Plasmids and phages for homologous recombination and methods of use
AssigneeThe United States of America as represented by the Department of Health and Human Services
InventorsDonald L. Court, Simanti Datta, Nina Costantino
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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.
Claims31
A plasmid, comprising an origin of replication (ori) selected from the group consisting of pSC101, 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, Exo and Gam and a terminator,
- wherein the selectable marker replaces the rexAB coding region in the lambda genome, wherein the lambda genome contains an N-kil deletion, 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, 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 plasmid comprises SEQ ID NO:
- 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.
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, Gam and Exo operably linked to PL, b) OL and OR;
- c) a nucleic acid encoding c1857;
- d) an N-kil deletion;
- and d) a nucleic acid encoding a selectable marker replacing a nucleic acid encoding rexAB;
- wherein the chimeric plasmid does not comprises the N-kil region of lambda phage, and wherein bacterial host cells transformed with the plasmid can perform homologous recombination.
Dependencies are inferred from the claim text.