Plasmids and phages for homologous recombination and methods of use
Inventors
Court, Donald L. • Nina, Costantino
Assignees
US Department of Health and Human Services
Publication Number
US-8541229-B2
Publication Date
2013-09-24
Expiration Date
2025-05-20
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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.
Core Innovation
This invention relates to plasmids and phages that introduce homologous recombination (recombineering) functions into host cells, particularly bacterial cells such as gram negative bacteria including E. coli, Salmonella, Pseudomonas, Cyanobacteria, and Spirochaetes. It utilizes lambda phage recombination functions, including proteins Beta, Exo, and Gam, under control of de-repressible promoters like PL, to catalyze efficient in vivo homologous recombination with short homology arms as short as 35 bases, enabling precise DNA manipulation of plasmids, BACs, and bacterial chromosomes without using restriction enzymes or DNA ligase.
The problem addressed arises from limitations of classical recombinant DNA techniques, which rely on restriction endonucleases and ligases to combine DNA sequences but require convenient restriction sites and are limited to fragments less than about 20 kilobases. While homologous recombination offers an alternative, bacterial transformation with linear DNA is inefficient due to exonuclease degradation, requiring special strains and extensive homology lengths. Recombineering has been developed to overcome these limitations in specific E. coli strains, but broader applicability to more E. coli strains and other bacteria is needed.
The disclosed solution includes plasmids with origins of replication specific to target bacteria, de-repressible promoters, and nucleic acid sequences encoding single-stranded DNA binding proteins such as Beta, optionally together with Exo and Gam. These plasmids can be mobilized and used to transform a range of gram negative bacteria. Additionally, lambda phages are engineered containing amber mutations in essential genes and selectable markers, allowing them to lysogenize suppressor minus cells without lysis, thereby transferring recombineering functions efficiently. These innovations facilitate homologous recombination in diverse bacterial hosts via tight regulated expression of recombination proteins, enabling genetic engineering beyond current strain limitations.
Claims Coverage
The patent contains multiple independent claims focusing on lambda phages engineered for controlled recombineering and methods of using these phages to induce homologous recombination in bacterial cells.
Lambda phage engineered with amber mutations for conditional recombineering function expression
A lambda phage comprising operably linked promoter PL controlling a heterologous nucleic acid and Beta, nucleic acid sequences encoding P, O, Cro with at least two of P, O, Cro including amber codons so their proteins are not produced in suppressor minus host cells, lacks a bacterial origin of replication, and is lysogenic in suppressor minus hosts but lytic at 42° C. in hosts producing amber suppressor tRNA.
Inclusion of cI857 repressor and phage replication/integration elements
The lambda phage includes a nucleic acid sequence encoding the temperature sensitive repressor cI857, phage origin of replication, integrase (int), excisionase (xis), and attachment site (att) enabling lysogeny control, with modified regulation of PL promoter.
Insertion of heterologous selectable markers in rexAB region
The phage includes a promoter operably linked to a heterologous nucleic acid (e.g., selectable marker conferring drug resistance such as tetracycline resistance) inserted into the rexAB gene region.
Optional encoding of recombination proteins Exo and Gam linked to PL promoter
The phage optionally encodes Exo and Gam proteins operably linked to PL promoter, or may exclude one or both of these proteins.
Method of homologous recombination induction in bacterial host cells using engineered phage
A method transferring the engineered lambda phage into suppressor minus bacterial host cells, introducing a homologous oligonucleotide at least 20 nucleotides long to a target nucleic acid, and inducing Beta expression from PL promoter to facilitate homologous recombination.
Method for lambda phage production via infection of suppressor plus bacterial host
Producing lambda phage by infecting a gram negative bacterial host cell having an amber suppressor with the engineered phage to express functional Cro, O, and P proteins enabling lytic phage production.
The independent claims cover engineered lambda phages containing amber mutations in replication and regulatory genes with conditional expression of recombineering proteins, incorporation of selectable markers in specific phage genes, and methods of using these phages to mediate homologous recombination and produce phage in suppressor-positive hosts. These features provide tools for targeted genetic engineering in bacterial cells.
Stated Advantages
Efficient and precise in vivo homologous recombination using short homology arms as short as 35 base pairs.
Extension of recombineering to a variety of gram negative bacteria beyond limited E. coli strains.
Tight and coordinated regulation of recombination gene expression to minimize toxicity and background recombination.
Ability to produce high-titer phage stocks capable of transferring recombineering functions selectively depending on host suppressor status.
Documented Applications
Cloning and genetic modification of DNA sequences on plasmids, BACs, bacterial chromosomes, and bacteriophage lambda without the need for restriction enzymes or ligases.
Introduction of recombineering functions into diverse bacterial species including different strains of E. coli, Salmonella, Pseudomonas, Cyanobacteria, and Spirochaetes.
Engineering bacterial strains, including those carrying complex genomic libraries, with recombineering functions by infection with engineered lambda phage.
Production of customized plasmids with different origins of replication by recombineering.
Generation of bacteriophage lambda derivatives with defined mutations for use in recombineering applications.
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