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-8859277-B2
Publication Date
2014-10-14
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 discloses lambda phages and plasmids designed to introduce homologous recombination functions, termed recombineering, into bacterial host cells. Recombineering utilizes recombination functions encoded by lambdoid bacteriophages, enabling efficient in vivo homologous recombination between DNA sequences with homologies as short as 20 to 100 bases, as opposed to classical genetic engineering methods that require longer homologies and restriction enzyme sites. These methods enable precise manipulation of DNA in host cells such as E. coli and other gram negative bacteria without the need for restriction enzymes or ligases.
The recombineering system involves plasmids and phages containing an origin of replication suitable for the bacterial host, a de-repressible promoter such as the lambda PL promoter, and nucleic acid sequences encoding single-stranded binding proteins like Beta, and optionally exonuclease (Exo) and Gam proteins. These plasmids and phages allow host cells to gain recombineering functions, facilitating homologous recombination of introduced nucleic acids bearing homology arms to target sequences. The plasmids include repressor genes such as cI857 to tightly regulate expression. Bacterial host cells transformed with such plasmids or lysogenized with such phages can perform homologous recombination efficiently.
The problem addressed is the limitation of classical restriction-ligation genetic engineering techniques, which rely on the presence of convenient restriction sites and restrict manipulation to DNA fragments typically less than 20 kb. Additionally, linear DNA fragments do not efficiently transform bacteria due to exonuclease degradation and require large homology regions for recombination. Previous recombineering methods were limited to certain E. coli strains, and there was a need to introduce recombineering functions broadly into diverse E. coli strains and other bacteria, including gram negative species such as Salmonella, Pseudomonas, Cyanobacteria, and Spirochaetes.
Claims Coverage
The patent includes multiple independent claims relating to plasmids that confer homologous recombination functions to bacterial host cells and methods for producing such host cells. The main inventive features pertain to plasmid compositions, specific genetic elements therein, and their functional capabilities.
Plasmid comprising lambda genome with N and kil deletions and regulated Beta expression
A plasmid comprising an origin of replication, a selectable marker, a promoter operably linked to a repressor binding the lambda PL promoter, and a lambda genome including the PL promoter with nucleic acid encoding Beta operably linked to the promoter and a terminator. The lambda genome contains deletions of the N and kil genes. The plasmid allows bacterial host cells transformed with it to perform homologous recombination.
Origin of replication supporting diverse bacterial hosts
The origin of replication (ori) in the plasmid can be selected from pSC101, colE1, pBR322, pBBR1, and RK2, enabling replication in various bacteria including Escherichia coli and Salmonella typhimurium.
Selectable marker replacing rexAB coding region in the lambda genome
The plasmid’s selectable marker can replace the rexAB coding region within the lambda genome, facilitating selection in host cells.
Inclusion of additional recombineering proteins Exo and Gam
The plasmid can further encode the recombineering proteins Exo and/or Gam operably linked to the de-repressible promoter.
Use of defined transcriptional terminators
Transcription termination after Beta (and optionally Exo and Gam) is effected by terminators chosen from TL3, TINT, TL1, TL2, TR1, TR2, T6S, or TOOP of lambda phage, with TL3 specified as preferred in some embodiments.
Use of temperature sensitive repressor cI857
The plasmid encodes a repressor such as cI857, which binds the PL promoter to tightly regulate expression of recombineering genes, providing inducible control.
Plasmid structural arrangement
The plasmid components are arranged in 5′ to 3′ order as origin of replication, a promoter operably linked to a repressor gene that binds the PL promoter, followed by the PL promoter operably linked to Beta gene.
Chimeric plasmid including lambda elements with specific deletions and selectable markers
A chimeric plasmid including an origin of replication from pSC101, colE1, pBBR1 or RK2, and lambda nucleic acid comprising Beta operably linked to PL, lambda OL and OR operator sites, a cI857 repressor, N and kil deletions, and a selectable marker. This plasmid enables homologous recombination in host cells.
Methods of producing bacteria with homologous recombination capability
Methods comprise introducing the described plasmids into bacterial host cells, thereby rendering the host cells capable of performing homologous recombination, particularly gram negative bacteria such as Escherichia coli.
The independent claims collectively protect plasmids encoding recombineering functions with tightly regulated expression of lambda Beta protein, deletions in lambda genes N and kil, inclusion of selectable markers, origins of replication suitable for diverse bacterial hosts, and methods using such plasmids to produce bacterial hosts capable of homologous recombination.
Stated Advantages
Provides a method for rapid and precise in vivo DNA manipulation using short homologous regions (as short as 20 base pairs) without reliance on restriction enzymes or DNA ligase.
Allows introduction of recombineering functions into a broad range of bacterial strains and species beyond limited E. coli strains, including other gram negative bacteria.
The de-repressible promoter system with temperature sensitive repressor ensures tight and coordinated induction minimizing background recombination and cytotoxicity.
Plasmids with broad host range origins and selectable markers permit ease of use in diverse bacteria and facilitate selection of transformed cells.
Lambda phages with amber mutations in essential genes enable controlled production of phage particles and stable lysogen formation in suppressor minus strains, allowing efficient delivery of recombineering functions via phage infection.
Documented Applications
Genetic engineering of bacterial artificial chromosomes (BACs), plasmids, chromosomes, and bacteriophage lambda DNA using homologous recombination mediated by lambda recombinases.
Introduction of precise mutations or gene insertions/deletions into bacterial genomes or extrachromosomal elements via recombineering in E. coli and other gram negative bacteria such as Salmonella typhimurium and Pseudomonas.
Isolation and construction of plasmids with different origins of replication by gap repair recombination in lambda prophage-containing strains.
Engineering of lambda phages carrying recombineering functions and selectable markers to infect bacterial strains, enabling lysogen formation and transient expression of recombineering genes.
Use of single-stranded oligonucleotides or double-stranded DNA substrates with short homology arms to perform base changes, insertions, deletions or replacements in bacterial genomes and phage genomes.
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