Synthetic phages with recombinant tailspike proteins and related methods

Abstract

Compositions, methods, systems, and kits related to synthetic phages with customized host ranges are described. The customized host range of the synthetic phages is conferred to the synthetic phages by one or more recombinant tail spike proteins. For example, the recombinant tail spike proteins may include a combination of N-terminal and C-terminal regions and are engineered in the laboratory.

Description

[Technical Field]

[0001]

[0001] There is strong interest in using phages for a variety of applications related to the detection of microorganisms in biological, food, water, and clinical samples, as well as for antimicrobial therapeutic applications, particularly when drug-resistant microorganisms are involved. The limited host range of a single phage often limits its usefulness for both diagnostic and therapeutic applications. Traditionally, combinations of various phages, or "cocktails," have been used to address the problem of a phage's narrow host range. However, using multi-phage combinations can complicate the interpretation of test results and the overall effectiveness of phage-based applications. The presence of multiple different phages in a cocktail further complicates reagent preparation and can burden quality control requirements for regulatory approval. The ability to control the host range of a phage to include or exclude a target host of interest can be advantageous. Summary of the Invention

[0002]

[0002] As used herein, the terms "invention," "this invention," "this invention," and "the present invention" are intended to broadly refer to the subject matter of this patent application and all of the claims that follow. Statements containing these terms should be understood neither to limit the subject matter described herein nor to limit the meaning or scope of the claims that follow. The applicable embodiments of the present invention are defined by the claims, not this summary. This summary is a high-level overview of various aspects of the present invention, illustrating some of the concepts described and illustrated in the specification and accompanying drawings. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used alone to determine the scope of the claimed subject matter. The subject matter should be understood by reference to the entire specification, any or all figures, and appropriate portions of each claim. Several exemplary embodiments of the present invention are described below.

[0003] Included among the embodiments of the present invention and described in this disclosure are, inter alia, the following non-limiting exemplary embodiments. An exemplary embodiment of the present invention is a synthetic phage comprising at least one recombinant tail spike protein (TSP) comprising an N-terminal region and a C-terminal region, wherein (a) a combination of at least a portion of the N-terminal region and the C-terminal region has been engineered in the laboratory, and / or (b) the C-terminal region comprises one or more engineered amino acid sequences, and wherein the synthetic phage is constructed from a parent phage that is an Ackermannviridae phage. In some embodiments of the synthetic phage, the C-terminal region is capable of recognizing a target host. In some embodiments of the synthetic phage, the at least one recombinant TSP confers on the synthetic phage the ability to recognize a target host, which ability was not present in the parent phage. In some embodiments of the synthetic phage, the C-terminal region of the at least one recombinant TSP comprises at least one amino acid sequence that occurs in the C-terminal region of a TSP derived from a phage that is different from the parent phage and is capable of recognizing a target host. In some embodiments of the synthetic phage, the phage that differs from the parent phage is a non-Ackermannviridae phage. In some embodiments of the synthetic phage, the C-terminal region of at least one recombinant TSP comprises at least one amino acid sequence that occurs in the C-terminal region of a TSP in the phage that is unable to infect at least one host recognized by the parent phage. In some embodiments of the synthetic phage, the parent phage is a recombinant phage. In some embodiments of the synthetic phage, the synthetic phage comprises at least one recombinant TSP that is a plurality of recombinant TSPs. In some embodiments of the synthetic phage, the synthetic phage comprises at least two recombinant TSPs that can recognize at least two different target hosts. In some embodiments of the synthetic phage, the at least one recombinant TSP has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity to SEQ ID NO: 6, 7, 8, 10, 12, 14, or 18.

[0004]

[0004] Some embodiments of the present invention include recombinant tail spike proteins (TSPs) comprising N-terminal and C-terminal regions comprising amino acid sequences derived from an Ackermannviridae phage, wherein (a) a combination of at least a portion of the N-terminal and C-terminal regions is engineered in the laboratory, and / or (b) the C-terminal region comprises one or more engineered amino acid sequences. In some embodiments, the C-terminal region of the recombinant TSP is capable of recognizing a target host. In some embodiments of the recombinant TSP, the amino acid sequences of the C-terminal and N-terminal regions are derived from the same Ackermannviridae phage. In some embodiments of the recombinant TSP, the amino acid sequences of the C-terminal and N-terminal regions are derived from different phages. In some embodiments of the recombinant TSP, the amino acid sequence of the C-terminal region is derived from a non-Ackermannviridae phage. In some embodiments of the recombinant TSP, the amino acid sequence of the C-terminal region comprises one or more amino acid sequences of a TSP of SPTD1 phage. In some embodiments of the recombinant TSP, the recombinant TSP has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity to SEQ ID NO: 6, 7, 8, 10, 12, 14, or 18. Also included in embodiments of the invention are nucleic acid sequences encoding recombinant TSPs according to the present disclosure.

