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WO2025059342A1 - Plateforme de synthèse et d'ingénierie de bactériophages diversifiés - Google Patents

Plateforme de synthèse et d'ingénierie de bactériophages diversifiés Download PDF

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WO2025059342A1
WO2025059342A1 PCT/US2024/046443 US2024046443W WO2025059342A1 WO 2025059342 A1 WO2025059342 A1 WO 2025059342A1 US 2024046443 W US2024046443 W US 2024046443W WO 2025059342 A1 WO2025059342 A1 WO 2025059342A1
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species
bacteriophage
phage
nucleic acid
genome
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Rani Brooks
Nicholas Sandoval
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Tulane University
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Tulane University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10211—Podoviridae
    • C12N2795/10251—Methods of production or purification of viral material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10211—Podoviridae
    • C12N2795/10251—Methods of production or purification of viral material
    • C12N2795/10252—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/22—Klebsiella
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/38—Pseudomonas
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/44—Staphylococcus

Definitions

  • coli lysates coli lysates
  • methods for using the same for cell-free bacteriophage synthesis (CFBS) to generate a broad range of bacteriophage species Also disclosed herein are phage engineering methods for converting a lysogenic bacteriophage into a lytic bacteriophage, as well as phage engineering methods for generating a genetically modified bacteriophage species that infects and kills a broader range of bacterial strains compared to the wild-type bacteriophage species.
  • BACKGROUND [0003] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.
  • Multidrug-resistant (MDR) bacteria pose one of the greatest emerging health threats. These pathogens possess intrinsic and acquired mechanisms of resistance to most common antibiotics, leaving few treatment options for the most at-risk patients.
  • Phage therapy a resurgent anti-bacterial treatment, has the potential to cure bacterial infections resistant to small molecule antibiotics. Unlike small molecule antibiotics, phage do not harm beneficial bacterial flora that protect against future infections. [0005] Despite a promising future, phage therapy is encumbered by logistical challenges in the manufacturing process.
  • phage preparations must be purified to remove endotoxin, a component of outer membranes of Gram-negative bacteria that induces septic shock. While several organic solvent 1 4859-9106-8900.1 Atty. Dkt.
  • the cell lysate composition comprises an effective amount of transcription/translation (TXTL) machinery that is configured to synthesize at least one bacteriophage that does not target E. coli under cell-free conditions.
  • TXTL transcription/translation
  • the mixture comprises additional bacterial cell lysates obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 distinct bacterial host species that are not E. coli.
  • the E. coli lysate comprises about 1% to about 99.9999% of the mixture. In certain embodiments, the E.
  • coli lysate comprises about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 2 4859-9106-8900.1 Atty.
  • the at least one additional bacterial cell lysate comprises about 0.0001%-99% of the mixture.
  • the at least one additional bacterial cell lysate comprises about 0.0001%, about 0.0005%, about 0.001%, about 0.005%, about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about
  • the present disclosure provides a modified bacterial cell lysate composition
  • a modified bacterial cell lysate composition comprising a mixture of an E. coli lysate and genomic DNA derived from a bacterial host species that is not E. coli, wherein the cell lysate composition comprises an effective amount of transcription/translation (TXTL) machinery that is configured to synthesize at least one bacteriophage that does not target E. coli under cell-free conditions.
  • TXTL transcription/translation
  • the bacterial host species may be selected from among Bacillus species, Klebsiella species, Pseudomonas species, Acinobacter species, Staphylococcus species, Shigella species, Xanthomonas species, Serratia species, Erwinia species, Ralstonia species, Candidatus Liberibacter species, Mycobacterium species, Escherichia species, Enterococcus species, Enterobacter species, 3 4859-9106-8900.1 Atty. Dkt.
  • the modified bacterial cell lysate compositions of the present technology may be prepared using one or more of French-press cell lysis, sonication, runoff reactions, or lysate dialysis.
  • the present disclosure provides an in vitro method for synthesizing bacteriophage virions comprising contacting a bacteriophage genome with any and all embodiments of the modified bacterial cell lysate compositions disclosed herein, and an energy buffer in vitro to obtain a reaction mixture, and incubating the reaction mixture under conditions to produce viable phage virions, wherein the energy buffer comprises canonical amino acids, phosphoenol pyruvate (PEP), nucleoside triphosphates (NTPs), cofactors, and coenzymes.
  • the reaction mixture is incubated at 16°C to 42°C for about 1-24 hours.
  • the reaction mixture is incubated at 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, or 42°C for about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours.
  • the bacteriophage genome may be isolated from a naturally occurring bacteriophage, or a genetically engineered bacteriophage. In other embodiments, the bacteriophage genome is a synthetic bacteriophage genome. [0013] In some embodiments, the bacteriophage genome is obtained from a bacteriophage that specifically infects bacterial host cells that are identical to the non- E. coli bacterial host species from which the genomic DNA is derived. In other embodiments, the bacteriophage genome is obtained from a bacteriophage that specifically infects bacterial host cells that are distinct from the non- E. coli bacterial host species from which the genomic DNA is derived.
  • the bacterial host cells may be selected from among Bacillus species, Klebsiella species, Pseudomonas species, Acinobacter species, Staphylococcus species, Shigella species, Xanthomonas species, Serratia species, Erwinia species, Ralstonia species, Candidatus Liberibacter species, Mycobacterium species, Escherichia species, Enterococcus species, Enterobacter species, Streptococcus species, Flavobacterium species, Borrelia species, Clostridioides species, Helicobacter species, Salmonella species, Clavibacter species, Campylobacter species, Agrobacterium species, Pectobacterium species, Burkholderia species, Xylella species, Yersinia 4 4859-9106-8900.1 Atty.
  • the bacteriophage genome is obtained from a bacteriophage selected from among T7 phage, T7-like phage, Lambda, K1E, T3, T5, T4, or PhiX174.
  • the energy buffer further comprises one or more of a polyethylene glycol (PEG) polymer (such as PEG-8000 or PEG- 6000), deoxynucleotide triphosphates (dNTPs), and stabilizers.
  • PEG polyethylene glycol
  • dNTPs deoxynucleotide triphosphates
  • the energy buffer further comprises one or more of Mg-glutamate, K-glutamate, tRNA, cAMP, folinic acid, spermidine, 3-PGA, HEPES, Nicotinamide adenine dinucleotide (NAD), coenzyme A (CoA), DTT, and maltodextrin.
  • the reaction mixture further comprises a vector including a nucleic acid sequence encoding a reporter gene, wherein the nucleic acid sequence encoding the reporter gene is operably linked to a promoter that is responsive to a RNA polymerase (RNAP) encoded by the bacteriophage genome.
  • RNAP RNA polymerase
  • the RNAP is selected from among SP6 RNA Polymerase, T3 RNA Polymerase and T7 RNA polymerase.
  • the reporter gene may encode a bioluminescent protein, a fluorescent protein, or a chemiluminescent protein.
  • bioluminescent proteins include, but are not limited to, Aequorin, firefly luciferase, Renilla luciferase, red luciferase, luxAB, or nanoluciferase.
  • chemiluminescent proteins include, but are not limited to, ⁇ -galactosidase, horseradish peroxidase (HRP), or alkaline phosphatase.
