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
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A method for producing a plasmid, comprising:
transfecting a bacterial cell comprising a lysogenic lambda prophage with a linear nucleic acid molecule, wherein the linear nucleic acid molecule comprises two homology arms that flank a plasmid origin of replication, wherein one of the homology arms is a homology arm of 30 to 1000 nucleotides in length that is homologous to a lambda nucleic acid sequence between a nucleic acid sequence encoding excisionase (xis) and a nucleic acid sequence encoding PL and another of the homology arms is a homology arm of 30 to 1000 nucleotides in length that is homologous to a lambda nucleic acid sequence encoding a c1857 repressor, PR and/or Cro; and
inducing expression of Beta from the lysogenic lambda prophage;
thereby inducing recombination of the linear nucleic acid molecule with the lambda prophage and producing a plasmid comprising the plasmid origin of replication and a lambda prophage nucleic acid sequence,
wherein the lysogenic lambda prophage comprises PL; the 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 encoding xis, and att;
a phage origin of replication, and
a nucleic acid sequence encoding c1857 repressor;
and the lambda nucleic acid sequence located between the nucleic acid sequence encoding xis and the nucleic acid sequence encoding Beta and the lambda nucleic acid sequence encoding the c1857 repressor, PR and Cro;
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 prophage does not comprise an active bacterial origin of replication; and wherein the lambda prophage is lysogenic in a suppressor minus host cell and is lytic and produces phage at 42° C. in a host cell that produces an amber suppressive tRNA.
- The method of claim 1, wherein the plasmid origin of replication is a conditional origin of replication.
- The method of claim 1, wherein the resultant plasmid can replicate at 32° C. but not at 37° C.
- The method of claim 1, wherein the bacterial cell is a gram negative bacterial cell.
- The method of claim 1, wherein the prophage comprises an amber mutation in P.
- The method of claim 1, wherein the prophage comprises an amber mutation in O.
- The method of claim 1, wherein the prophage further comprises a promoter operably linked to a nucleic acid sequence encoding a heterologous nucleic acid sequence.
- The method of claim 7, wherein the promoter operably linked to the heterologous nucleic acid is inserted into a nucleic acid encoding rexAB of the phage.
- The method of claim 8, wherein the heterologous nucleic acid confers drug resistance to a host cell.
- The method of claim 9, wherein the heterologous nucleic acid confers resistance of the host cell to kanamycin, ampicillin, tetracycline, chloramphenicol, neomycin, hygromycin or zeocin.
- The method of claim 1, wherein the prophage comprises an amber mutation in P and an amber mutation in O.
- The method of claim 1, wherein the prophage comprises a nucleic acid sequence encoding Exo operably linked to the PL, promoter.
- The method of claim 1, wherein the prophage comprises a nucleic acid sequence encoding Gam operably linked to the PL, promoter.
- The method of claim 1, wherein the prophage nucleic acid sequence does not encode Gam or Exo.
- The method of claim 1, wherein the plasmid origin of replication is a pBR322, pBBR1, IncQ, pSC101, pUC5, pUC8, RK2, or P1 origin of replication.
- The method of claim 1, wherein the lambda nucleic acid sequence between the nucleic acid encoding xis and the nucleic acid sequence encoding PL, comprises a nucleic acid sequence encoding tL3, and wherein one of the homologoy arms comprises a nucleic acid sequence homologous to tL3.
- The method of claim 1, wherein the one of the two homology arms is homologous to pR.
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The method of claim 1, further comprising
replacing the plasmid origin of replication with a different origin of replication.
- The method of claim 1, wherein the lysogenic lambda prophage comprises a nucleic acid encoding Exo.
- The method of claim 19, wherein the lysogenic lambda prophage comprises a nucleic acid sequence encoding Gam.