[0005]

[0005] Some embodiments of the present invention include methods for modifying phage host range. An exemplary method includes modifying a parent phage, an Ackermannviridae phage, to include at least one recombinant tail spike protein (TSP) according to the present disclosure, thereby generating a synthetic phage with a modified host range. In some embodiments, the parent phage is capable of recognizing at least one host, and the synthetic phage with the modified host range is unable to recognize the at least one host. In some embodiments, the parent phage is capable of recognizing at least one host, and the synthetic phage with the modified host range is capable of recognizing at least one target host that is different from the at least one host. In some embodiments, the modified host range is broader or narrower than the host range of the parent phage. In some embodiments, modifying the parent phage includes modifying the C-terminal region of at least one tail spike protein (TSP) of the parent phage. In some embodiments, modifying the C-terminal region of at least the TSP of the parent phage includes replacing at least a portion of the C-terminal region of the TSP of the parent phage with the C-terminal region of the recombinant TSP. In some embodiments, the exchange is performed using homologous recombination.

[0006]

[0006] Some embodiments of the present invention include methods for detecting a target host using a synthetic phage according to the present disclosure. In some embodiments, such methods include contacting a sample with the synthetic phage for a time sufficient for the synthetic phage to infect the target host, and detecting the synthetic phage or progeny phage of the synthetic phage, where positive detection of the synthetic phage or progeny phage of the synthetic phage indicates the presence of the target host in the sample. In some embodiments, the synthetic phage includes an indicator gene, and detecting includes detecting an indicator protein product produced by the synthetic phage or progeny phage of the synthetic phage, where positive detection of the indicator protein product indicates the presence of the target host in the sample.

[0007]

[0007] Some embodiments of the present invention include kits and systems for practicing the methods of the present disclosure, where the kit or system comprises a synthetic phage. Some embodiments of the present invention also include methods of detecting target hosts using a recombinant TSP according to the present disclosure. Some embodiments of the present invention include methods of controlling microorganisms using a synthetic phage according to the present disclosure. Such methods may include administering to a subject or contacting a sample, object, device, or material with the synthetic phage, where the synthetic phage is lytic. In any of the methods according to the present disclosure, the sample may be (but is not limited to) a food sample, an environmental sample, a water sample, a commercial sample, or a clinical sample.

[0008]

[0008] Some embodiments of the present invention include a method for constructing a synthetic phage, comprising: selecting a parent phage capable of infecting a first microorganism but not a second microorganism, where the parent phage is an Ackermannviridae phage; transforming the first microorganism with a homologous recombination (HR) plasmid containing a nucleic acid sequence encoding a C-terminal region of a tail spike protein (TSP) capable of recognizing the second microorganism and an HR sequence flanked by nucleic acid sequences complementary to the corresponding sequence in the TSP of the parent phage, thereby generating a transformed microorganism; infecting the first microorganism with the parent phage to cause HR between the HR plasmid and the genome of the parent phage, thereby generating a plurality of synthetic phage clones; and isolating the synthetic phage clones containing the recombinant TSP, thereby constructing a synthetic phage capable of infecting the second microorganism.

[0009]

[0009] An embodiment of the present invention includes a method for constructing a synthetic phage having a desired host recognition ability, the method comprising the steps of: (a) providing a virion of a recombinant phage constructed from an Ackermannviridae phage, wherein the virion lacks a tail spike protein-encoding gene (TSP-encoding gene) and comprises a tail spike protein (TSP) capable of recognizing a bacterial host; (b) providing a bacterial host comprising one or more plasmids encoding a TSP having the desired host recognition ability; (c) infecting the bacterial host with the virion of the recombinant phage; and (d) producing progeny phage in the bacterial host, wherein the progeny phage virions lack the TSP-encoding gene and comprise TSPs encoded by one or more plasmids in the bacterial host, and the progeny phage are synthetic phage having the desired host recognition ability. In an embodiment of the above method, step (a) comprises the steps of selecting a parent phage capable of infecting a bacterial host suitable for homologous recombination (HR), wherein the parent phage is an Ackermannviridae phage or an engineered phage constructed from an Ackermannviridae phage; transforming the bacterial host suitable for HR with an HR plasmid comprising a nucleic acid sequence encoding a tractable marker and sequences flanking nucleic acid sequences that are complementary to corresponding sequences flanking the TSP-encoding gene cluster of the parent phage; infecting the bacterial host suitable for HR with the parent phage to cause HR between the HR plasmid and the genome of the parent phage, thereby generating a plurality of phage clones; and isolating a phage clone comprising the tractable marker from the plurality of phage clones, thereby constructing a recombinant phage lacking the TSP-encoding genes.

