Method for constructing chimeric plasmid library

Patent metadata

Field	Value
Publication number	US-11643648-B2
Application number	US-202017600836-A
Country	US
Kind code	B2
Filing date	Mar 24, 2020
Priority date	Apr 1, 2019
Publication date	May 9, 2023
Grant date	May 9, 2023

Abstract

The present invention addresses the problem of providing a novel method which is for preparing a DNA fragment for microbial cell transformation, and by which the combinatorial library of a long-chain DNA can be efficiently constructed and confirmation of the genotype of the obtained clone is facilitated. The present invention is a method for preparing a DNA fragment, which is for microbial cell transformation and has at least one insert DNA unit that includes a DNA containing an effective replication origin in a host microorganism and an insert DNA in which unit DNAs are linked, the method being characterized by including: (A) a step for preparing, through an OGAB method, a plurality of types of plasmids having an insert DNA unit in which a plurality of types of unit DNAs capable of being linked in a specific linking order are linked; (B) a step for decomposing a plasmid into unit DNAs by treating the plurality of types of plasmids prepared in the step (A) with a restriction enzyme suitable for each plasmid and preparing a mixed liquid of a plurality of types of unit DNAs; and (C) a step for preparing a long-chain DNA fragment by re-assembling the unit DNAs through the OGAB method by using the mixed liquid of a plurality of types of unit DNAs obtained in the step (B).

First claim

The invention claimed is:

1. A preparation method of a DNA fragment, the DNA fragment having at least one insert DNA unit comprising: a DNA comprising a replication origin effective in a host; and an insert DNA in which unit DNAs are linked, characterized in that the method comprises:
(A) processing a plurality of types of plasmids, wherein the plasmids comprise an insert DNA unit in which a plurality of types of unit DNAs capable of being linked in a specific linking order are linked, with a restriction enzyme suitable for each plasmid to prepare a plurality of types of unit DNA mixture solutions each of which comprises the plurality of types of unit DNAs linked on each of the plasmid; and
(B) re-assembling the plurality of types of unit DNAs by OGAB method using the plurality of types of unit DNA mixture solutions obtained in step (A) to prepare a long-chain DNA fragment.

2. The preparation method of a DNA fragment of claim 1 , wherein the DNA fragment is a DNA fragment for cell transformation.

3. The preparation method of a DNA fragment of claim 2 , wherein the replication origin is effective in a host microorganism, and the DNA fragment for cell transformation is for microbial cell transformation.

4. The preparation method of a DNA fragment of claim 1 , further comprising preparing the plurality of types of plasmids prior to step (A).

5. The preparation method of a DNA fragment of claim 1 , wherein the plurality of types of plasmids are prepared by OGAB method.

6. The preparation method of a DNA fragment of claim 1 , wherein all the ratios between molar concentrations for DNA fragments in the plurality of types of unit DNA mixture solutions obtained in step (A) are 0.8 to 1.2.

7. The preparation method of a DNA fragment of claim 1 , wherein that in the plurality of types of plasmids, the number of types of unit DNAs comprised in one type of insert DNA unit is 3 to 60.

8. The preparation method of a DNA fragment of claim 1 , wherein the number of types of the restriction enzymes used in step (A) is three or less.

9. The preparation method of a DNA fragment of claim 1 , wherein the restriction enzyme is a restriction enzyme that produces an overhang end.

10. A preparation method of a DNA fragment, the DNA fragment having at least one insert DNA unit comprising: a DNA comprising a replication origin effective in a host; and an insert DNA in which unit DNAs are linked, characterized in that the method comprises:
(A′) preparing a plurality of types of plasmids comprising a DNA fragment for cell transformation by the preparation method of a DNA fragment of claim 1 ;
(B′) processing the plurality of types of plasmids obtained in step (A′) with a restriction enzyme suitable for each plasmid to cleave the plasmids into a plurality of types of unit DNAs and preparing a plurality of types of unit DNA mixture solutions; and
(C) re-assembling the plurality of types of unit DNAs by OGAB method using the plurality of types of unit DNA mixture solutions obtained in step (B′) to prepare a long-chain DNA fragment.

11. The preparation method of a DNA fragment of claim 10 , comprising selecting a plurality of types of plasmids comprising the obtained long-chain DNA fragment and reusing the plasmids as the plasmids in step (B′).

12. A preparation method of a plasmid, the method comprising:
preparing a DNA fragment by the preparation method of a DNA fragment of claim 1 ; and
including the DNA fragment in a plasmid.

13. A method for constructing a chimeric plasmid library, using the preparation method of a DNA fragment of claim 1 .

14. A method for constructing a chimeric plasmid library, using the preparation method of a plasmid of claim 12 .

15. A cell transformation method comprising:
preparing a DNA fragment by the preparation method of a DNA fragment of claim 1 ; and
transforming a cell with the DNA fragment or the plasmid.

16. A cell transformation method comprising:
preparing a plasmid by the preparation method of a plasmid of claim 12 ; and
transforming a cell with the DNA fragment or the plasmid.

17. A preparation method of a composition for cell transformation, the method comprising:
preparing a DNA fragment by the preparation method of a DNA fragment of claim 1 ; and
preparing a composition for cell transformation using the DNA fragment or the plasmid.

18. A preparation method of a composition for cell transformation, the method comprising:
preparing a plasmid by the preparation method of a plasmid of claim 12 ; and
preparing a composition for cell transformation using the DNA fragment or the plasmid.

19. A method for transforming a cell with a DNA fragment of interest,
wherein the DNA fragment has at least one insert DNA unit comprising: a DNA comprising a replication origin effective in a host that is the cell; and an insert DNA,
wherein the insert DNA is formed by linking a plurality of types of unit DNAs capable of being linked in a specific linking order,
wherein the method comprises:
(A) processing a plurality of types of plasmids comprising the insert DNA unit with a restriction enzyme suitable for each plasmid to prepare a plurality of types of unit DNA mixture solutions each of which comprises the plurality of types of unit DNAs linked on the insert DNA of each of the plasmid;
(B) re-assembling the plurality of types of unit DNAs by OGAB method using the plurality of types of unit DNA mixture solutions obtained in step (A) to prepare a long-chain DNA fragment; and
(C) transforming the cell with the long-chain DNA fragment.

20. A method for producing a cell comprising a DNA fragment of interest,
wherein the DNA fragment has at least one insert DNA unit comprising: a DNA comprising a replication origin effective in a host that is the cell; and an insert DNA,
wherein the insert DNA is formed by linking a plurality of types of unit DNAs capable of being linked in a specific linking order,
wherein the method comprises:
(A) processing a plurality of types of plasmids comprising the insert DNA unit with a restriction enzyme suitable for each plasmid to prepare a plurality of types of unit DNA mixture solutions each of which comprises the plurality of types of unit DNAs linked on the insert DNA of each of the plasmid;
(B) re-assembling the plurality of types of unit DNAs by OGAB method using the plurality of types of unit DNA mixture solutions obtained in step (A) to prepare a long-chain DNA fragment; and
(C) transforming the cell with the long-chain DNA fragment.

Assignees

• Univ Kobe Nat Univ Corp

Inventors

• Tsuge Kenji
• Ishii Jun
• Kondo Akihiko

Classifications

• C40B50/06 Primary

  Biochemical methods, e.g. using enzymes or whole viable microorganisms · CPC title

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12N15/1027

  by DNA shuffling, e.g. RSR, STEP, RPR · CPC title

• C12N15/1037 Primary

  Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display · CPC title

• C12N15/63 Primary

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title