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A method for producing a plasmid, comprising:
transfecting a bacterial cell comprising a lysogenic lambda prophage with a linear nucleic acid molecule,
wherein the lysogenic lambda prophage comprises a nucleic acid sequence encoding Beta operably linked to PL; a nucleic acid sequence encoding c1857; a nucleic acid sequence encoding P; a nucleic acid sequence encoding O; and a nucleic acid sequence encoding Cro;
wherein the linear nucleic acid molecule comprises first and second homology arms that flank a plasmid origin of replication, wherein each of the first and second homology arms is a homology arm of 30 to 1000 nucleotides in length that is homologous to the lysongenic prophage;
wherein the lambda prophage does not comprise an active bacterial origin of replication; and
wherein the lambda prophage is lysogenic in a suppressor minus host cell and is lytic and produces phage at 42° C. in a host cell that produces an amber suppressive tRNA; and
inducing expression of Beta from the lysogenic lambda prophage in the bacterial cell, thereby inducing recombination of the linear nucleic acid molecule with the lambda prophage and producing a plasmid comprising the plasmid origin of replication and a lambda prophage nucleic acid sequence, wherein the lambda prophage nucleic acid sequence comprises the nucleic acid sequence encoding Beta.
- The method of claim 21, wherein the plasmid origin of replication is a conditional origin of replication.
- The method of claim 21, wherein the resultant plasmid can replicate at 32° C. but not at 37° C.
- The method of claim 21, wherein the bacterial cell is a gram negative bacterial cell.
- The method of claim 21, wherein the prophage comprises an amber mutation in P.
- The method of claim 25, wherein the prophage comprises an amber mutation in O.
- The method of claim 21, wherein the prophage further comprises a promoter operably linked to a nucleic acid sequence encoding a heterologous nucleic acid sequence.
- The method of claim 27, wherein the promoter operably linked to the heterologous nucleic acid is inserted into a nucleic acid encoding rexAB of the phage.
- The method of claim 28, wherein the heterologous nucleic acid confers drug resistance to a host cell.
- The method of claim 29, wherein the heterologous nucleic acid confers resistance of the host cell to kanamycin, ampicillin, tetracycline, chloramphenicol, neomycin, hygromycin or zeocin.
- The method of claim 21, wherein the prophage comprises an amber mutation in P and an amber mutation in O.
- The method of claim 21, wherein the prophage comprises a nucleic acid sequence encoding Exo operably linked to the PL promoter.
- The method of claim 21, wherein the prophage comprises a nucleic acid sequence encoding Gam operably linked to the PL promoter.
- The method of claim 21, wherein the prophage nucleic acid sequence does not encode Gam or Exo.
- The method of claim 21, wherein the plasmid origin of replication is a pBR322, pBBR1, IncQ, pSC101, pUC5, pUC8, RK2, or P1 origin of replication.
- The method of claim 21, wherein the lambda prophage comprises a nucleic acid sequence encoding tL3, and the first homology arm comprises sequence homologous to tL3.
- The method of claim 21, wherein the lambda prophage comprises a nucleic acid sequence encoding pR, and the second homology arm comprises sequence homologous to pR.
- The method of claim 21, further comprising replacing the plasmid origin of replication with a different origin of replication.
- The method of claim 21, wherein the lysogenic lambda prophage comprises a nucleic acid encoding Exo.
- The method of claim 39, wherein the lysogenic lambda prophage comprises a nucleic acid sequence encoding Gam.
Owners (US)
Applicants
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Us Health
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Inventors
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Court Donald L
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Datta Simanti
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Costantino Nina
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CPC Classifications
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C12N15/74
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C12N7/00
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C12N15/70
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C12N15/73
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C12N2795/10321
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C12N2795/10343
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IPC Classifications
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C12N15/74
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A01H1/00
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A01K67/00
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A01K67/033
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C12N5/00
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C12N5/02
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C12N7/00
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C12N7/01
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C12N15/70
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C12N15/73
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C12N15/82
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C12N15/87
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Document Preview
- Publication: Jun 21, 2016
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Application:
Sep 9, 2014
US 201414481795 A
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Priority:
Sep 9, 2014
US 201414481795 A
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Jun 26, 2013
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Jan 15, 2010
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