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US 11,891,630 B2Granted
Bacteriophage engineering via semi-synthesis
AssigneeThe Charles Stark Draper Laboratory, Inc.
InventorsKirsty A. McFarland, Miles T. Rogers, Connor McBrine, Jason W. Holder
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Abstract
The present disclosure provides methods of generating recombinant bacteriophage genomes via semi-synthesis. Specifically, the present technology provides methods of integrating a heterologous nucleic acid sequence into a bacteriophage genome, and isolating recombinant bacteriophages that express the heterologous nucleic acid sequence.
Claims8
A method for generating a recombinant Klebsiella phage K11 bacteriophage genome comprising:
- (a) generating a plurality of PCR fragments that are no more than 15 kilobases in length from a template comprising a first Klebsiella phage K11 bacteriophage DNA genome using polymerase chain reaction (PCR), wherein the plurality of PCR fragments collectively span the entire length of the first Klebsiella phage K11 bacteriophage DNA genome, wherein at least one end of each PCR fragment comprises a sequence that is homologous to an opposite end of another PCR fragment and wherein each PCR fragment is no more than 15 kilobases in length;
- and
- (b) recombining in vitro the plurality of PCR fragments with a heterologous nucleic acid in the presence of a recombination system under conditions to produce a recombinant Klebsiella phage K11 bacteriophage genome, wherein the recombinant Klebsiella phage K11 bacteriophage genome comprises the nucleic acid sequence of SEQ ID NO:
- 23.
1 more element
A method for generating a recombinant Enterobacteria phage T7 bacteriophage genome comprising:
- (a) generating a plurality of PCR fragments that are no more than 15 kilobases in length from a template comprising a first Enterobacteria phage T7 bacteriophage DNA genome using polymerase chain reaction (PCR), wherein the plurality of PCR fragments collectively span the entire length of the first Enterobacteria phage T7 bacteriophage DNA genome, wherein at least one end of each PCR fragment comprises a sequence that is homologous to an opposite end of another PCR fragment and wherein each PCR fragment is no more than 15 kilobases in length;
- and
- (b) recombining in vitro the plurality of PCR fragments with a heterologous nucleic acid in the presence of a recombination system under conditions to produce a recombinant Enterobacteria phage T7 bacteriophage genome, wherein the recombinant Enterobacteria phage T7 bacteriophage genome comprises the nucleic acid sequence of SEQ ID NO:
- 2.
1 more element
Dependencies are inferred from the claim text.