Plasmids and phages for homologous recombination and methods of use
AssigneeThe United States of America, as represented by the Secretary, Department of Health and Human Services
InventorsDonald L. Court, Simanti Datta, Nina Costantino
Life of this patent
Estimating the term…
Abstract
Lambda phages that can be used to introduce recombineering functions into host cells are disclosed. Also disclosed are plasmids that can be used to confer recombineering functions to a variety of strains of E. coli and to other bacteria, including Salmonella, Pseudomonas, Cyanobacteria, Spirochaetes. These plasmids and phages can be isolated in vitro and can be used to transform bacterial cells, such as gram negative bacteria.
Claims21
A plasmid, comprising an origin of replication (ori), a nucleic acid encoding a selectable marker, a nucleic acid encoding a promoter operably linked to a nucleic acid sequence encoding a repressor that specifically binds a PL promoter of lambda phage, and a lambda genome comprising the PL promoter of lambda phage, and a nucleic acid encoding Beta operably linked to a promoter and a terminator,
- wherein the lambda genome contains an N deletion and a kil deletion, and
- wherein bacterial host cells transformed with the plasmid can perform homologous recombination.
A plasmid, comprising an origin of replication (ori), a nucleic acid encoding a selectable marker, a nucleic acid encoding a promoter operably linked to a nucleic acid sequence encoding a repressor that specifically binds a PL promoter of lambda phage, and a lambda genome comprising the PL promoter of lambda phage, and a nucleic acid encoding Beta operably linked to a promoter and a terminator,
- wherein the lambda genome contains an N deletion and a kil deletion, and
- wherein bacterial host cells transformed with the plasmid can perform homologous recombination;
- and
- wherein the lambda genome encodes rexA and rexB.
A chimeric plasmid, comprising an origin of replication (ori) selected from the group consisting of pSC101, colE1, pBBR1 and RK2;
- and
- λ phage nucleic acid, wherein the λ phage nucleic acid comprises
- a) a nucleic acid encoding Beta operably linked to PL, b) OL and OR;
- c) a nucleic acid encoding cI857;
- d) an N deletion, e) a kil deletion;
- and f) a nucleic acid encoding a selectable marker;
- and wherein bacterial host cells transformed with the plasmid can perform homologous recombination.
A plasmid, comprising an origin of replication selected from the group consisting of pSC101, pBR322, colE1, pBBR1 and RK2, a nucleic acid encoding a selectable marker, a nucleic acid encoding a promoter operably linked to a nucleic acid sequence encoding a repressor that specifically binds a PL promoter of lambda phage, and a lambda genome comprising the PL promoter of lambda phage operably linked to a nucleic acid encoding Beta and a terminator 3′ of the nucleic acid encoding Beta, wherein the lambda genome contains an N deletion and a kil deletion.
Dependencies are inferred from the claim text.