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Publication Number
US-10781441-B2
Publication Date
2020-09-22
Expiration Date
Abstract
A method for producing one or more hybrid bacteriophage host range determinant (HRD) sequences, which comprises: (1) identifying at least two DNA sequences, each encoding an HRD in a series of regions in the DNA sequence, wherein the HRDs are different from one another, (2) incorporating each region into a vector in which each region is flanked by a recognition site of a restriction enzyme capable of cutting DNA at a specific cleavage site outside of the recognition sequence, so that the cleavage site of the restriction enzyme is situated at the boundary of each region, wherein the cleavage site sequences of the regions from an individual series are different from one another and wherein the cleavage site sequences at the boundaries of corresponding regions from different series are the same; (3) treating the vectors with a restriction enzyme capable of cutting DNA at a specific cleavage site outside of the recognition sequence so as to generate a mixture of the regions; and (4) treating the mixture of the regions with a ligase to ligate them to form an array of DNA sequences encoding an array of hybrid HRDs.
Core Innovation
The invention provides a method for producing one or more hybrid bacteriophage host range determinant (HRD) sequences. At least two DNA sequences are identified, each encoding an HRD in a series of regions, and each region is incorporated into a vector in which the region is flanked by a restriction enzyme recognition site with a cleavage boundary outside the recognition sequence.
The cleavage site sequences at the boundaries of corresponding regions from different series are the same, while the cleavage site sequences of the regions within an individual series are different from one another. Vectors are treated with a restriction enzyme to generate a mixture of the regions, and the mixture is then treated with a ligase to ligate them and form an array of DNA sequences encoding an array of hybrid HRDs.
The disclosed approach enables combinatorial mixing of HRD regions into chimeric HRD proteins, including tail fibre proteins, in which a receptor binding region and a linking or N-terminal region are represented as ordered regions. The document further describes selection and screening of recombinant phage carrying the hybrid HRDs, with emphasis on altered or broadened host range.
Claims Coverage
The document includes one independent claim directed to producing hybrid bacteriophage HRD sequences, with multiple dependent claims. Overall, the claim set centers on region-based HRD assembly using restriction enzyme cleavage boundaries and ligase-mediated formation of an array encoding hybrid HRDs, together with optional selection criteria for recombinant phage.
Region-flanked HRD DNA assembly with out-of-recognition cleavage boundaries
A method for producing one or more hybrid bacteriophage HRD sequences by identifying at least two DNA sequences each encoding an HRD in a series of regions, incorporating each region into a vector flanked by a restriction enzyme recognition site capable of cutting at a specific cleavage site outside the recognition sequence, such that the cleavage site is situated at the boundary of each region with corresponding-region boundaries across different series having the same cleavage-site sequences.
Restriction digestion to generate a region mixture
Treating the vectors with a restriction enzyme capable of cutting at a specific cleavage site outside of the recognition sequence to generate a mixture of the regions.
Ligation to form an array encoding hybrid HRDs
Treating the mixture of the regions with a ligase to ligate them to form an array of DNA sequences encoding an array of hybrid HRDs.
Hybrid HRD boundaries defined across corresponding regions
The cleavage-site sequences of the regions from an individual series are different from one another, while the cleavage site sequences at the boundaries of corresponding regions from different series are the same, enabling combination of regions from different HRD donors into hybrid HRD sequences.
Cleavage-site sequence alteration without changing encoded amino acids
Forming the cleavage site sequence of at least one region by altering the nucleotide base sequence while keeping the encoded amino acid sequence unchanged.
Tail fibre region boundary definition for a Phi33 tail fibre protein
Defining the N-terminal region and C-terminal region of a bacteriophage Phi33 tail fibre protein by amino-acid boundaries based on the Phi33 amino acid sequence.
Using specified Type IIS and Type IIB restriction enzymes
Selecting restriction enzymes constrained to specified Type IIS restriction enzymes and specified Type IIB restriction enzymes or isoschizomers thereof.
Selecting recombinant phage with quantitatively defined broad host range
Selecting a recombinant phage with hybrid HRDs and a broad host range defined as more than 50% across at least 35 clinical isolates, preferably more than 50%, derived from multiple infection sites and exhibiting a range of antibiotic resistance phenotypes.
Array workflow for transforming host cells, infecting, screening, and selecting recombinant phage
Contacting an array of delivery vectors with first host cells to create an array of transformed first host cells, infecting them with a target phage, performing phage replication and recombination, screening recombinant phage, and selecting recombinant phage bearing hybrid HRDs.
The claim set defines a ligation-based strategy for producing arrays of hybrid bacteriophage HRD sequences by assembling HRD-encoding regions in vectors using restriction recognition sites with cleavage boundaries outside the recognition sequences, generating region mixtures by restriction digestion, and forming hybrid HRD-encoding arrays through ligase-mediated joining at matching boundaries. Dependent claims further specify cleavage-site generation constraints, tail fibre region boundary definitions, restriction enzyme lists, and optional host-range breadth selection criteria for recombinant phage carrying the hybrid HRDs.
Stated Advantages
Enables production of an array of DNA sequences encoding an array of hybrid HRDs.
Supports generation of recombinant phage with altered or broadened host range.
Provides quantitative definition of broad host range for selecting recombinant phage.
Allows selection of recombinant phage based on infection-site diversity and range of antibiotic resistance phenotypes.
Documented Applications
Engineering bacteriophage host range by incorporating chimeric HRD protein tail fibre regions into recombinant phage, including selection of recombinant phage with dual host infectivity.
Use in the context of SASPject-like phage vectors by incorporating chimeric tail fibre genes to broaden delivery.
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