Synthetic genes

• US 20080261300A1
• Filed: 08/20/2007
• Published: 10/23/2008
• Est. Priority Date: 09/26/2002
• Status: Abandoned Application

• Current Assignee: Kosan Biosciences, Inc. (Bristol-Myers Squibb Company)
• Original Assignee: Kosan Biosciences, Inc. (Bristol-Myers Squibb Company)
• Inventors: Reid, Ralph C., Santi, Daniel V., Jayaraj, Sebastian, Kodumal, Sarah J.
• Application Number:
  US11/894,753
• Publication Number:
  US 20080261300A1
• Time in Patent Office: Days
• US Class Current: 435/320.1
• 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 15/52 Genes encoding for enzymes or proenzymes
• 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: C12N 15/70 Vectors or expression systems specially adapted for E. coli

Abstract

The invention provides strategies, methods, vectors, reagents, and systems for production of synthetic genes, production of libraries of such genes, and manipulation and characterization of the genes and corresponding encoded polypeptides. In one aspect, the synthetic genes can encode polyketide synthase polypeptides and facilitate production of therapeutically or commercially important polyketide compounds.

Claims

1-29. -29. (canceled)

30. A composition comprising a cognate pair of vectors, wherein said cognate pairs are:

a) a first vector comprising SM42S₁-Sy₁-2S₂-SM2-R₁ digested with a Type IIS restriction enzyme that recognizes 2S₂, and

a second vector comprising SM5-2S₃-Sy₂-2S₄-SM3-R₁ digested with a Type IIS restriction enzyme that recognizes 2S₃;

or

b) a first vector comprising L-2S₁-Sy₁-2S₂-SM2-R₁ digested with a Type IIS restriction enzyme that recognizes 2S₂, and

a second vector comprising L′

-2S₃-Sy₂-2S₄-SM3-R₁ digested with a Type IIS restriction enzyme that recognizes 2S₃;

wherein SM1, SM2, SM3, SM4 are sequences encoding different selection markers, R₁ is a recognition site for a restriction enzyme, L and L′

are recognition sites that are the same or the same or different, and each different from R₁, 2S₁, 2S₂, 2S₃, and 2S₄ are recognition sites for Type IIS restriction enzymes, wherein 2S₁, 2S₂ are not the same, 2S₃, and 2S₄ are not the same, and digestion of the first vector with 2S₂ and the second vector with 2S₃ results in compatible ends.

31. The composition of claim 30 wherein 2S₁, and 2S₃ are the same and 2S₂ and 2S₄ are the same.

32. The composition of claim 30 wherein Sy₁ and Sy₂ encode polypeptide segments of a polyketide synthase.

33. (canceled)

34. A method for joining a series of DNA units using a vector pair comprising

a) providing a first set of DNA units, each in a first-type selectable vector comprising a first selectable marker and providing a second set of DNA units, each in a second-type selectable vector comprising a second selectable marker different from the first,

wherein said first-type and second-type selectable vectors can be selected based on the different selectable markers,

b) recombinantly joining a DNA unit from the first set with an adjacent DNA unit from the second set to generate a first-type selectable vector comprising a third DNA unit, and obtaining a desired clone by selecting for the first selectable marker

c) recombinantly joining the third DNA unit with an adjacent DNA unit from the second set to generate a first-type selectable vector comprising a fourth DNA unit, and obtaining a desired clone by selecting for the first selectable marker, or

recombinantly joining the third DNA unit with an adjacent DNA unit from the second series to generate a second-type selectable vector comprising a fourth DNA unit, and obtaining a desired clone by selecting for the second selectable marker.

35. The method of claim 34 wherein step (c) comprises recombinantly joining the third DNA unit with an adjacent DNA unit from the second set to generate a first-type selectable vector comprising a fourth DNA unit, and obtaining a desired clone by selecting for the first selectable marker, said method further comprising

recombinantly combining the fourth DNA unit with an adjacent DNA unit from the second series to generate a first-type selectable vector comprising a fifth DNA unit, and obtaining a desired clone by selecting for the first selection marker, or

recombinantly combining the third DNA unit with an adjacent DNA unit from the second set to generate a second-type selectable vector comprising a fifth DNA unit, and obtaining a desired clone by selecting for the second selection marker.

36. The method of claim 34 wherein step (c) comprises recombinantly joining the third DNA unit with an adjacent DNA unit from the second series to generate a second-type selectable vector comprising a fourth DNA unit, and obtaining a desired clone by selecting for the second selectable marker, said method further comprising

recombinantly joining the fourth DNA unit with an adjacent DNA unit from the first set to generate a first-type selectable vector comprising a fifth DNA unit, and obtaining a desired clone by selecting for the first selection marker, or

recombinantly joining the third DNA unit with an adjacent DNA unit from the first set to generate a second-type selectable vector comprising a fifth DNA unit and obtaining a desired clone by selecting for the second selection marker.

37. The method of claim 34 wherein the desired clone comprises a sequence encoding a PKS domain.

38-60. -60. (canceled)

61. A system for high through-put synthesis of synthetic genes comprising:

at least one source microwell plate containing oligonucleotides for assembly PCR

a source for an assembly PCR amplification mixture

a source for LIC extension primer mixture

at least one PCR microwell plate for amplification of oligonucleotides

a liquid handling device which

retrieves a plurality of predetermined sets of oligonucleotides from the source microwell plate(s)

combines the predetermined sets and the amplification mixture in wells of the at least one PCR microwell plate;

retrieves LIC extension primer mixture; and

combines the LIC extension primer mixture and amplicons in a well of the at least one PCR microwell plate; and

a heat source for PCR amplification configured to accept the at least one PCR microwell plate.

63-64. -64. (canceled)