  • fluorescent proteins include, but are not limited to, sfGFP, TagBFP, Azurite, EBFP2, mKalamal, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3A, mTurquoise, monomeric Midoriishi- Cyan, TagCFP, mTFP1, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, EYFP, Citrine, Venus, SYFP2, TagYFP, Monomeric Kusabira- Orange, mKO ⁇ , mKO2, mOrange, mOrange2, mRaspberry, mCherry, dsRed, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mPlum, HcRed-Tandem, mKate2, mNeptune, Nir
  • the present disclosure provides a method for converting a lysogenic bacteriophage into a lytic bacteriophage comprising: generating a plurality of PCR fragments from a DNA template comprising a prophage DNA genome using polymerase chain reaction (PCR), wherein the plurality of PCR fragments collectively span the entire length of the prophage DNA genome except for a gene region encoding integrase; and recombining in vitro the plurality of PCR fragments with a donor heterologous nucleic acid comprising a 5’ flanking 5 4859-9106-8900.1 Atty. Dkt.
  • PCR polymerase chain reaction
  • each PCR fragment comprises a sequence that is homologous to (i) an opposite end of another PCR fragment or (ii) the 5’ flanking region of the donor heterologous nucleic acid or (iii) the 3’ flanking region of the donor heterologous nucleic acid.
  • the donor heterologous nucleic acid replaces the gene region encoding integrase in the recombinant lytic bacteriophage genome.
  • the methods of the present technology further comprise propagating the recombinant lytic bacteriophage genome in a bacterial host.
  • the methods of the present technology further comprise contacting the recombinant lytic bacteriophage genome with a bacterial cell lysate, and an energy buffer in vitro to obtain a reaction mixture, and incubating the reaction mixture under conditions to produce viable phage virions, wherein the energy buffer comprises canonical amino acids, phosphoenol pyruvate (PEP), nucleoside triphosphates (NTPs), cofactors, and coenzymes and wherein the cell lysate comprises an effective amount of transcription/translation (TXTL) machinery that is configured to synthesize bacteriophage under cell-free conditions.
  • TXTL transcription/translation
  • the present disclosure provides a method for generating a genetically modified bacteriophage species comprising generating a plurality of PCR fragments from a DNA template comprising a bacteriophage DNA genome using polymerase chain reaction (PCR), wherein the plurality of PCR fragments collectively span the entire length of the bacteriophage DNA genome except for a first gene region; and recombining in vitro the plurality of PCR fragments with a donor heterologous nucleic acid comprising a 5’ flanking region and a 3’ flanking region in the presence of a recombination system under conditions to produce a genetically modified bacteriophage genome, wherein at least one end of each PCR fragment comprises a sequence that is homologous to (i) an opposite end of another PCR fragment or (ii) the 5’ flanking region of the donor heterologous nucleic acid or (iii) the 3’ flanking region of the donor heterologous nucleic acid, and wherein the donor heterologous
  • the genetically modified bacteriophage species is genetically modified T7 phage.
  • the genetically modified bacteriophage species lacks endotoxin genes (e.g., LpxL1, LpxL2).
  • the method further comprises propagating the genetically modified bacteriophage genome in a bacterial host.
  • the method further comprises contacting the genetically modified bacteriophage genome with a bacterial cell lysate, 6 4859-9106-8900.1 Atty. Dkt.
  • the energy buffer comprises canonical amino acids, phosphoenol pyruvate (PEP), nucleoside triphosphates (NTPs), cofactors, and coenzymes and wherein the cell lysate comprises an effective amount of transcription/translation (TXTL) machinery that is configured to synthesize bacteriophage under cell-free conditions.
  • the bacterial cell lysate is obtained from naturally occurring bacterial host cell or a genetically modified bacterial host cell.
  • the genetically modified bacterial host cell overexpresses one or more of translation initiation factor IF-3 (infC), OxyS and CyaR and/or represses RecC subunit exonuclease RecBCD.
  • the bacterial cell lysate comprises any and all embodiments of the modified bacterial cell lysate compositions disclosed herein.
  • the energy buffer further comprises one or more of a polyethylene glycol (PEG) polymer (such as PEG-8000 or PEG- 6000), deoxynucleotide triphosphates (dNTPs), and stabilizers.
  • PEG polyethylene glycol
  • the energy buffer further comprises one or more of Mg-glutamate, K-glutamate, tRNA, cAMP, folinic acid, spermidine, 3-PGA, HEPES, Nicotinamide adenine dinucleotide (NAD), coenzyme A (CoA), DTT, and maltodextrin.
  • the reaction mixture further comprises a vector including a nucleic acid sequence encoding a reporter gene, wherein the nucleic acid sequence encoding the reporter gene is operably linked to a promoter that is responsive to a RNA polymerase (RNAP) encoded by the bacteriophage genome.
  • RNAP RNA polymerase
  • the RNAP is selected from among SP6 RNA Polymerase, T3 RNA Polymerase and T7 RNA polymerase.
  • the reporter gene may encode a bioluminescent protein, a fluorescent protein, or a chemiluminescent protein.
  • bioluminescent proteins include, but are not limited to, Aequorin, firefly luciferase, Renilla luciferase, red luciferase, luxAB, or nanoluciferase.
  • chemiluminescent proteins include, but are not limited to, ⁇ -galactosidase, horseradish peroxidase (HRP), or alkaline phosphatase.
  • fluorescent proteins include, but are not limited to, sfGFP, TagBFP, Azurite, EBFP2, mKalamal, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3A, mTurquoise, monomeric Midoriishi- Cyan, TagCFP, mTFP1, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, EYFP, Citrine, Venus, SYFP2, TagYFP, Monomeric Kusabira- Orange, mKO ⁇ , mKO2, mOrange, mOrange2, mRaspberry, mCherry, dsRed, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mPlum, HcRed-Tandem, mKate2, mNeptune, Nir
  • the recombination system comprises a 5’-3’ exonuclease, a DNA polymerase, and a DNA ligase.
  • the donor heterologous nucleic acid comprises an open reading frame that encodes a bioluminescent protein, a fluorescent protein, a chemiluminescent protein, a heterologous bactericidal gene, a phage protein that modifies host range, or any combination thereof.
  • the open reading frame of the donor heterologous nucleic acid is operably linked to an expression control sequence that is capable of directing expression of the bioluminescent protein, the fluorescent protein, the chemiluminescent protein, the heterologous bactericidal gene, a phage protein that modifies host range, or any combination thereof.
  • the expression control sequence may be an inducible promoter or a constitutive promoter.
  • FIG.1 Schematic of Cell-free bacteriophage synthesis
  • FIGs.2A-2J Approaches to diversified cell-free bacteriophage synthesis.
  • FIG. 2A Rebooting phage with standardized E. coli-TXTL from phage genomes (gDNA);
  • FIG.2B Establish a standard Gram (-) TXTL (e.g. E. coli BL21) and equivalent high-productivity Gram (+) TXTL (e.g. B. megaterium) for rebooting corresponding Gram (-) and (+) phage respectively;
  • FIG.2C Standardized E.
  • FIG.2D Standardized E. coli-TXTL supplemented with non-E. coli cell-lysate to provide non-preidentified host factors to reboot non-coliphage
  • FIG.2E representation of diverse bacteria specifically targeted by diverse phage
  • FIG.2F Establish a Genus-level CFBS
  • FIG.2G “Universal” CFBS comprised of a mixture of diverse cell-lysates
  • FIG.2H in vitro phage self-assembly by mixing phage gDNA with pre-expressed capsid protein in buffer + ATP
  • FIG.2I CFBS by decoupled transcription (TX) and translation (TL) by generating phage mRNAs through in vitro transcription to then be added to standardized cell- extracts for translation and phage assembly
  • FIG.2J Establish a cell-free mimic proteome to phage host to provide essential host factors
  • FIG.2A, FIG.2C, FIG.2H, and FIG.2I have been demonstrated as feasible in peer reviewed research. See Mark Rustad et al., Synthetic Biology, Volume 3, Issue 1, 2018, ysy002, https://doi.org/10.1093/synbio/ysy002; Cell-free production of personalized therapeutic phages 8 4859-9106-8900.1 Atty. Dkt. No.: 136669-0119 targeting multidrug-resistant bacteria. https://doi.org/10.1016/j.chembiol.2022.06.003; Lauren R.H. Krumpe, et al.
