Plasmids and phages for homologous recombination and methods of use
Inventors
Court, Donald L. • Datta, Simanti • Costantino, Nina
Assignees
US Department of Health and Human Services
Publication Number
US-9371532-B2
Publication Date
2016-06-21
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
The invention relates to the development of plasmids and lambda phages that confer homologous recombination functions, specifically recombineering functions, to bacterial host cells. These plasmids and phages enable efficient catalysis of homologous recombination in vivo between DNA sequences with homologies as short as 35 bases. The approach allows for cloning and precise modification of genes on plasmids, BACs, bacterial chromosomes, and bacteriophage lambda without relying on restriction sites or DNA ligase, enabling rapid and precise in vivo manipulation of DNA.
The problem addressed is the limitation of classical recombinant DNA techniques that rely on restriction endonucleases and ligases, restricting recombination to convenient restriction sites and limiting the size of DNA fragments handled, typically to less than about 20 kilobases. Moreover, linear DNA fragments are inefficiently introduced into bacteria due to degradation by bacterial exonucleases, requiring large homology arms and poorly growing specialized bacterial strains. Current recombineering methods are effective but limited to a small number of E. coli strains, creating a need for methods to transfer recombineering functions to a broader variety of bacterial strains, including other gram negative bacteria.
The invention discloses plasmids containing an origin of replication specific to target bacterial cells, a de-repressible promoter operably linked to nucleic acids encoding lambda recombination proteins such as Beta, and optionally Exo and Gam, enabling recombineering functions in a variety of bacteria including E. coli, Salmonella, Pseudomonas, Cyanobacteria, and Spirochaetes. Lambda phages are also engineered with amber mutations in essential genes and selectable markers such that they can lysogenize suppressor minus host cells, transferring recombineering functions without causing lysis, while retaining the ability to be induced to the lytic cycle in suppressor plus strains for high phage production.
Claims Coverage
The claims primarily cover methods for producing plasmids by recombineering a linear nucleic acid molecule containing a plasmid origin of replication and homology arms with a lysogenic lambda prophage that has specific genetic features including amber mutations in essential genes. These claims describe inventive features of the genetic components of the prophage, their regulation, and the method steps inducing recombination to produce the plasmid.
Use of lysogenic lambda prophage with de-repressible PL promoter and Beta recombination function
The lysogenic lambda prophage includes the PL promoter operably linked to nucleic acid encoding Beta, together with nucleic acids encoding P, O, Cro, integrase (int), excisionase (xis), and a c1857 repressor, enabling de-repressible expression of Beta and associated recombination functions.
Inclusion of amber codons in essential prophage genes controlling lytic replication
The lambda prophage genes encoding Cro and one or both of P and O contain amber codons that prevent production of these proteins in suppressor minus host cells, rendering the prophage lysogenic and defective for lytic replication in such hosts, but allowing lytic replication in hosts that produce amber suppressor tRNAs.
Production of recombinant plasmids containing lambda nucleic acid and plasmid origin of replication
Method inducing expression of Beta initiates recombination between the linear nucleic acid molecule flanked by homology arms and the lambda prophage, producing a plasmid comprising the plasmid origin of replication and selected lambda prophage sequences without an active bacterial origin of replication.
Use of conditional and specific origins of replication in plasmids
The plasmid origin of replication can be conditional or chosen from origins such as pBR322, pBBR1, IncQ, pSC101, pUC5, pUC8, RK2, or P1, controlling replication properties including temperature sensitivity (e.g., replicating at 32°C but not 37°C).
Insertion of heterologous nucleic acid encoding selectable marker into prophage rexAB genes
A promoter operably linked to a heterologous nucleic acid sequence, such as a drug resistance marker conferring resistance to antibiotics like kanamycin, ampicillin, tetracycline, chloramphenicol, neomycin, hygromycin, or zeocin, is inserted into the rexAB region of the prophage.
Optional inclusion of Exo and Gam recombination proteins operably linked to the PL promoter
The prophage optionally encodes the exonuclease Exo and the Gam protein operably linked to the PL promoter for enhanced recombination functionality.
Method steps for replacing plasmid origins of replication
The method includes steps for replacing the plasmid origin of replication with alternative origins using recombineering to generate plasmids with desired replication properties.
The claims cover a method of producing plasmids through recombineering involving a lysogenic lambda prophage equipped with regulated expression of recombineering proteins and engineered amber mutations that control lytic functions, enabling recombination with homologous linear DNA containing plasmid origins, and optionally including selectable markers and various replicons. This system allows generation of plasmids tailored for specific bacterial hosts and controlled replication, expanding the utility of recombineering.
Stated Advantages
Allows for efficient homologous recombination with short homologies, as short as 35 bases, enabling precise in vivo DNA manipulation without dependency on restriction sites.
Enables the use of recombineering functions in a broad variety of bacterial strains beyond limited E. coli strains, including gram negative bacteria like Salmonella, Pseudomonas, Cyanobacteria, and Spirochaetes.
The engineered phage system permits high yield lytic phage production in suppressor strains and dormant lysogen formation in other strains, enabling efficient transfer of recombineering functions without host cell killing.
The plasmid systems employ de-repressible promoters such as PL and temperature-sensitive repressors for tight regulation and inducible expression of recombination proteins, reducing toxicity and unwanted recombination.
Documented Applications
Use in generating plasmids with specified origins of replication through recombineering in bacterial cells containing lysogenic lambda prophages.
Introduction of recombineering functions into various gram negative bacterial cells to catalyze homologous recombination for genetic engineering.
Construction and modification of BACs, plasmids, bacterial chromosomes, and bacteriophage DNA without reliance on restriction enzymes or DNA ligase.
Generating bacterial strains lysogenized with engineered defective lambda prophages for use in functional genomic analysis and genetic manipulation.
Use of recombineering in Salmonella typhimurium and other gram negative bacteria for precise genetic modifications using oligonucleotides.
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