[0010]

[0010] These and other embodiments of the present disclosure are described in detail below. For example, some other embodiments relate to systems, devices, and computer-readable media associated with the methods described herein.

[0011]

[0011] This disclosure includes the following figures. These figures illustrate particular embodiments and / or features of embodiments of the present invention and are intended to supplement any description(s) of embodiments of the present invention. These figures do not limit the scope of embodiments of the present invention unless the written description expressly indicates that this is the case. [Brief explanation of the drawings]

[0012] [Figure 1] Schematic diagram of the four tail spike proteins (TSPs) of phage CBA120, a member of the Ackermannviridae family. Each of TSPs 1-4 recognizes a unique bacterial host, as indicated. The TSPs form a complex with TSP4, which attaches to the baseplate. [Figure 2] Schematic diagram of the TSPs of an exemplary phage of the Ackermannviridae family, which has four TSPs. Each TSP can recognize a different surface host receptor, typically allowing infection of all hosts that display that particular receptor. [Figure 3] FIG. 1 is a schematic diagram of the use of Ackermannviridae phages as a customizable modular platform for constructing conditionally replicating synthetic phages for the detection of Gram-negative bacteria. [Figure 4]This is a schematic diagram of a complementing bacterial host that expresses a TSP in trans via a plasmid to complement a recombinant phage lacking the TSP-encoding gene, resulting in the production of a viable, infectious recombinant that can then be subjected to a single round of infection in wild-type bacteria. The use of different plasmids encoding unique TSPs allows for the assembly of recombinants with customizable host ranges. Wild-type bacteria lacking the complementing plasmid are unable to support the production of infectious progeny due to the lack of a TSP. [Figure 5] Electron microscope images of SPTD1 and CBA120 phages, both of which are members of the Ackermannviridae family. [Figure 6] FIG. 1 is a schematic diagram of the virion of an Ackermannviridae phage. [Figure 7] FIG. 1 is a schematic diagram of the homologous recombination process used to generate the synthetic phage CBA120-SPTD1.chiTSP2 (RBP-CBA120-2) from the CBA120 phage by exchanging CBA120 TSP2 with a recombinant TSP containing the N-terminal domain from CBA TSP2 and the C-terminal domain from SPTD1 TSP2 ("CBA120-SPTD1 TSP2"). [Figure 8]

[0023] Figure 1 shows a pairwise alignment of the amino acid sequences of CBA120 TSP2 (SEQ ID NO: 9) and recombinant CBA120-SPTD1 TSP2 (SEQ ID NO: 10) performed with the EMBOSS Needle tool available from the European Molecular Biology Laboratory (EMBL) European Bioinformatics Institute "EMBOSS Needle." The "neck" region is highlighted and boxed. As shown, the two amino acid sequences are identical up to two amino acids upstream of the neck (as indicated by the arrow). [Figure 9]In the left panel, an alignment of the amino acid sequences around the junction ("splice") site (indicated by the arrow between amino acids 244 and 245) of CBA120 TSP2 (amino acids (aa) 241-272 of SEQ ID NO: 9) and recombinant CBA120-SPTD1 TSP2 (amino acids (aa) 241-272 of SEQ ID NO: 10) is shown. In the right panel, a schematic diagram of the hypothetical structure of the recombinant CBA120-SPTD1 TSP2 protein is shown. [Figure 10]