  • FIGs.3A-3B Conversion of lysogenic to lytic phage by genome engineering.
  • FIG. 3A Synthetic phage genomes assembled in vitro. Prophage sequences are identified in lysogens and lysogeny genes (e.g.
  • FIG.3B Engineered lytic phage rebooted from synthetic genomes by (4a) transformation into competent host/stepping-stone bacteria or (4b) in vitro by cell-free bacteriophage synthesis.
  • FIGs.4A-4B Rebooting phage from synthetic genomes.
  • FIG.4A Phage genome fragments are PCR amplified with flanking DNA homologous to adjacent genomic regions (here fragments A, B, C, D). Synthetic genomes are combined by Gibson assembly.
  • FIG.4B Synthetic genomes are rebooted into active phage in vivo by transformation into competent host bacteria (top) or in vitro using cell-free bacteriophage synthesis (bottom). Created with Biorender.com.
  • FIGs.5A-5C Rapid rebooting of T7_ClyF and T7_sfGFP from synthetic genomes.
  • FIG.5A Progress of Gibson assembly of synthetic T7 genomes (from PCR fragments A, B, E, expression cassette, F, and D) from 0-60 min.
  • FIG.5B Quick confirmation of successful CFBS rebooting of synthetic T7 by detecting sfGFP expression after infection of BL21 with rebooted phage. Fluorescence was measured as an endpoint after 30 min infection of BL21 with phage taken directly from CFBS reactions. LB medium was used as a negative control.
  • FIG.5C Spot tests of antimicrobial activity of synthetic T7 against MRSA, E. coli BL21, and co-culture overlay plates. Purified T7 stocks (T7_WT) were included as a control. RFU Differences between LB and synthetic phage cultures were evaluated by unpaired t-test (significance threshold p ⁇ 0.05, *; p ⁇ 0.0001, ****).
  • FIG.6 Recent non-model bacteria TXTL cell-free expression system setup, processing, and protein yields. 9 4859-9106-8900.1 Atty. Dkt. No.: 136669-0119
  • FIG.7 Recent non-model bacteria TXTL cell-free expression system setup, processing, and protein yields.
  • FIG.8 Cell-free Bacteriophage Synthesis: Phage Characteristics, Yields, and Efficiency.
  • PCR 1 A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No.4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds.
  • the modified bacterial cell lysate compositions of the present technology confers the following advantages over conventional phage engineering techniques: (1) the modified bacterial cell lysate compositions enable the study pathogenic or fastidious phage transcription/translation and promotes high-yield protein expression; (2) the modified bacterial cell lysate compositions can be used to screen for and optimize gene expression using endogenous transcription regulatory elements; and (3) the modified bacterial cell lysate compositions promotes expression of endogenous gene clusters for chemical synthesis without the need for extensive genetic engineering. 10 4859-9106-8900.1 Atty. Dkt.
  • amino acid refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally encoded amino acids are the 20 canonical amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrolysine and selenocysteine.
  • canonical amino acids alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine
  • pyrolysine and selenocysteine pyrolysine and selenocystein
  • Amino acid analogs refer to agents that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ⁇ carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (such as, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • amino acids forming a polypeptide are in the D form.
  • the amino acids forming a polypeptide are in the L form.
  • a first plurality of amino acids forming a polypeptide is in the D form and a second plurality is in the L form.
  • Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter code.
  • bacteria or “phage” refers to a virus that infects bacteria. Bacteriophages are obligate intracellular parasites that multiply inside bacteria by co-opting 11 4859-9106-8900.1 Atty. Dkt.
  • the host biosynthetic machinery i.e., viruses that infect bacteria.
  • viruses that infect bacteria.
  • different bacteriophages may contain different materials, they all contain nucleic acid and protein, and can under certain circumstances be encapsulated in a lipid membrane.
  • the nucleic acid can be either DNA or RNA (but not both) and can exist in various forms.
  • the term “effective amount” refers to a quantity sufficient to achieve a desired effect or outcome, e.g., an amount which results in the manufacturing or synthesis of bacteriophage under in vitro cell-free conditions, e.g., cell-free bacteriophage synthesis (CFBS).
  • CFBS cell-free bacteriophage synthesis
  • expression includes one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.
  • an “expression control sequence” refers to polynucleotide sequences which are necessary to affect the expression of coding sequences to which they are operably linked. Expression control sequences are sequences which control the transcription, post- transcriptional events and translation of nucleic acid sequences.
  • Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion.
  • RNA processing signals such as splicing and polyadenylation signals
  • sequences that enhance translation efficiency e.g., ribosome binding sites
  • sequences that enhance protein stability e.g., ribosome binding sites
  • heterologous nucleic acid sequence is any sequence placed at a location in the genome where it does not normally occur.
  • a heterologous nucleic acid sequence may comprise a sequence that does not naturally occur in a bacteriophage, or it may comprise only sequences naturally found in the bacteriophage, but placed at a non-normally occurring location in the genome.
  • the heterologous nucleic acid sequence is not a natural phage sequence.
  • the heterologous nucleic acid sequence is a 12 4859-9106-8900.1 Atty. Dkt. No.: 136669-0119 natural phage sequence that is derived from a different phage.
  • the heterologous nucleic acid sequence is a sequence that occurs naturally in the genome of a wild- type phage but is then relocated to another site where it does not naturally occur, rendering it a heterologous sequence at that new site.
  • “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same nucleobase or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.
  • a polynucleotide or polynucleotide region has a certain percentage (for example, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences.
  • This alignment and the percent homology or sequence identity can be determined using software programs known in the art. In some embodiments, default parameters are used for alignment.
  • One alignment program is BLAST, using default parameters.
  • Biologically equivalent polynucleotides are those having the specified percent homology and encoding a polypeptide having the same or similar biological activity.
  • a “host cell” is a bacterial cell that can be infected by a phage to yield progeny phage particles.
  • a host cell can form phage particles from a particular type of phage genomic DNA.
  • the phage genomic DNA is introduced into the host cell by infecting the host cell with a phage.
  • the phage genomic DNA is introduced into the host cell using transformation, electroporation, or any other suitable technique.
  • the phage genomic DNA is substantially pure when introduced into the host cell.
  • the phage genomic DNA is present in a vector when introduced into the host cell.
  • the definition of host cell can vary from one phage to 13 4859-9106-8900.1 Atty. Dkt. No.: 136669-0119 another.
  • E. coli may be the natural host cell for a particular type of phage, but Klebsiella pneumoniae is not.
  • isolated refers to a substance or entity that has been separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting).
  • Isolated substances and/or entities may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated.
  • isolated substances and/or entities are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.
  • a substance is “pure” if it is substantially free of other components.
  • phage genome includes naturally occurring phage genomes and derivatives thereof. Generally, the derivatives possess the ability to propagate in the same hosts as the naturally occurring phage. In some embodiments, the only difference between a naturally occurring phage genome and a derivative phage genome is at least one of a deletion and an addition of nucleotides from at least one end of the phage genome (if the genome is linear) or at least one point in the genome (if the genome is circular).
