Compositions and Methods for the Regulation of Multiple Genes of Interest in a Cell

• US 20120315670A1
• Filed: 11/02/2010
• Published: 12/13/2012
• Est. Priority Date: 11/02/2009
• Status: Abandoned Application

• Current Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Original Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Inventors: Jacobson, Joseph, Church, George
• Application Number:
  US13/505,376
• Publication Number:
  US 20120315670A1
• Time in Patent Office: Days
• US Class Current: 435/69.1
• CPC Class Codes: C12N 15/1051 Gene trapping, e.g. exon-, intron-, IRES-, signal sequence-trap cloning, trap vectors
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Abstract

Methods and compositions are provided for manipulating the genome of host cell to produce at least one exogenous gene product. Also provided are methods and composition for producing a programmable cell comprising a plurality of exogenous genes, wherein each exogenous gene is under the control of a disrupted regulatory sequence and wherein the disrupted regulatory sequences are restored by in vivo recombination. Preferably, the gene of interest is under the control of a genetically altered promoter which sequence recombination effects the expression of the exogenous gene(s).

Claims

1. A method for expressing at least one polypeptide of interest in a host cell, the method comprising:

a. introducing a set of genetic elements in a host cell, the set of genetic elements comprising at least one regulatory sequence and at least one coding nucleic acid sequence of interest, wherein the set of genetic elements comprises recombination sites therebetween;

b. exposing the cell under conditions promoting recombination;

c. rearranging the set of genetic elements by allowing recombination between recombination sites; and

d. selecting the host cell having expressing the at least one polypeptide of interest.

2. The method of claim 1 wherein the regulatory sequence is a promoter sequence.

3. The method of claim 1 wherein the genetic elements are on the same nucleic acid or on different nucleic acids.

5. The method of claim 1 wherein the genetic elements are on a plasmid, a vector or are integrated in the genome of the host cell.

7. The method of claim 1 wherein the expression of the at least one polypeptide of interest is modulated by rearranging the regulatory sequence.

8. The method of claim 1 wherein at least one regulatory sequence is disrupted.

9. The method of claim 1 wherein expression of selected coding sequences is modulated by restoring the activity of the at least one regulatory sequence.

10. The method of claim 9 wherein the disrupted regulatory sequence comprises a 3′

segment having a recombination site at its 5′

end and a 5′

segment having a recombination site at its 3′

end and wherein the activity of the regulatory sequence is restored by recombination.

12. The method of claim 1 wherein the at least one regulatory sequence is a library of promoters.

14. The method of claim 12 wherein the library of promoters is a library of promoter variants.

15. The method of claim 1 wherein the at least one coding sequence is a library of unrelated coding sequences.

4. (canceled)

6. (canceled)

11. (canceled)

13. (canceled)

16-22. -22. (canceled)

23. A method for manipulating the genome of host cell to produce at least one exogenous gene product, the method comprising:

a. providing a host cell capable of performing site directed recombination;

b. providing two or more genetic elements, wherein a first genetic element comprises a genetically disrupted promoter sequence that is operably linked to a second genetic element comprising at least part of a coding sequence;

c. contacting the host cell with the genetic elements;

d. restoring the activity of the disrupted promoter sequence by site directed recombination;

e. selecting a host cell in which the recombination has occurred; and

f. producing the at least one exogenous gene product.

24. The method of claim 23 wherein the gene product is not expressed from said genetic element when the promoter is disrupted.

25. The method of claim 24 wherein the promoter is disrupted by a sequence alteration.

49. The method of claim 23 wherein the second genetic element comprises a cluster of genes.

50. The method of claim 49 wherein the cluster of gene codes for metabolic enzymes from a metabolic pathway.

26-48. -48. (canceled)

51-86. -86. (canceled)

87. A method of producing a programmable engineered cell capable of expressing at least one nucleic acid sequence, the method comprising:

a. providing a cell comprising at least one exogenous nucleic acid sequence wherein the at least one nucleic acid sequence is linked to a regulatory nucleic acid sequence;

b. providing at least one predefined oligonucleotide sequence homologous to the at least part of the regulatory nucleic acid sequence;

c. exposing the cell to conditions promoting oligonucleotide-directed recombination; and

d. selecting a cell expressing the at least one nucleic acid sequence.

88. The method of claim 87 wherein the at least one nucleic acid sequence is operably linked to the regulatory sequence.

89. The method of claim 88 wherein the regulatory sequence is a promoter sequence and the regulatory sequence is disrupted.

92. The method of claim 87 wherein the at least one nucleic acid sequence is a library of genes and the at least one oligonucleotide is a library of predefined oligonucleotides sequences.

93. The method of claim 87 wherein at least part of the regulatory sequence is replaced by homologous recombination.

94. The method of claim 93 wherein recombination between homologous sequences occurs in parallel or serially.

90-91. -91. (canceled)

95-97. -97. (canceled)