Bacteriophage engineering via semi-synthesis
Inventors
McFarland, Kirsty A. • Rogers, Miles T. • McBrine, Connor • Holder, Jason W.
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Assignees
Charles Stark Draper Laboratory Inc
Member
DraperDraper is an independent nonprofit engineering innovation company with a legacy spanning over 90 years, dedicated to delivering transformative solutions for national security, prosperity, and global challenges. Renowned for its pioneering work in guidance, navigation, and control (GN&C) systems, Draper partners with government, industry, and academia to engineer advanced technologies in space, defense, biotechnology, and electronic systems. The company leverages multidisciplinary expertise, digital engineering, and a collaborative approach to provide field-ready prototypes, mission-critical systems, and innovative research. Draper’s mission is to ensure the nation's security and prosperity by delivering sustainable, cutting-edge solutions that address the toughest problems of today and tomorrow, while fostering an inclusive and diverse workforce. Draper also invests in the next generation of innovators through robust educational programs, including internships, co-ops, and the Draper Scholars Program, integrating academic research with real-world problem-solving.
Draper is an independent nonprofit engineering innovation company with a legacy spanning over 90 years, dedicated to delivering transformative solutions for national security, prosperity, and global challenges. Renowned for its pioneering work in guidance, navigation, and control (GN&C) systems, Draper partners with government, industry, and academia to engineer advanced technologies in space, defense, biotechnology, and electronic systems. The company leverages multidisciplinary expertise, digital engineering, and a collaborative approach to provide field-ready prototypes, mission-critical systems, and innovative research. Draper’s mission is to ensure the nation's security and prosperity by delivering sustainable, cutting-edge solutions that address the toughest problems of today and tomorrow, while fostering an inclusive and diverse workforce. Draper also invests in the next generation of innovators through robust educational programs, including internships, co-ops, and the Draper Scholars Program, integrating academic research with real-world problem-solving.
Publication Number
US-11891630-B2
Publication Date
2024-02-06
Expiration Date
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.
Core Innovation
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.
The background identifies challenges addressed by the present technology, including that phage genomes have relatively few restriction sites and are heavily modified, making traditional cloning techniques challenging, that phage genomes are compact with very little non-coding DNA, that many existing phage engineering technologies that rely on in vitro strategies are generally inefficient and challenging to scale up, and that engineering phages within bacteria can be problematic due to toxicity of phages to bacteria and difficulty in maintaining stability of large engineered genomes. The summary states that the methods disclosed herein permit higher recovery of recombinant bacteriophage genomes that express the phenotypic properties associated with the heterologous nucleic acid sequence relative to that observed with other phage engineering methods (for example, overall yield about 20-100% in stated examples), and that the methods are useful for integrating heterologous nucleic acids into bacteriophage genomes to generate recombinant bacteriophage genomes.
Claims Coverage
The patent includes two independent method claims directed to semi-synthetic generation of recombinant bacteriophage genomes. The main inventive features extracted from those independent claims are listed below.
Generation of PCR fragments spanning the Klebsiella phage K11 genome
Generating a plurality of PCR fragments from a template comprising a first Klebsiella phage K11 bacteriophage DNA genome, 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 [procedural detail omitted for safety].
In vitro recombination to produce a recombinant Klebsiella phage K11 genome
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 [procedural detail omitted for safety].
Generation of PCR fragments spanning the Enterobacteria phage T7 genome
Generating a plurality of PCR fragments from a template comprising a first Enterobacteria phage T7 bacteriophage DNA genome, 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 [procedural detail omitted for safety].
In vitro recombination to produce a recombinant Enterobacteria phage T7 genome
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 [procedural detail omitted for safety].
The independent claims cover semi-synthetic methods that assemble genome-spanning PCR fragments with homologous ends and perform in vitro recombination with a heterologous nucleic acid to produce recombinant bacteriophage genomes exemplified by K11 (SEQ ID NO:23) and T7 (SEQ ID NO:2).
Stated Advantages
Permits higher recovery of recombinant bacteriophage genomes that express the phenotypic properties associated with the heterologous nucleic acid sequence relative to other phage engineering methods.
Demonstrated higher overall yield in stated examples (about 20-100% in reported examples) compared to referenced alternative methods (e.g., BAR 3.0 showed 0% recovery in the disclosed comparison).
Enables integration of heterologous nucleic acids into bacteriophage genomes to generate recombinant bacteriophage genomes useful for the disclosed purposes.
Documented Applications
Identifying bacteria present within a biological sample by contacting the sample with a recombinant bacteriophage that encodes a detectable gene product and detecting bacterial host cells infected by the recombinant phage.
Profiling antibiotic susceptibility of bacteria present within a biological sample by contacting the sample with an antibiotic and a recombinant bacteriophage that encodes a detectable gene product, detecting infected bacterial host cells, and determining antibiotic effectiveness based on reduction of recombinant phage infected bacterial host cells relative to an untreated control.
Use for diagnostic and therapeutic purposes as noted in the background as potential utilities of engineered phages.
Kits for integrating a heterologous nucleic acid sequence into a bacteriophage genome as explicitly disclosed in the document.
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