# US 7,608,448 B2 · Method and cloning vectors utilizing intergeneric conjugation for manipulation of actinomycetes biosynthesis genes > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 7,608,448 B2 - Document ID: US7608448B2 - Status: Granted - Kind: B2 - Application number: 10/402,841 - Filed: 2003-03-28 - Published: 2003-12-04 - Granted: 2009-10-27 - Earliest claimed: 2002-03-29 - Assignees: Wyeth - Inventors: Nathan Magarvey - Web page: https://patentgrep.com/patent/US7608448B2 ## CPC - C12N 15/70: Vectors or expression systems specially adapted for E. coli - C12N 15/74: Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The present invention relates to a vector comprising sequences that permit direct transfer of the vector from one prokaryotic cell to another, such as by intergeneric conjugation. The invention also relates to methods of making and using the vector. ## Claims ### Claim 1 An intergeneric shuttle vector comprising an expression control sequence; a multiple cloning site into which a nucleic acid encoding a polypeptide can be inserted that is positioned such that when the nucleic acid is inserted into the multiple cloning site the nucleic acid is operatively associated with the expression control sequence; an E. coli origin of replication; an actinomycetes origin of replication from pJV1; a cos cosmid cloning site; and an origin of transfer (OriT) that directs transfer of the vector from an E. coli cell to an actinomycetes cell during conjugation, and wherein the shuttle vector has a vector map according to pNWA200. ### Claim 2 (depends on 1) A host cell comprising the vector of claim 1. ### Claim 3 (depends on 2) The host cell of claim 2, wherein the host cell is an E. coli cell. ### Claim 4 (depends on 1) The intergeneric shuffle vector of claim 1 further comprising said nucleic acid encoding a polypeptide inserted into said multiple cloning site such that said nucleic acid is operatively associated with said expression control sequence. ### Claim 5 An intergeneric shuttle vector comprising an expression control sequence; a multiple cloning site into which a nucleic acid encoding a polypeptide can be inserted that is positioned such that when the nucleic acid is inserted into the multiple cloning site said nucleic acid is operatively associated with the expression control sequence; an E. coli origin of replication; an actinomycetes origin of replication from pJV1; a cos cosmid cloning site; and an origin of transfer (OriT) that directs transfer of the shuttle vector from an E. coli cell to an actinomycetes cell during conjugation, and wherein the shuttle vector has a vector map according to pNWA115. ### Claim 6 (depends on 5) A host cell comprising the vector of claim 5. ### Claim 7 (depends on 6) The host cell of claim 6, wherein the host cell is an E. coli cell. ### Claim 8 (depends on 5) The intergeneric shuttle vector of claim 5 further comprising said nucleic acid encoding a polypeptide inserted into said multiple cloning site such that said nucleic acid is operatively associated with said expression control sequence. ### Claim 9 (depends on 4) A method of expressing a polypeptide comprising (i) culturing an E. coli cell comprising the shuttle vector of claim 4 with an actinomycetes cell; (ii) allowing direct transfer of the vector from said E. coli cell to said actinomycetes cell to occur via conjugation; and (iii) expressing the polypeptide in said actinomycetes cell. ### Claim 10 (depends on 9) The method according to claim 9, wherein the nucleic acid encoding a polypeptide comprises a modified nucleotide sequence and the expressed polypeptide is a modified polypeptide. ### Claim 11 (depends on 10) The method according to claim 10, wherein the modification comprises insertion of a heterologous nucleotide sequence, deletion of a nucleotide sequence, or alteration of a least one nucleotide within the sequence. ### Claim 12 (depends on 9) The method according to claim 9, further comprising in step ii, identifying a recipient cell wherein the nucleic acid encoding the polypeptide is incorporated into the genome of said recipient cell by homologous recombination and wherein said step (iii) of expressing the polypeptide comprises expressing the polypeptide in said recipient cell. ### Claim 13 (depends on 9) The method according to claim 9, wherein the actinomycetes cell is Acetobacter xylinum, Achrombacter parvulus, Acinetobacter spp., Aeromonas spp., Agrobacterium spp., Alcaligenes spp., Anabaena spp., Azospirrillum brazilense, Azotobacter spp., Bordetella spp., Caulobater spp., Enterobacteriaceae, Haemophilus influenzae, Hypomycrobium X, Legionella pneumophila, Methylophilus methyltrophus, Ethylosinus trichosporium, Myxococcus xanthus, Neisseria spp., Paracoccus denitrificans, Pseudomonas spp., Rhizobium spp., Rhodopseudomonas spp., Rhodospirillum spp., Thiobacillus spp., Vibrio cholerae, Xanthomonas spp., Yersinia enterocolitica, Myxococcus or Bacteroides. ### Claim 14 (depends on 8) A method of expressing a polypeptide comprising: (i) culturing an E. coli cell comprising the shuttle vector of claim 8 with an actinomycetes cell; (ii) allowing direct transfer of the vector from said E. coli cell to said actinomycetes cell to occur via conjugation; and (iii) expressing the polypeptide in said actinomycetes cell. ### Claim 15 (depends on 14) The method according to claim 14, wherein the nucleic acid encoding a polypeptide comprises a modified nucleotide sequence, and whereby the expressed polypeptide is a modified polypeptide. ### Claim 16 (depends on 15) The method according to claim 15, wherein the modification comprises insertion of a heterologous nucleotide sequence, deletion of a nucleotide sequence, or alteration of a least one nucleotide within the sequence. ### Claim 17 (depends on 14) The method according to claim 14, further comprising in step ii, identifying a recipient cell wherein the nucleic acid encoding the polypeptide is incorporated into the genome of said recipient cell by homologous recombination and wherein said step (iii) of expressing the polypeptide comprises expressing the polypeptide in said recipient cell. ### Claim 18 (depends on 14) The method according to claim 14, wherein said actinomycetes cell is Acetobacter xylinum, Achrombacter parvulus, Acinetobacter spp., Aeromonas spp., Agrobacterium spp., Alcaligenes spp., Anabaena spp., Azospirrillum brazilense, Azotobacter spp., Bordetella spp., Caulobater spp., Enterobacteriaceae, Haemophilus influenzae, Hypomycrobium X, Legionella pneumophila, Methylophilus methyltrophus, Ethylosinus trichosporium, Myxococcus xanthus, Neisseria spp., Paracoccus denitrificans, Pseudomonas spp., Rhizobium spp., Rhodopseudomonas spp., Rhodospirillum spp., Thiobacillus spp., Vibrio cholerae, Xanthomonas spp., Yersinia.