# US 8,541,229 B2 · Plasmids and phages for homologous recombination and methods of use > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,541,229 B2 - Document ID: US8541229B2 - Status: Granted - Kind: B2 - Application number: 12/688,764 - Filed: 2010-01-15 - Published: 2010-06-03 - Granted: 2013-09-24 - Earliest claimed: 2004-05-21 - Assignees: The United States of America, as represented by the Secretary, Department of Health and Human Services - Applicants: Donald L. Court; Costantino Nina - Inventors: Donald L. Court; Costantino Nina - Web page: https://patentgrep.com/patent/US8541229B2 ## CPC - C12N 15/74: Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora - C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) - C12N 15/70: Vectors or expression systems specially adapted for E. coli - C12N 15/73: Expression systems using phage (lambda) regulatory sequences - C12N 2795/10321: Viruses as such, e.g. new isolates, mutants or their genomic sequences - C12N 2795/10343: viral genome or elements thereof as genetic vector - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## 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. ## Claims ### Claim 1 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. ### Claim 2 (depends on 1) The lambda phage of claim 1, comprising an amber mutation in P. ### Claim 3 (depends on 1) The phage of claim 1, comprising an amber mutation in O. ### Claim 4 (depends on 1) The phage of claim 1, wherein the promoter operably linked to the heterologous nucleic acid is inserted into a nucleic acid encoding rexAB of the phage. ### Claim 5 (depends on 1) The phage of claim 1, wherein the heterologous nucleic acid confers drug resistance to a host cell. ### Claim 6 (depends on 5) The phage of claim 5, wherein the heterologous nucleic acid encodes tetracycline resistance. ### Claim 7 (depends on 1) The page of claim 1, comprising an amber mutation in P and an amber mutation in O. ### Claim 8 (depends on 1) The phage of claim 1, comprising a nucleic acid sequence encoding Exo operably linked to the PL promoter. ### Claim 9 (depends on 1) The phage of claim 1, comprising a nucleic acid sequence encoding Gam operably linked to the PL promoter. ### Claim 10 (depends on 1) The phage of claim 1, wherein the phage nucleic acid does not encode Gam or Exo. ### Claim 11 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. ### Claim 12 (depends on 1) A method for inducing homologous recombination in a bacterial host cell, comprising transferring the phage of claim 1 into a suppressor minus bacterial host cell of interest, introducing into the bacterial host cell an oligonucleotide at least 20 nucleotides in length sufficiently homologous for recombination to occur with a target nucleic acid, but not identical to the target nucleic acid; inducing expression of Beta from PL, thereby inducing homologous recombination between the oligonucleotide and the target nucleic acid, to introduce the oligonucleotide into the target nucleic acid. ### Claim 13 (depends on 12) The method of claim 12, wherein the bacterial host cell is a gram negative bacterial host cell. ### Claim 14 (depends on 12) The method of claim 12, wherein an extrachromosomal element comprises the target nucleic acid sequence. ### Claim 15 (depends on 1) A method of producing lambda phage, comprising transferring the phage of claim 1 into a gram negative bacterial host cell comprising an amber suppressor such that functional Cro and functional O and P proteins are produced, thereby producing lambda phage.