  • polynucleotide or “nucleic acid” means any RNA or DNA, which may be unmodified or modified RNA or DNA.
  • Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, RNA that is mixture of single- and double- stranded regions, and hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • the term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the material is derived from a cell so modified. Thus, for 14 4859-9106-8900.1 Atty. Dkt.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
  • an endogenous nucleic acid sequence in the genome of an organism is deemed “recombinant” herein if a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence, such that the expression of this endogenous nucleic acid sequence is altered.
  • a heterologous sequence is a sequence that is not naturally adjacent to the endogenous nucleic acid sequence, whether or not the heterologous sequence is itself endogenous to the organism (originating from the same organism or progeny thereof) or exogenous (originating from a different organism or progeny thereof).
  • a promoter sequence can be substituted (e.g., by homologous recombination) for the native promoter of a gene in the genome of an organism, such that this gene has an altered expression pattern. This gene would be “recombinant” because it is separated from at least some of the sequences that naturally flank it.
  • a nucleic acid is also considered “recombinant” if it contains any modifications that do not naturally occur in the corresponding nucleic acid in a genome.
  • an endogenous coding sequence is considered “recombinant” if it contains an insertion, deletion or a point mutation introduced artificially, e.g., by human intervention.
  • a “recombinant nucleic acid” also includes a nucleic acid integrated into a host cell chromosome at a heterologous site and a nucleic acid construct present as an episome.
  • a “recombinant bacteriophage genome” is a bacteriophage genome that has been genetically modified by the insertion of a heterologous nucleic acid sequence into the bacteriophage genome.
  • a “recombinant bacteriophage” means a bacteriophage that comprises a recombinant bacteriophage genome.
  • the bacteriophage genome is modified by recombinant DNA technology to introduce a heterologous nucleic acid sequence into the genome at a defined site.
  • the heterologous nucleic acid sequence is introduced with no corresponding loss of endogenous phage genomic nucleotides.
  • the heterologous nucleic acid sequence is inserted between N1 and N2.
  • the heterologous nucleic acid sequence is flanked by nucleotides N1 and N2.
  • endogenous phage nucleotides are removed or replaced during the insertion of the heterologous nucleic acid sequence.
  • the heterologous nucleic acid sequence is inserted in place of some or all of the endogenous phage sequence which is removed.
  • endogenous phage 15 4859-9106-8900.1 Atty. Dkt.
  • sample refers to clinical samples obtained from a subject or isolated microorganisms.
  • a sample is obtained from a biological source (i.e., a "biological sample"), such as tissue, bodily fluid, or microorganisms collected from a subject.
  • Sample sources include, but are not limited to, mucus, sputum, bronchial alveolar lavage (BAL), bronchial wash (BW), whole blood, bodily fluids, cerebrospinal fluid (CSF), urine, plasma, serum, or tissue.
  • Bacteriophage are obligate intracellular parasites that multiply inside bacteria by co- opting some or all of the host biosynthetic machinery. Phages contain nucleic acid and protein, and may be enveloped by a lipid membrane. Depending upon the phage, the nucleic acid genome can be either DNA or RNA but not both, and can exist in either circular or linear forms. The size of the phage genome varies depending upon the phage.
  • Phage genomes come in a variety of sizes and shapes (e.g., linear or circular). Most phages range in size from 24-200 nm in diameter.
  • the capsid is composed of many copies of one or more phage proteins, and acts as a protective envelope around the phage genome.
  • phages have tails attached to the phage capsid.
  • the tail is a hollow tube through which the phage nucleic acid passes during infection.
  • the size of the tail can vary and some phages do not even have a tail structure.
  • the tail is surrounded by a contractile sheath which contracts during infection of the bacterial host cell.
  • phages At the end of the tail, phages have a base plate and one or more tail fibers attached to it. The base plate and tail fibers are involved in the binding of the phage to the host cell.
  • Lytic or virulent phages are phages which can only multiply in bacteria and lyse the bacterial host cell at the end of the life cycle of the phage.
  • the lifecycle of a lytic phage begins with an eclipse period. During the eclipse phase, no infectious phage particles can be found either inside or outside the host cell.
  • the phage nucleic acid takes over the host biosynthetic machinery and phage specific mRNAs and proteins are produced.
  • Early phage mRNAs code for 16 4859-9106-8900.1 Atty. Dkt. No.: 136669-0119 early proteins that are needed for phage DNA synthesis and for shutting off host DNA, RNA and protein biosynthesis. In some cases, the early proteins actually degrade the host chromosome. After phage DNA is made late mRNAs and late proteins are made.
  • the late proteins are the structural proteins that comprise the phage as well as the proteins needed for lysis of the bacterial cell.
  • the phage nucleic acid and structural proteins are assembled and infectious phage particles accumulate within the cell.
  • the bacteria begin to lyse due to the accumulation of the phage lysis protein, leading to the release of intracellular phage particles.
  • the number of particles released per infected cell can be as high as 1000 or more.
  • Lytic phage may be enumerated by a plaque assay. The assay is performed at a low enough concentration of phage such that each plaque arises from a single infectious phage.
  • the infectious particle that gives rise to a plaque is called a PFU (plaque forming unit).
  • Lysogenic phages are those that can either multiply via the lytic cycle or enter a quiescent state in the host cell.
  • the phage genome exists as a prophage (i.e., it has the potential to produce phage).
  • the phage DNA actually integrates into the host chromosome and is replicated along with the host chromosome and passed on to the daughter cells.
  • the host cell harboring a prophage is not adversely affected by the presence of the prophage and the lysogenic state may persist indefinitely. The lysogenic state can be terminated upon exposure to adverse conditions.
  • Conditions which favor the termination of the lysogenic state include: desiccation, exposure to UV or ionizing radiation, exposure to mutagenic chemicals, etc.
  • Adverse conditions lead to the production of proteases (rec A protein), the expression of the phage genes, reversal of the integration process, and lytic multiplication.
  • a phage genome comprises at least 5 kilobases (kb), at least 10 kb, at least 15 kb, at least 20 kb, at least 25 kb, at least 30 kb, at least 35 kb, at least 40 kb, at least 45 kb, at least 50 kb, at least 55 kb, at least 60 kb, at least 65 kb, at least 70 kb, at least 75 kb, at least 80 kb, at least 85 kb, at least 90 kb, at least 95 kb, at least 100 kb, at least 105 kb, at least 110 kb, at least 115 kb, at least 120 kb, at least 125 kb, at least 130 kb, at least 135 kb, at least 140 kb, at least 145 kb, at least 150 kb, at least 175 kb, at least 200 kb, at least 225
  • the present disclosure provides a modified bacterial cell lysate composition comprising a mixture of an E. coli lysate and at least one additional bacterial cell lysate obtained from a bacterial host species that is not E. coli, wherein the cell lysate composition comprises an effective amount of transcription/translation (TXTL) machinery that is 17 4859-9106-8900.1 Atty. Dkt. No.: 136669-0119 configured to synthesize at least one bacteriophage that does not target E. coli under cell-free conditions.
  • TXTL transcription/translation
  • the at least one additional bacterial cell lysate may be obtained from a bacterial host species selected from among Bacillus species, Klebsiella species, Pseudomonas species, Acinobacter species, Staphylococcus species, Shigella species, Xanthomonas species, Serratia species, Erwinia species, Ralstonia species, Candidatus Liberibacter species, Mycobacterium species, Escherichia species, Enterococcus species, Enterobacter species, Streptococcus species, Flavobacterium species, Borrelia species, Clostridioides species, Helicobacter species, Salmonella species, Clavibacter species, Campylobacter species, Agrobacterium species, Pectobacterium species, Burkholderia species, Xylella species, Yersinia species, Neisseria species, and Listertia species.