[0023] Figure 1 shows a pairwise alignment of amino acid sequences from SPTD1 TSP2 (SEQ ID NO: 3) and recombinant CBA120-SPTD1 TSP2 (SEQ ID NO: 10) proteins, performed in EMBOSS Needle. The predicted "neck" region is highlighted and boxed. [Figure 11] FIG. 1 is a schematic diagram of the HR.CBA120-SPTD1. chimeric TSP2 plasmid insert. [Figure 12] These photographic images show the results of plating experiments testing the properties of synthetic phage CBA120-SPTD1.chiTSP2 (RBP-CBA120-2; also designated ChiTSP2) compared with CBA120 and SPTD1 (also designated MTSP1) phages. Culture medium alone was also tested as a negative control. The left image shows plate cultures of E. coli O157:H7 ATCC 43888 infected with the phages, as indicated. The right image shows plate cultures of Citrobacter sedlakii ATCC 51493 infected with the phages, as indicated. The lower left and right portions of each plate are uninfected controls. [Figure 13] FIG. 1 is a schematic diagram of the homologous recombination process used to generate the synthetic phage CBA120-SPTD1.chiTSP3 from CBA120 by replacing CBA120 TSP3 with a recombinant TSP containing the N-terminal domain from CBA120 TSP3 and the C-terminal domain from SPTD1 TSP3 ("CBA120-SPTD1 TSP3"). [Figure 14]1 shows a pairwise alignment of the amino acid sequences of CBA120 TSP3 (SEQ ID NO: 11) and SPTD1 TSP3 (SEQ ID NO: 4) performed with Emboss Needle. The "neck" region is indicated by a box. [Figure 15]

[0023] Figure 1 shows a pairwise alignment performed with Emboss Needle of the amino acid sequences of CBA120 TSP3 (SEQ ID NO: 11) and recombinant CBA120-SPTD1 TSP3 (SEQ ID NO: 12). The junction site between amino acids 158 and 159 in the "neck" region (boxed) of recombinant CBA120-SPTD1 TSP3 is indicated by an arrow. [Figure 16]

[0023] Figure 1 shows a pairwise alignment performed with Emboss Needle of the amino acid sequences of SPTD1 TSP3 (SEQ ID NO: 4) and recombinant CBA120-SPTD1 TSP3 (SEQ ID NO: 12). The "neck" region is clearly marked and boxed. [Figure 17] FIG. 1 is a schematic diagram of the HR.CBA120-SPTD1. chimeric TSP3 plasmid insert. [Figure 18] 10A-10C are photographic images of plate cultures showing the characteristics of synthetic phages CBA120-SPTD1.chiTSP3 (RBP-CBA120-1) and CBA120-SPTD1.chiTSP2 (RBP-CBA120-2) compared to the parental phage. Plate sections are labeled as follows: 1—CBA120; 2—SPTD1; 3—CBA120-SPTD1.chiTSP2 (RBP-CBA120-2); 4—CBA120-SPTD1.chiTSP3 (RBP-CBA120-1) lysate made in Salmonella 19585; 5—CBA120-SPTD1.chiTSP3 (RBP-CBA120-1) lysate made in E. coli 43888; 6—no-phage negative control. [Figure 19]FIG. 1 is a schematic diagram of the homologous recombination process used to generate the SPTD1-derived synthetic phage SPTD1-CBA120.chiTSP4(RBP-SPTD1-1) by exchanging SPTD1 TSP4 with a recombinant TSP4 containing the N-terminal domain from SPTD1 TSP4 and the C-terminal domain from CBA120 TSP4 ("SPTD1-CBA120 TSP4"). [Figure 20]

