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US 2011/0236927 A1Published application
Genetically stabilized tandem gene duplication
AssigneeMassachusetts Institute of Technology
InventorsGregory Stephanopoulos, Keith Edward Jaggard
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Abstract
The invention provides constructs and methods for producing genetically stabilized tandem gene duplications.
Claims35
A method for producing a genetically stable tandem gene duplication comprising
- integrating into a chromosome of a host cell a nucleic acid construct comprising a nucleic acid sequence that encodes one or more proteins operably linked to one or more promoter sequences, a nucleic acid sequence encoding a selectable marker, and homologous nucleic acid segments flanking the nucleic acid sequences that encode the one or more proteins and the selectable marker, wherein the host cell comprises a functional recombinase,
- selecting for tandem gene duplication (TGD) of the nucleic acid sequences that encode the one or more proteins and the selectable marker by culturing the host cell under selective conditions in which the selectable marker confers a growth advantage to the host cell, wherein the TGD is mediated by the functional recombinase, and
- stabilizing the TGD by deleting the recombinase or disabling the recombinase.
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A nucleic acid construct comprising
- a nucleic acid sequence that encodes one or more proteins operably linked to one or more promoter sequences functional in a host cell,
- a nucleic acid sequence that encodes a selectable marker, and
- homologous nucleic acid segments flanking the nucleic acid sequence that encodes the one or more proteins and the nucleic acid sequence that encodes the selectable marker,
- wherein the nucleic acid sequence that encodes the one or more proteins is operably linked to the one or more promoter sequences on a multicopy number plasmid vector having an origin of replication during construction of the construct.
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Dependencies are inferred from the claim text.