  • a bacterial host species selected from among Bacillus species, Klebsiella species, Pseudomonas species,
  • the mixture comprises additional bacterial cell lysates obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 distinct bacterial host species that are not E. coli.
  • the E. coli lysate comprises about 1% to about 99.9999% of the mixture. In certain embodiments, the E.
  • coli lysate comprises about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%
  • the at least one additional bacterial cell lysate comprises about 0.0001%-99% of the mixture.
  • the at least one additional bacterial cell lysate comprises about 0.0001%, about 0.0005%, about 0.001%, about 0.005%, about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, 18 4859-9106-8900.1 Atty. Dkt.
  • the present disclosure provides a modified bacterial cell lysate composition
  • a modified bacterial cell lysate composition comprising a mixture of an E. coli lysate and genomic DNA derived from a bacterial host species that is not E. coli, wherein the cell lysate composition comprises an effective amount of transcription/translation (TXTL) machinery that is configured to synthesize at least one bacteriophage that does not target E. coli under cell-free conditions.
  • TXTL transcription/translation
  • the bacterial host species may be selected from among Bacillus species, Klebsiella species, Pseudomonas species, Acinobacter species, Staphylococcus species, Shigella species, Xanthomonas species, Serratia species, Erwinia species, Ralstonia species, Candidatus Liberibacter species, Mycobacterium species, Escherichia species, Enterococcus species, Enterobacter species, Streptococcus species, Flavobacterium species, Borrelia species, Clostridioides species, Helicobacter species, Salmonella species, Clavibacter species, Campylobacter species, Agrobacterium species, Pectobacterium species, Burkholderia species, Xylella species, Yersinia species, Neisseria species, and Listertia species.
  • the modified bacterial cell lysate compositions of the present technology may be prepared using one or more of French-press cell lysis, sonication, runoff reactions, or lysate dialysis.
  • the present disclosure provides an in vitro method for synthesizing bacteriophage virions comprising contacting a bacteriophage genome with any and all embodiments of the modified bacterial cell lysate compositions disclosed herein, and an energy buffer in vitro to obtain a reaction mixture, and incubating the reaction mixture under conditions to produce viable phage virions, wherein the energy buffer comprises canonical amino acids, phosphoenol pyruvate (PEP), nucleoside triphosphates (NTPs), cofactors, and coenzymes.
  • PEP phosphoenol pyruvate
  • NTPs nucleoside triphosphates
  • the reaction mixture is incubated at 16°C to 42°C for about 1-24 hours. In certain embodiments, the reaction mixture is incubated at 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, or 42°C for about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours.
  • the bacteriophage genome may be isolated from a naturally occurring bacteriophage, or a genetically engineered bacteriophage. In other embodiments, the bacteriophage genome is a synthetic bacteriophage genome. [0064] In some embodiments, the bacteriophage genome is obtained from a bacteriophage that specifically infects bacterial host cells that are identical to the non- E. coli bacterial host species from which the genomic DNA is derived. In other embodiments, the bacteriophage genome is obtained from a bacteriophage that specifically infects bacterial host cells that are distinct from the non- E. coli bacterial host species from which the genomic DNA is derived.
  • the bacterial host cells may be selected from among Bacillus species, Klebsiella species, Pseudomonas species, Acinobacter species, Staphylococcus species, Shigella species, Xanthomonas species, Serratia species, Erwinia species, Ralstonia species, Candidatus Liberibacter species, Mycobacterium species, Escherichia species, Enterococcus species, Enterobacter species, Streptococcus species, Flavobacterium species, Borrelia species, Clostridioides species, Helicobacter species, Salmonella species, Clavibacter species, Campylobacter species, Agrobacterium species, Pectobacterium species, Burkholderia species, Xylella species, Yersinia species, Neisseria species, and Listertia species.
  • the bacteriophage genome is obtained from a bacteriophage selected from among T7 phage, T7-like phage, Lambda, K1E, T3, T5, T4, or PhiX174.
  • the energy buffer further comprises one or more of a polyethylene glycol (PEG) polymer (such as PEG-8000 or PEG- 6000), deoxynucleotide triphosphates (dNTPs), and stabilizers.
  • PEG polyethylene glycol
  • dNTPs deoxynucleotide triphosphates
  • the energy buffer further comprises one or more of Mg-glutamate, K-glutamate, tRNA, cAMP, folinic acid, spermidine, 3-PGA, HEPES, Nicotinamide adenine dinucleotide (NAD), coenzyme A (CoA), DTT, and maltodextrin.
  • the reaction mixture further comprises a vector including a nucleic acid sequence encoding a reporter gene, wherein the nucleic acid sequence encoding the reporter gene is operably linked to a promoter that is responsive to a RNA polymerase (RNAP) encoded by the bacteriophage genome.
  • RNAP RNA polymerase
  • the RNAP is selected from among SP6 RNA Polymerase, T3 RNA Polymerase and T7 RNA polymerase.
  • the reporter gene may encode a bioluminescent protein, a fluorescent protein, or a chemiluminescent protein. Examples of bioluminescent proteins include, but are not limited to, Aequorin, firefly luciferase, Renilla luciferase, red luciferase, luxAB, or nanoluciferase.
  • chemiluminescent proteins include, but are not limited to, ⁇ -galactosidase, horseradish peroxidase (HRP), or alkaline phosphatase.
  • fluorescent proteins include, but are not limited to, sfGFP, TagBFP, Azurite, EBFP2, mKalamal, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3A, mTurquoise, monomeric Midoriishi- Cyan, TagCFP, mTFP1, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, EYFP, Citrine, Venus, SYFP2, TagYFP, Monomeric Kusabira- Orange, mKO ⁇ , mKO2, mOrange, mOrange2, mRaspberry, mCherry, dsRed, mStrawberry, mTangerine, td
  • the present disclosure provides a method for converting a lysogenic bacteriophage into a lytic bacteriophage comprising: generating a plurality of PCR fragments from a DNA template comprising a prophage DNA genome using polymerase chain reaction (PCR), wherein the plurality of PCR fragments collectively span the entire length of the prophage DNA genome except for a gene region encoding integrase; and recombining in vitro the plurality of PCR fragments with a donor heterologous nucleic acid comprising a 5’ flanking region and a 3’ flanking region in the presence of a recombination system under conditions to produce a recombinant lytic bacteriophage genome, wherein at least one end of each PCR fragment comprises a sequence that is homologous to (i) an opposite end of another PCR fragment or (ii) the 5’ flanking region of the donor heterologous nucleic acid or (iii) the 3’ flanking region
  • PCR polymerase chain
  • the donor heterologous nucleic acid replaces the gene region encoding integrase in the recombinant lytic bacteriophage genome.
  • the methods of the present technology further comprise propagating the recombinant lytic bacteriophage genome in a bacterial host.
  • the methods of the present technology further comprise contacting the recombinant lytic bacteriophage genome with a bacterial cell lysate, and an energy buffer in vitro to obtain a reaction mixture, and incubating the reaction mixture under conditions to produce viable phage virions, wherein the energy buffer comprises canonical 21 4859-9106-8900.1 Atty. Dkt.
