 5′

promoter sequence, and a coding sequence of a second marker gene 3′

of the fourth restriction site, wherein the second marker gene is regulated by the 3′

promoter sequence,

selecting for ligated second nucleic acid vectors that express the first and the second marker genes, wherein expression of the first and the second marker genes is indicative of correct assembly of the third and fourth populations of nucleic acid segments, digesting the ligated second nucleic acid vector with restriction enzymes that cleave at the second and fourth restriction sites to release a fifth population of nucleic acid segments lacking a 5′

promoter sequence and comprising a 3′

promoter sequence,

digesting the ligated first nucleic acid vector with restriction enzymes that cleave at the first and third restriction sites to release a sixth population of nucleic acid segments comprising a 5′

promoter sequence and lacking a 3′

promoter sequence, and combining the fifth and sixth populations of nucleic acid segments with a third nucleic acid vector digested with restriction enzymes that cleave at the first and fourth restriction sites and having a third marker gene coding sequence 5′

of the first restriction site, wherein the third marker gene expression is regulated by the 3′

promoter sequence, and a fourth marker gene coding sequence 3′

of the fourth restriction site, wherein the fourth marker gene is regulated by the 5′

promoter sequence, and

selecting for ligated third nucleic acid vectors that express the third and fourth marker genes, wherein expression of the third and fourth marker genes is indicative of correct assembly of the fifth and sixth populations of nucleic acid segments.

5. The method of claim 1, wherein the first and second marker genes are antibiotic resistance genes.

6. The method of claim 2, wherein the first, second, third and fourth marker genes are antibiotic resistance genes.

7. The method of claim 1, wherein the restriction enzymes are type II restriction enzymes or type IIS restriction enzymes.

8. The method of claim 1, wherein the restriction enzymes that cleave the first restriction and fourth restriction sites are type II restriction enzymes, and the restriction enzymes that cleave the second and third restriction sites are type IIS restriction enzymes.

9. The method of claim 1, wherein the restriction enzymes that cleave the first, second, third, and fourth sites are type II restriction enzymes.

10. The method of claim 1, wherein the first, second, third and fourth populations of nucleic acids are cloned nucleic acids or PCR-derived nucleic acids.

11. The method of claim 1, wherein the first, second, third and fourth populations of nucleic acids comprise nucleic acids of about 1 kb in length.

12. The method of claim 2, further comprising digesting the ligated first nucleic acid vectors that express the first and the second marker genes using restriction enzymes that cleave at the first and fourth restriction sites in order to release an assembled nucleic acid.

13. The method of claim 2, further comprising digesting the ligated third nucleic acid vectors that express the third and fourth marker genes using restriction enzymes that cleave at the first and fourth restriction sites in order to release an assembled nucleic acid.

14. The method of claim 12, wherein the assembled nucleic acid is about 50 kb in length.

15. The method of claim 12, wherein the assembled nucleic acid is about 100 kb in length.

16. The method of claim 1, wherein the first activation sequence is a promoter, terminator or other activation sequence or fragment thereof.

17. The method of claim 1, wherein the second activation sequence is a promoter, terminator or other activation sequence or fragment thereof.

18. The method of claim 1, wherein the first marker gene is a selectable marker.

19. The method of claim 1, wherein the second marker gene is a selectable marker.

3. A method for assembling nucleic acid segments, the method comprising

providing a first and second population of nucleic acids, each population having at least first, second, third and fourth restriction sites, wherein each population comprises at its 5′

end a 5′

promoter sequence located between the first and second restriction sites and at its 3′

end a 3′

promoter sequence located between the third and fourth restriction sites, wherein the 5′ and

3′

promoter sequences are in opposite orientation to each other;

generating a first population of nucleic acid segments having the 5′

promoter sequence but lacking the 3′

promoter sequence by digesting the first population of nucleic acids using a first set of restriction enzymes that cleave the nucleic acids at the first and third restriction sites,

generating a second population of nucleic acid segments having the 3′

promoter sequence but lacking the 5′

promoter sequence by digesting the second population of nucleic acids using a second set of restriction enzymes that cleave the nucleic acids at the second and fourth restriction sites,

combining in the presence of a ligase the first and second populations of nucleic acid segments with a first linearized nucleic acid vector that is digested with restriction enzymes that cleave at the first and fourth restriction sites, wherein the first linearized nucleic acid vector comprises (i) at its 3′

end a coding sequence of a first marker gene 5′

of the first restriction site, wherein the first marker gene expression is regulated by the 5′

promoter sequence, and (ii) at its 5′

end a coding sequence of a second marker gene 3′

of the fourth restriction site, wherein the second marker gene is regulated by the 3′

promoter sequence, and

selecting for ligated first nucleic acid vectors that express the first and the second marker genes, wherein expression of the first and the second marker genes is indicative of correct assembly of the first and second populations of nucleic acid segments.

20. A method of assembling a nucleic acid, the method comprising a plurality of consecutive alternating assembly cycles, wherein each alternating assembly cycle comprises:

a) combining a first nucleic acid insert with a second nucleic acid insert and a first vector, wherein the first nucleic acid insert comprises a 5′

first portion of a target sequence and at its 5′

end a first activation sequence or fragment thereof, the second nucleic acid insert comprises a second portion of the target sequence and at its 3′

end a second activation sequence or fragment thereof, and the first linearized vector comprises a first activatable marker at its 3′

end and a second activatable marker at its 5′

end;

b) selecting for correct assembly of the first and second nucleic acid inserts in the first vector by selecting for activation of the first and second activatable markers, wherein correct assembly results in activation of the first activatable marker by the first activation sequence and activation of the second activatable marker by the second activation sequence;

c) isolating from step b) an assembled nucleic acid comprising the first and second portions of the target sequence and the first activation sequence, but not the second activation sequence;

d) combining the nucleic acid of step c) with a third nucleic acid insert and a second vector, wherein the third nucleic acid insert comprises a third portion of the target sequence and the second activation sequence at its 3′

end, and the second linearized vector comprises a third activatable marker at its 3′

end and a fourth activatable markers at its 5′

end;

e) selecting for correct assembly of the nucleic acid of step c) and the third nucleic acid insert in the second vector by selecting for activation of the third and fourth activatable markers, wherein correct assembly results in activation of the third activatable marker by the first activation sequence and activation of the fourth activatable marker by the second activation sequence; and

f) isolating from step e) an assembled nucleic acid comprising the first, second, and third portions of the target sequence and the first activation sequence, but not the second activation sequence;

wherein the nucleic acid of step f) can be combined in a subsequent assembly cycle starting at step a) with a with a fourth nucleic acid insert and the first linearized vector, wherein the fourth nucleic acid insert comprises a 3′

fourth portion of the target sequence and the second activation sequence.

21. The method of claim 20 wherein at least one nucleic acid insert comprises a non-functional activation sequence and wherein correct assembly of the target sequence results in the assembly of a functional activation sequence.