[0033] Figure 1 shows a pairwise alignment of the amino acid sequences of CBA120 TSP4 (SEQ ID NO: 13) and SPTD1 TSP4 (SEQ ID NO: 5) performed with Emboss Needle. The "neck" region (based on the published TSP structure) is marked with a box. The arrow indicates the junction site between amino acid 479 of SPTD1 TSP4 and amino acid 480 of CBA120 TSP4. [Figure 21] FIG. 1 is a schematic diagram of the structure of the N-terminally truncated CBA120 TSP4 protein. [Figure 22] FIG. 1 is a schematic diagram of the hypothetical structure of the recombinant SPTD1 CBA120 TSP4 protein. [Figure 23] 1 shows a pairwise alignment performed with Emboss Needle of the amino acid sequences of SPTD1 TSP4 (SEQ ID NO: 5) and recombinant SPTD1-CBA120 TSP4, also referred to as SPTD1.ChiTSP4 (SEQ ID NO: 14). The junction site between amino acid 479 of SPTD1 TSP4 and amino acid 480 of CBA120 TSP4 is indicated by an arrow. The "neck" region (based on the published TSP structure) is indicated by a box. [Figure 24] 1 shows a pairwise alignment performed with Emboss Needle of the amino acid sequences of CBA120 TSP4 (SEQ ID NO: 13) and recombinant SPTD1-CBA120 TSP4, also referred to as SPTD1.ChiTSP4 (SEQ ID NO: 14). The junction site between amino acid 479 of SPTD1 and amino acid 480 of recombinant SPTD1-CBA120-TSP4 is indicated by an arrow. The neck region (based on the published TSP structure) is indicated by a box. [Figure 25]Schematic diagram of the HR.SPTD1-CBA120chiTSP4 plasmid insert. The upstream homologous recombination region is part of the N-terminal region of SPTD1 TSP4. [Figure 26] 10 is a photographic image of a plate culture showing the results of plating synthetic SPTD1-CBA120.chiTSP4(RBP-SPTD1-1) onto E. coli O78 (ECOR70). [Figure 27] FIG. 1 is a schematic diagram of the homologous recombination process used to generate the SPTD1-derived synthetic phage SPTD1-CBA120.chiTSP1(RBP-SPTD1-2) by exchanging SPTD1 TSP1 with a recombinant TSP1 containing the N-terminal domain from SPTD1 TSP1 and the C-terminal domain from CBA120 TSP1 ("SPTD1-CBA120 TSP1"). [Figure 28] 1 shows a pairwise alignment performed with Emboss Needle of the amino acid sequences of CBA120 TSP1 (SEQ ID NO: 15) and SPTD1 TSP1 (SEQ ID NO: 2). The arrow indicates the junction site between amino acid 148 of SPTD1 TSP1 and amino acid 152 of CBA120 TSP1. The "neck" region (based on the published TSP structure) is indicated by a box. [Figure 29] FIG. 1 is a schematic diagram of the structure of the CBA120 TSP1 protein. [Figure 30] 1 shows a pairwise alignment performed with Emboss Needle of the amino acid sequences of SPTD1 TSP1 (SEQ ID NO: 2) and recombinant SPTD1-CBA120 TSP1, also referred to as SPTD1.ChiTSP1 (SEQ ID NO: 7). The junction between amino acid SPTD1 TSP1 and amino acid 149 of CBA120 TSP1 is marked with an arrow. [Figure 31]1 shows a pairwise alignment performed with Emboss Needle of the amino acid sequences of CBA120 TSP1 (SEQ ID NO: 15) and recombinant SPTD1-CBA120 TSP1, which may also be referred to as SPTD1.ChiTSP1 (SEQ ID NO: 7). The junction between amino acid SPTD1 TSP1 and amino acid 149 of CBA120 TSP1 is marked with an arrow. [Figure 32] Schematic diagram of the HR.SPTD1-CBA120chiTSP1 plasmid insert. The upstream homologous recombination region is the N-terminal region of TSP1 of SPTD1, which contains the TD1 and TD2 domains. [Figure 33] FIG. 1 is a schematic diagram of the homologous recombination process used to generate the synthetic phage SPTD1-CBA120.chiTSP1-Det7.chiTSP2(RBP-SPTD1-3) derived from SPTD1-CBA120.chiTSP1(RBP-SPTD1-2) by replacing TSP2 in SPTD1-CBA120.chiTSP1(RBP-SPTD1-2) with a recombinant TSP2 containing the N-terminal domain from SPTD1 TSP2 and the C-terminal domain from Det7 TSP2 ("SPTD1-Det7 TSP2"). [Figure 34] 1 shows a sequence alignment of the amino acid sequences of Det7 TSP2 (SEQ ID NO: 16), CBA120 TSP2 (SEQ ID NO: 11), and SPTD1 TSP2 (SEQ ID NO: 3) performed in Clustal Omega. The arrow indicates the junction site between amino acid 252 of SPTD1 TSP2 and amino acid 256 of Det7 TSP2. The "neck" region, based on published TSP structures, is indicated by a box. [Figure 35] FIG. 1 is a schematic diagram of the structure of the CBA120 TSP2 protein. [Figure 36] FIG. 1 is a schematic diagram of the predicted monomeric structure of a recombinant TSP containing the N-terminus of SPTD1 TSP2 and the C-terminus of Det7 TSP2 (SPTD1-Det7 TSP2 or SPTD1.Chi.TSP2). [Figure 37]