  • the present disclosure provides a method for generating a genetically modified bacteriophage species comprising generating a plurality of PCR fragments from a DNA template comprising a bacteriophage DNA genome using polymerase chain reaction (PCR), wherein the plurality of PCR fragments collectively span the entire length of the bacteriophage DNA genome except for a first gene region; and recombining in vitro the plurality of PCR fragments with a donor heterologous nucleic acid comprising a 5’ flanking region and a 3’ flanking region in the presence of a recombination system under conditions to produce a genetically modified bacteriophage genome, wherein at least one end of each PCR fragment comprises a sequence that is homologous to (i) an opposite end of another PCR fragment or (ii) the 5’ flanking region of the donor heterologous nucleic acid or (iii) the 3’ flanking region of the donor heterologous nucleic acid, and wherein the donor heterologous
  • the genetically modified bacteriophage species is genetically modified T7 phage.
  • the genetically modified bacteriophage species lacks endotoxin genes (e.g., LpxL1, LpxL2).
  • the homologous 5’ flanking region of the heterologous nucleic acid has a length of about 20-30 base pairs (bps), 30- 40 bps, 40-50 bps, 50-60 bps, 60-70 bps, 70-80 bps, 80-90 bps, 90-100 bps, 100-110 bps, 110- 120 bps, 120-130 bps, 130-140 bps, 140-150 bps, 150-160 bps, 160-170 bps, 170-180 bps, 180- 190 bps, 190-200 bps, 200-210 bps, 210-220 bps, 220-230 bps, 230-240 bps, 240-250 bps, 250- 260 bps, 260-270 bps, 270-280 bps, 280-290 bps, 290-300 bps, 300-310 bps, 310- 320 bps, 320- bps, 330-340 bps, 340-350 bps, 350
  • the homologous 3’ flanking region of the heterologous nucleic acid has a length of about 20-30 base pairs (bps), 30-40 bps, 40-50 bps, 50-60 bps, 60-70 bps, 70-80 bps, 80-90 bps, 90-100 bps, 100-110 bps, 110-120 bps, 120-130 bps, 130-140 bps, 140-150 bps, 150-160 bps, 160-170 bps, 170-180 bps, 180-190 bps, 190-200 bps, 200-210 bps, 210-220 bps, 220-230 bps, 230-240 bps, 240-250 bps, 250-260 bps, 260-270 bps, 270-280 bps, 280-290 bps, 290-300 22 4859-9106-8900.1 Atty.
  • the method further comprises propagating the genetically modified bacteriophage genome in a bacterial host.
  • the method further comprises contacting the genetically modified bacteriophage genome with a bacterial cell lysate, and an energy buffer in vitro to obtain a reaction mixture, and incubating the reaction mixture under conditions to produce viable phage virions, wherein the energy buffer comprises canonical amino acids, phosphoenol pyruvate (PEP), nucleoside triphosphates (NTPs), cofactors, and coenzymes and wherein the cell lysate comprises an effective amount of transcription/translation (TXTL) machinery that is configured to synthesize bacteriophage under cell-free conditions.
  • TXTL transcription/translation
  • the bacterial cell lysate is obtained from naturally occurring bacterial host cell or a genetically modified bacterial host cell.
  • the genetically modified bacterial host cell overexpresses one or more of translation initiation factor IF-3 (infC), OxyS and CyaR and/or represses RecC subunit exonuclease RecBCD.
  • the bacterial cell lysate comprises any and all embodiments of the modified bacterial cell lysate compositions disclosed herein.
  • the energy buffer further comprises one or more of a polyethylene glycol (PEG) polymer (such as PEG-8000 or PEG- 6000), deoxynucleotide triphosphates (dNTPs), and stabilizers.
  • PEG polyethylene glycol
  • dNTPs deoxynucleotide triphosphates
  • the energy buffer further comprises one or more of Mg-glutamate, K-glutamate, tRNA, cAMP, folinic acid, spermidine, 3-PGA, HEPES, Nicotinamide adenine dinucleotide (NAD), coenzyme A (CoA), DTT, and maltodextrin.
  • the reaction mixture further comprises a vector including a nucleic acid sequence encoding a reporter gene, wherein the nucleic acid sequence encoding the reporter gene is operably linked to a promoter that is responsive to a RNA polymerase (RNAP) encoded by the bacteriophage genome.
  • RNAP RNA polymerase
  • the RNAP is selected from among SP6 RNA Polymerase, T3 RNA Polymerase and T7 RNA polymerase.
  • the reporter gene may encode a bioluminescent protein, a fluorescent protein, or a chemiluminescent protein.
  • bioluminescent proteins include, but are not limited to, Aequorin, firefly luciferase, Renilla luciferase, red luciferase, luxAB, or nanoluciferase.
  • chemiluminescent proteins include, but are not limited 23 4859-9106-8900.1 Atty. Dkt. No.: 136669-0119 to, ⁇ -galactosidase, horseradish peroxidase (HRP), or alkaline phosphatase.
  • fluorescent proteins include, but are not limited to, sfGFP, TagBFP, Azurite, EBFP2, mKalamal, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3A, mTurquoise, monomeric Midoriishi- Cyan, TagCFP, mTFP1, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, EYFP, Citrine, Venus, SYFP2, TagYFP, Monomeric Kusabira- Orange, mKO ⁇ , mKO2, mOrange, mOrange2, mRaspberry, mCherry, dsRed, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mPlum, HcRed-Tandem, mKate2, mNeptune, Nir
  • the recombination system comprises a 5’-3’ exonuclease, a DNA polymerase, and a DNA ligase.
  • the donor heterologous nucleic acid comprises an open reading frame that encodes a bioluminescent protein, a fluorescent protein, a chemiluminescent protein, a heterologous bactericidal gene, a phage protein that modifies host range, or any combination thereof.
  • the phage protein that modifies host range is a tail spike protein (e.g., gp11, gp12, and gp17) or a structural phage virion protein that is involved with bacterial cell attachment or degradation of bacterial cell wall components.
  • the encoded gene product(s) produces a detectable signal upon exposure to the appropriate stimuli, and the resulting signal permits detection of bacterial host cells infected by the recombinant phage.
  • the open reading frame of the donor heterologous nucleic acid is operably linked to an expression control sequence that is capable of directing expression of the bioluminescent protein, the fluorescent protein, the chemiluminescent protein, the heterologous bactericidal gene, a phage protein that modifies host range, or any combination thereof.
  • the open reading frame may be inserted into the phage genome downstream of or in the 24 4859-9106-8900.1 Atty. Dkt. No.: 136669-0119 place of an endogenous phage open reading frame sequence.
  • the expression control sequence is an inducible promoter or a constitutive promoter (e.g., sarA promoter or lpp promoter).
  • the inducible promoter or constitutive promoter may be an endogenous phage promoter sequence, a non-endogenous phage promoter sequence, or a bacterial host promoter sequence. Additionally or alternatively, in some embodiments, the inducible promoter is a pH-sensitive promoter, or a temperature sensitive promoter.
  • the heterologous nucleic acid sequence is inserted into the phage genome with no loss of endogenous phage genomic sequence. In some embodiments, the heterologous nucleic acid sequence replaces an endogenous phage genomic sequence.
  • the heterologous nucleic acid sequence includes an endogenous phage genomic sequence that was previously excised from the phage genome. [0079] In certain embodiments, the heterologous nucleic acid sequence replaces an endogenous phage genomic sequence that is less than the length of the heterologous nucleic acid sequence. Accordingly, in some embodiments, the length of the recombinant phage genome is longer than the length of the wild-type phage genome. In some embodiments, the heterologous nucleic acid sequence replaces an endogenous phage genomic sequence that is greater than the length of the heterologous nucleic acid sequence.
