Lambda phages useful in introducing recombineering functions into host cells; origin of replication (ori) selected from pSC101, colE1, pBBR1 and RK2
Inventors
Court, Donald L. • Datta, Simanti • Costantino, Nina
Assignees
US Department of Health and Human Services
Publication Number
US-7674621-B2
Publication Date
2010-03-09
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 useful for introducing recombineering functions into host bacterial cells. These recombineering functions utilize the recombination proteins encoded by lambdoid bacteriophages, such as Beta, Exo, and Gam, to efficiently catalyze homologous recombination in vivo between DNA sequences with short homologies as low as 20 to 50 bases. The plasmids include origins of replication specific for bacterial hosts, a de-repressible promoter controlling the expression of recombineering proteins, and selectable markers to facilitate selection of recombinants.
The problem solved by this invention addresses limitations in classical recombinant DNA technology, which depends on restriction endonucleases and DNA ligases, constraining DNA manipulation by availability of restriction sites and fragment size (<20 kb). Homologous recombination offers an alternative but faces difficulties in bacteria due to degradation of linear DNA and rarity of recombinants. Prior recombineering methods using lambda proteins were limited to certain E. coli strains, and methods for introducing these functions into other bacterial strains, including different E. coli strains and other gram-negative bacteria, were lacking.
The disclosed plasmids and phages encompass modular constructs with origins of replication from diverse plasmids such as pSC101, colE1, pBBR1, and RK2 to confer replication ability in various gram-negative bacteria including Escherichia, Salmonella, Pseudomonas, Cyanobacteria, and Spirochaetes. The lambda genome in these plasmids contains the minimal set of genes essential for recombineering (Beta, Exo, Gam) under control of a de-repressible promoter such as the lambda PL promoter regulated by a temperature-sensitive cI repressor. The phages contain amber mutations in essential genes to allow lytic growth only in suppressor strains, enabling controlled propagation of phage stocks while allowing lysogenization and recombineering function transfer in wild-type host strains. These innovations provide tight control and broad applicability of recombineering systems.
Claims Coverage
The claims include one independent claim directed to plasmids and methods for producing bacterial host cells capable of homologous recombination.
Plasmids comprising specific origins of replication and lambda Red genes under control
The plasmid comprises an origin of replication selected from pSC101, colE1, pBBR1, and RK2; a selectable marker replacing the rexAB coding region; a promoter and repressor system operably linked to the lambda PL promoter; and the lambda genome including PL promoter operably linked to nucleic acids encoding Beta, Exo and Gam, with an N-kil deletion.
Broad host range replication and regulatory components
The origin of replication enables plasmid replication in Salmonella typhimurium, Escherichia coli or both. The plasmid includes lambda operators OR, OL, promoters PL and PR, and a repressor binding PR, arranged as in lambda to allow tight regulation.
Specific plasmids as embodiments
Plasmids exemplified include pSIM2, pSIM4, pSIM5, pSIM6, pSIM7, pSIM8, and pSIM9, each comprising various replication origins and lambda genome components as specified.
Method for producing recombination-competent bacterial host cells
Introducing the disclosed plasmid into a bacterial host, particularly gram-negative bacteria such as E. coli, renders the host capable of performing homologous recombination mediated by lambda Red functions.
Chimeric plasmids with lambda nucleic acid and defined deletions
Chimeric plasmids combining an origin of replication with lambda nucleic acid encoding Beta, Gam, and Exo under PL promoter control, the presence of OL and OR operators, temperature sensitive cI857 repressor, an N-kil deletion, and selectable marker replacing rexAB, capable of supporting homologous recombination in transformed cells.
The independent claims cover plasmids containing specific origins of replication with lambda recombineering genes Beta, Exo, Gam under regulated control, selectable markers replacing rexAB, and deletions of N-kil genes, and methods of transforming host bacterial cells with such plasmids to confer homologous recombination ability.
Stated Advantages
Allows precise and efficient in vivo genetic engineering of DNA using short homologies as short as 20 to 50 bases without the need for restriction sites or ligation.
Permits homologous recombination in a broad range of gram-negative bacterial strains including diverse E. coli strains and other species such as Salmonella and Pseudomonas.
Provides tight control of recombination gene expression through temperature-sensitive repressors and de-repressible promoters, reducing toxicity and background recombination.
Enables generation of high titer phage stocks via amber mutations suppressible in select host strains, facilitating easy introduction of recombineering functions by phage infection.
Documented Applications
Use for cloning and modification of genes on plasmids, BACs, chromosomal DNA of enteric bacteria, and bacteriophage lambda via recombineering.
Introduction of recombineering functions into gram-negative bacteria to perform homologous recombination with short homology arms.
Generation of mutant and recombinant lambda phages using recombineering to create amber mutations for controlled lytic cycle induction.
Use in production and manipulation of plasmid vectors with broad host range replication origins such as pSC101, colE1, pBBR1, and RK2 for bacterial strains including Salmonella typhimurium and Escherichia coli.
Modification of Salmonella typhimurium galK gene to generate selectable phenotypes using plasmids encoding recombineering functions.
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