[0023] Figure 1 shows a pairwise alignment performed with Emboss Needle of the amino acid sequences of SPTD1.Chi.TSP2 (SEQ ID NO: 8) and recombinant SPTD1-Det7 TSP2, which may also be referred to as SPTD1 TSP2 (SEQ ID NO: 3). The arrow indicates the junction site between amino acid 252 of SPTD1 TSP2 and amino acid 256 of Det7 TSP2. [Figure 38] 1 shows a pairwise alignment performed with Emboss Needle of the amino acid sequences of recombinant SPTD1-Det7 TSP2 (SEQ ID NO: 8) and Det7 TSP2 (SEQ ID NO: 16). The arrow indicates the junction site between amino acid 252 of SPTD1 TSP2 and amino acid 256 of Det7 TSP2. [Figure 39] Schematic diagram of the HR.SPTD1-CBA120chiTSP2 plasmid insert. The upstream homologous recombination region is the N-terminal region of SPTD1 TSP2, which contains the attachment domain (AD), XD2, XD3, and TD1 domains. [Figure 40] FIG. 1 is a schematic diagram of the homologous recombination process used to generate the synthetic phage RBP-SPTD1-5 derived from SPTD1-CBA120.chiTSP1-Det7.chiTSP2(RBP-SPTD1-3) by replacing the TSP4 in SPTD1-CBA120.chiTSP1-Det7.chiTSP2(RBP-SPTD1-3) with a recombinant TSP4 containing the N-terminal domain from SPTD1 TSP4 and the C-terminal domain from TR2 TSP ("SPTD1.TSP4-TR2.TSP"). [Figure 41] 1 shows a pairwise alignment of the amino acid sequences of SPTD1 TSP4 (SEQ ID NO: 5) and TR2 TSP (SEQ ID NO: 18) performed with Emboss Needle. The junction site between amino acid 479 of SPTD1 TSP4 and amino acid 363 of TR2 TSP is indicated by an arrow. The "neck" region (based on AlphaFold2 structure prediction and published homology) is indicated by a box. [Figure 42] Schematic of the predicted structure generated by AlphaFold2 of SPTD1 TSP4. [Figure 43] Schematic of the predicted structure generated by AlphaFold2 of the TR2 TSP. [Figure 44] Schematic of the predicted structure generated by AlphaFold2 of a recombinant TSP containing the N-terminus of SPTD1 TSP4 and the C-terminus of TR2 TSP. Protein structure of chimeric TSP4. [Figure 45]

[0023] Figure 1 shows a pairwise alignment performed with Emboss Needle of the amino acid sequence of SPTD1 TSP4 (SEQ ID NO: 5) and a recombinant TSP comprising the N-terminal domain of SPTD1 TSP4 and the C-terminal domain of TR2 TSP (which can be referred to as "SPTD1.TSP4-TR2.TSP" or "chi.TSP4" (SEQ ID NO: 18)). The junction site between amino acid 479 of SPTD1 TSP4 and amino acid 363 of TR2 TSP is indicated by an arrow. The "neck" region (based on AlphaFold2 structure prediction and published homology) is indicated by a box. [Figure 46]

[0023] Figure 1 shows a pairwise alignment performed with Emboss Needle of the amino acid sequence of TR2 TSP (SEQ ID NO: 17) and a recombinant TSP comprising the N-terminal domain of SPTD1 TSP4 and the C-terminal domain of TR2 TSP (which can be referred to as "SPTD1.TSP4-TR2.TSP" or "chi.TSP4" (SEQ ID NO: 18)). The junction site between amino acid 479 of SPTD1 TSP4 and amino acid 363 of TR2 TSP is indicated by an arrow. The "neck" region (based on AlphaFold2 structure prediction and published homology) is indicated by a box. [Figure 47] Schematic diagram of the HR.SPTD1.TSP4-TR2.TSP plasmid insert. The upstream homologous recombination region is the N-terminal region of SPTD1 TSP4. [Figure 48] 1 is a photographic image of a plate culture showing the results of plating synthetic bacteriophage RBP-SPTD1-5 on Salmonella Kentucky. DETAILED DESCRIPTION OF THE INVENTION

[0013]