  • the length of the recombinant phage genome is shorter than the length of the wild-type phage genome.
  • the heterologous nucleic acid sequence replaces an endogenous phage genomic sequence that is equal to the length of the heterologous nucleic acid sequence.
  • the length of the heterologous nucleic acid sequence is at least 100 bases, at least 200 bases, at least 300 bases, at least 400 bases, at least 500 bases, at least 600 bases, at least 700 bases, at least 800 bases, at least 900 bases, at least 1 kilobase (kb), at least 1.1 kb, at least 1.2 kb, at least 1.3 kb, at least 1.4 kb, at least 1.5 kb, at least 1.6 kb, at least 1.7 kb, at least 1.8 kb, at least 1.9 kb, at least 2.0 kb, at least 2.1 kb, at least 2.2 kb, at least 2.3 kb, at least 2.4 kb, at least 2.5 kb, at least 2.6 kb, at least 2.7 kb, at least 2.8 kb, at least 2.9 kb, at least 3.0 kb, at least 3.1 kb, at least 3.2
  • the heterologous nucleic acid sequence comprises a length that is less than or equal to a length selected from the group consisting of 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, and 10 kb.
  • the heterologous nucleic acid sequence 25 4859-9106-8900.1 Atty. Dkt. No.: 136669-0119 comprises a length that is less than or equal to the maximum length of heterologous nucleic acid sequence that can be packaged into a phage particle comprising the phage genome.
  • the length of the heterologous nucleic acid sequence is from 100 to 500 bases, from 200 to 1,000 bases, from 500 to 1,000 bases, from 500 to 1,500 bases, from 1 kb to 2 kb, from 1.5 kb to 2.5 kb, from 2.0 kb to 3.0 kb, from 2.5 kb to 3.5 kb, from 3.0 kb to 4.0 kb, from 3.5 kb to 4.5 kb, from 4.0 kb to 5.0 kb, from 4.5 kb to 5.5 kb, from 5.0 kb to 6.0 kb, from 5.5 kb to 6.5 kb, from 6.0 kb to 7.0 kb, from 6.5 kb to 7.5 kb, from 7.0 kb to 8.0 kb, from 7.5 kb to 8.5 kb, from 8.0 kb to 9.0 kb, from 8.5 kb to 9.5 kb, or from 9.0 k
  • kits for preparing donor bacterial host cell lysates for cell-free bacteriophage synthesis (CFBS).
  • CFBS cell-free bacteriophage synthesis
  • the present disclosure provides a kit comprising a modified bacterial cell lysate composition comprising a mixture of an E. coli lysate and at least one additional bacterial cell lysate obtained from a bacterial host species that is not E. coli.
  • the at least one additional bacterial cell lysate may be obtained from a bacterial host species selected from among Bacillus species, Klebsiella species, Pseudomonas species, Acinobacter species, Staphylococcus species, Shigella species, Xanthomonas species, Serratia species, Erwinia species, Ralstonia species, Candidatus Liberibacter species, Mycobacterium species, Escherichia species, Enterococcus species, Enterobacter species, Streptococcus species, Flavobacterium species, Borrelia species, Clostridioides species, Helicobacter species, Salmonella species, Clavibacter species, Campylobacter species, Agrobacterium species, Pectobacterium species, Burkholderia species, Xylella species, Yersinia species, Neisseria species, and Listertia species.
  • a bacterial host species selected from among Bacillus species, Klebsiella species, Pseudomonas species,
  • kits further comprise a vector including a nucleic acid sequence encoding a reporter gene, wherein the nucleic acid sequence encoding the reporter gene is operably linked to a promoter that is responsive to a phage RNA polymerase (RNAP), such as SP6 RNA Polymerase, T3 RNA Polymerase and T7 RNA polymerase.
  • a phage RNA polymerase such as SP6 RNA Polymerase, T3 RNA Polymerase and T7 RNA polymerase.
  • the reporter gene encodes a bioluminescent protein, a fluorescent protein, or a chemiluminescent protein.
  • the kit is stored under conditions that permit the preservation of the bacteriophage genomes for extended periods, such as under bacteriophage-specific, controlled temperature, moisture, and pH conditions.
  • the kit may further comprise one or more of: wash buffers and/or reagents, hybridization buffers and/or reagents, labeling buffers and/or reagents, and detection means.
  • the buffers and/or reagents are usually optimized for the particular detection technique for which the kit is intended. Protocols for using these buffers and reagents for performing different steps of the procedure may also be included in the kit.
  • FIGs.6-7 summarize all non-model (re: non-E. coli) coupled transcription/translation CFES reported in peer-reviewed publications for which reporter protein (usually green fluorescent protein (GFP) a derivative) yields are given in readily comparable units (molarity or mass concentration).
  • reporter protein usually green fluorescent protein (GFP) a derivative
  • chassis strains for certain genera or species with suitable metabolism, the identification of positive and negative effector genes for strain engineering, and the overall knowledge base supporting a potential non-model TXTL donor.
  • Acinetobacter baumannii CFES would be challenging because Acinetobacter species often lack several enzymes to complete canonical glycolysis pathway (glucose ⁇ ⁇ pyruvate), which provide sustained ATP in modern CFES 22, 23 .
  • the gaps in glycolysis could be filled by plasmid-based expression of the missing enzymes in Acintobacter prior to lysis or exogenously adding purified enzymes to CFES reactions.
  • a chassis strain may be engineered for chromosomal expression of missing enzymes.
  • Non-model CFES have expanded in diversity and productivity, especially in the last 10 years. In most cases, standard CFES optimization workflows have resulted in sustained improvements in CFES yields and variety of protein or chemical products. Some intractable 30 4859-9106-8900.1 Atty. Dkt. No.: 136669-0119 organisms may be engineered for ease of use or to establish minimum metabolic function consistent with standard CFES processes and reagents. Multi-omic Interrogation of especially low-yield CFES may reveal bottlenecks and indicate appropriate direction of optimization efforts. TXTL chassis strain engineering should be preceded by screening of potential positive and negative effectors to avoid unfocused effort.
  • phage is able to deliver its genome into the bacteria and the bacterial TXTL machinery is able to replicate new functional phage particles in contrast to an unproductive infection in which phage genomes enter a cell, but new viable phage are not produced.
  • Phage host-specificity implies that most phage receptor binding proteins do not interact with most bacterial surface receptors. In other words, it is difficult to differentiate between phage-bacteria interactions in which infection fails (phage genomes never enter the cell) or if infections are unproductive (phage-mediated transcription, translation, genome replication, or genome packaging and capsid assembly fail).
  • phage engineering methods require well-characterized and highly-competent hosts.
  • Common phage engineering relies on homologous recombination of phage DNA while within a living bacterium isolating engineered phage from a mixed population of wild-type and modified phage by plaque assay 34 .
  • the primary limitation of these methods is that the recombination host is either permissive to phage infection (i.e. DNA entry through infection) or competent for transformation (amenable to naked DNA entry through heat-shock or electroporation). Competence is required for infection-based methods as well to provide a recombination template for the phage DNA 34 .
  • Nutrient Broth (NB), brain-heart infusion (BHI), Terrific Broth (TB), or other rich medium may be used as alternatives to 2xYTP.
  • OD600 OD600 reached 2.0-3.0
  • flasks were immediately placed on ice and gently mixed to quickly bring down temperature. Samples were kept cold from this step onward. After 15 min on ice, each culture was centrifuge at 10,000 x g for 5 min at 4 ⁇ C. The supernatants were removed, and pellets washed three times with ice-cold S30B buffer (10 mM Tris-Cl, pH 8.2, 14 mM magnesium glutamate, 60 mM potassium glutamate, 2 mM DTT).
