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US 8,541,229 B2Granted
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, Costantino Nina
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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.
Claims15
A lambda phage comprising,
- a promoter operably linked to a nucleic acid sequence encoding a heterologous nucleic acid sequence;
- PL;
- a nucleic acid sequence encoding Beta operably linked to PL;
- a nucleic acid sequence encoding P;
- a nucleic acid sequence encoding;
- a nucleic acid sequence encoding Cro;
- a nucleic acid sequence encoding integrase (int), a nucleic acid excisionase (xis) and att;
- a phage origin of replication, and
- a nucleic acid sequence encoding cI857;
- wherein the nucleic acid sequence encoding Cro comprises an amber codon, the nucleic acid sequence encoding P, the nucleic acid sequence encoding O, or both the nucleic acid sequence encoding P and the nucleic acid sequence encoding O comprise an amber codon, such that Cro and P, Cro and O, or Cro, P and O are not produced when the lambda phage is introduced into a suppressor minus host cell;
- wherein the lambda phage does not comprise a bacterial origin of replication;
- and
- wherein the lambda phage is lysogenic in a suppressor minus host cell and lytic at 42° C. in a host cell that produces an amber suppressive tRNA.
10 more elements
A lambda phage comprising,
- a promoter operably linked to a nucleic acid sequence encoding a heterologous nucleic acid sequence;
- PL;
- a nucleic acid sequence encoding Beta operably linked to PL;
- a nucleic acid sequence encoding P;
- a nucleic acid sequence encoding O;
- a nucleic acid sequence encoding Cro;
- a nucleic acid sequence encoding integrase (int), a nucleic acid excisionase (xis) and att;
- a phage origin of replication, and
- a nucleic acid sequence encoding cI857;
- wherein the nucleic acid sequence encoding Cro comprises an amber codon, the nucleic acid sequence encoding P, the nucleic acid sequence encoding O, or both the nucleic acid sequence encoding P and the nucleic acid sequence encoding O comprise an amber codon, such that Cro and P, Cro and O, or Cro, P and O are not produced when the lambda phage is introduced into a suppressor minus host cell;
- wherein the lambda phage does not comprise a bacterial origin of replication;
- and
- wherein the lambda phage is lysogenic in a suppressor minus host cell and lytic at 42° C. in a host cell that produces an amber suppressive tRNA.
10 more elements
Dependencies are inferred from the claim text.