  • FIG.2B Establish a standard Gram (-) TXTL (e.g. E. coli BL21) and equivalent high-productivity Gram (+) TXTL (e.g. B. megaterium) for rebooting corresponding Gram (-) and (+) phage respectively;
  • FIG.2C Standardized E. coli-TXTL supplemented with pre-identified essential host protein (co-expressed or exogenous);
  • FIG.2D Standardized E. coli-TXTL supplemented with non-E.
  • FIG.2E representation of diverse bacteria specifically targeted by diverse phage
  • FIG.2F Establish a Genus-level CFBS
  • FIG.2G “Universal” CFBS comprised of a mixture of diverse cell-lysates
  • FIG.2H in vitro phage self- assembly by mixing phage gDNA with pre-expressed capsid protein in buffer + ATP
  • FIG.2I CFBS by decoupled transcription (TX) and translation (TL) by generating phage mRNAs through in vitro transcription to then be added to standardized cell-extracts for translation and phage assembly
  • FIG.2J Establish a cell-free mimic proteome to phage host to provide essential host factors by (1) cell-free expression of the host proteome directed by its whole genome then (2) rebooting phage from its gDNA.
  • CFBS reaction conditions may be optimized for phage yield with respect to the following factors: bacterial growth conditions (media and growth temperature), sonication lysis energy input, runoff reactions, reaction composition (buffer pH 34 4859-9106-8900.1 Atty. Dkt. No.: 136669-0119 and concentrations of K(glu), Mg(glu) 2, DTT, PEG-8000, phage DNA, cell lysate, nuclease- inhibitors, protease-inhibitors, RNase-inhibitors, or other reaction enhancers or supplements), and reaction conditions (e.g. reaction volume, aerobic/anaerobic condition, incubation temperature, and incubation time).
  • reaction composition buffer pH 34 4859-9106-8900.1 Atty. Dkt. No.: 136669-0119 and concentrations of K(glu), Mg(glu) 2, DTT, PEG-8000, phage DNA, cell lysate, nuclease- inhibitors, protease-inhibitors, RNase
  • FIG.2B Standard Gram (-) TXTL (e.g. E. coli BL21) and equivalent Standard high- productivity Gram (+) TXTL (e.g. Bacillus megaterium or Streptomyces) for rebooting Gram (-) and (+) phage respectively. Synthesize Gram (+) phage using standard TXTL from Gram (+) bacterial lysates. Lysate preparation methods would be like description above. The Gram (+) lysate CFBS reaction conditions may be optimized as described above. [00112] FIG.2D: Hybrid cell-lysates comprised of standard E. TXTL supplemented with any non-E. coli cell-lysate from bacteria described herein.
  • Hybrid lysates may be prepared using different proportions of non-E. coli lysate for example 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 10%-99%.
  • Hybrid lysate CFBS reaction conditions may be optimized as described above.
  • FIG.2F Non-model lysate-based TXTL CFBS Reactions. Prepare cell lysates relatives of host bacteria at the Genus level.
  • lysates may be derived from and not limited to the following genera: Shigella, Xanthomonas, Serratia, Erwinia, Ralstonia, Candidatus Liberibacter, Mycobacterium, Escherichia, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas, Enterococcus, Enterobacter, Streptococcus, Flavobacterium, Borrelia, Clostridioides, Helicobacter, Salmonella, Clavibacter, Campylobacter, Agrobacterium, Pectobacterium, Burkholderia, Xylella, Yersinia, Neisseria, Listeria, Bacillus, Nocardia, Chlamydia.
  • FIG.2G “Universal” CFBS comprised of a mixture of cell lysates including 2-10 cell- lysates derived from different non-E. coli strains of bacteria. Diverse lysates contribute host factors to enable synthesis of diverse phage by CFBS without elucidating “essential” host factors. Universal CFBS reactions may or may not include an E. coli lysate component. 35 4859-9106-8900.1 Atty. Dkt. No.: 136669-0119 Universal CFBS lysates may be prepared using different proportions of non-E.
  • FIG.2J CFBS supplemented with synthetic host proteome.
  • a cell- free reaction is prepared for CFBS omitting phage DNA. Instead, the reaction is initiated by adding purified host bacteria gDNA to recapitulate the host proteome in situ (See IGeTT method).
  • iGeTT reaction will be carried out for 0-8h prior to addition of phage DNA to transition to phage synthesis.
  • a CFBS reaction can be prepared as normal but supplemented with a portion of a separate iGeTT reaction directed host DNA.
  • CFBS supplemented with synthetic host proteome maybe supplemented using different proportions of iGeTT reactions containing host gDNA: 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 10%-99%.
  • No.: 136669-0119 discussed herein can be readily broken down into a lower third, middle third and upper third, etc.
  • all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above.
  • a range includes each individual member.
  • a group having 1-3 cells refers to groups having 1, 2, or 3 cells.
  • a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.
  • Emslander Q Vogele K, Braun P, Stender J, Willy C, Joppich M, Hammerl JA, Abele M, Meng C, Pichlmair A, Ludwig C, Bugert JJ, Simmel FC, Westmeyer GG. Cell-free production of personalized therapeutic phages targeting multidrug-resistant bacteria. Cell Chem Biol. 2022;29(9):1434-1445 e1437. Epub 2022/07/13. doi: 10.1016/j.chembiol.2022.06.003. Available from: https://www.ncbi.nlm.nih.gov/pubmed/35820417. 30.

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Abstract

La présente divulgation concerne des compositions de lysat de cellules bactériennes modifiées et des procédés d'utilisation de celles-ci pour la synthèse de bactériophages acellulaires (CFBS, « cell-free bacteriophage synthesis ») pour générer une large gamme d'espèces de bactériophages. Sont également divulgués des procédés d'ingénierie de phage pour convertir un bactériophage lysogène en un bactériophage lytique, ainsi que des procédés pour générer une espèce de bactériophage génétiquement modifiée qui cible une plage plus large de souches hôtes bactériennes par rapport à l'espèce bactériophage de type sauvage.
PCT/US2024/046443 2023-09-13 2024-09-12 Plateforme de synthèse et d'ingénierie de bactériophages diversifiés Pending WO2025059342A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220259571A1 (en) * 2019-07-18 2022-08-18 Technische Universität München Host-independent expression of bacteriophages
WO2023205267A1 (fr) * 2022-04-20 2023-10-26 The Administrators Of The Tulane Educational Fund Synthèse de bactériophage acellulaire améliorée par modulation génétique de machinerie de transcription/traduction (txtl) bactérienne

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220259571A1 (en) * 2019-07-18 2022-08-18 Technische Universität München Host-independent expression of bacteriophages
WO2023205267A1 (fr) * 2022-04-20 2023-10-26 The Administrators Of The Tulane Educational Fund Synthèse de bactériophage acellulaire améliorée par modulation génétique de machinerie de transcription/traduction (txtl) bactérienne

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BROOKS RANI, MORICI LISA, SANDOVAL NICHOLAS: "Cell Free Bacteriophage Synthesis from Engineered Strains Improves Yield", ACS SYNTHETIC BIOLOGY, AMERICAN CHEMICAL SOCIETY, WASHINGTON DC ,USA, vol. 12, no. 8, 1 August 2023 (2023-08-01), Washington DC ,USA , pages 2418 - 2431, XP093294820, ISSN: 2161-5063, DOI: 10.1021/acssynbio.3c00239 *

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