[0060] overview Described herein are compositions, methods, systems, and kits related to synthetic phages with customized host ranges and recombinant phage tail spike proteins (TSPs) with customized microbial recognition capabilities. Customizing the host range of a synthetic phage according to the present disclosure can include imparting the synthetic phage with the ability to recognize one or more target hosts and / or eliminating the recognition of one or more hosts from the synthetic phage. The ability of a synthetic phage to recognize or not recognize one or more hosts may be referred to in the present disclosure as "host range," "host recognition ability," or "host recognition capability." In some embodiments, the customized host range of a synthetic phage according to the present disclosure can be modified compared to the natural or recombinant parent phage from which the synthetic phage is constructed. In some other embodiments, synthetic phage with customized host ranges may be constructed de novo using the principles and techniques described in this disclosure along with general principles of synthetic biology, genetic engineering, and other related fields. Exemplary synthetic phages are constructed from or based on Ackermannviridae phages. Customizing the ability of a recombinant TSP to recognize a microorganism according to the present disclosure can include modifying the ability of the TSP to bind to a surface receptor of the microorganism, which can include modifying a TSP domain (e.g., the C-terminal domain) involved in binding to the surface receptor. In some embodiments, the ability of a recombinant TSP to recognize a microorganism can be modified compared to the native or recombinant parent TSP from which the recombinant TSP is constructed. In some other embodiments, a recombinant TSP can be constructed de novo using the principles and techniques described in this disclosure along with general principles of synthetic biology, genetic engineering, and other related fields. An exemplary recombinant TSP is constructed from or based on a TSP derived from an Ackermannviridae phage.

[0014]

[0061] As envisioned by the inventors and described herein, in some embodiments, a customized host range is imparted to a synthetic phage, which may be constructed from or based on Ackermannviridae phage, by one or more recombinant TSPs. Some synthetic phage embodiments incorporate one or more recombinant TSPs containing a combination of laboratory-engineered N- and C-terminal regions. As discussed in more detail in this disclosure, the C-terminal region of the TSP is responsible for target host recognition. Thus, in one example, synthetic phage according to embodiments of the invention can be endowed with the ability to recognize a target host by including a C-terminal region capable of recognizing the target host in the synthetic phage's recombinant TSP. Other examples and embodiments are envisioned and described in more detail in this disclosure, along with methods of constructing synthetic phage according to embodiments of the invention and other related methods, methods of using synthetic phage according to embodiments of the invention, and systems and kits related to synthetic phage according to embodiments of the invention.

[0015]

[0062] As conceived and envisioned by the inventors, embodiments of the present invention, examples of which are described in this disclosure, have various advantages. For example, embodiments of the present invention provide a versatile, highly customizable, and easy-to-use platform for producing synthetic phage with customized host ranges, as well as recombinant TSPs with customized abilities to recognize microorganisms. For example, based on embodiments of the present invention, various plasmids encoding various recombinant TSPs with different abilities to recognize microorganisms may be produced and then used to engineer highly efficient (time- and cost-saving) synthetic phage with desired customized host ranges. In another example, phage specific for highly pathogenic bacteria, such as Burkholderia pseudomallei or Yersinia pestis, can be engineered to also infect non-pathogenic hosts, allowing phage production to be carried out at lower biosafety levels with reduced risk to personnel and associated costs. In another example, embodiments of the present invention may enable the production of synthetic phage capable of infecting a variety of bacteria without the need to perform phage production in phage hosts that are difficult to culture or that are antibiotic-resistant.

[0016]

[0063] In some exemplary embodiments, Ackermannviridae phage TSPs are used to construct recombinant TSPs. TSPs are structurally similar across different Ackermannviridae subcategories, allowing recombinant TSPs to be used to engineer synthetic phages according to embodiments of the present invention from a wide range of Ackermannviridae phages. The methods for constructing synthetic phages envisioned by the inventors may use TSP amino acid sequences from public databases, thereby reducing or eliminating the need for laboratory experimentation to identify phages with desired properties for constructing recombinant TSPs and / or synthetic phages with customized host ranges according to embodiments of the present invention. There is evidence that horizontal gene transfer of TSP-encoding nucleic acid sequences occurs between Ackermannviridae phages and non-Ackermannviridae phages, such as with podoviruses (bacteriophages of the Podoviridae family). For example, the podovirus bacteriophage P22 TSP is believed to be related to the Ackermannviridae Salmonella phage Det7 TSP. Thus, it is contemplated that amino acid sequences derived from TSPs or short-tail fiber proteins from non-Ackermannviridae phages, such as, but not limited to, Podoviridae or Siphoviridae, can be used in recombinant TSPs and / or synthetic phages with customized host range according to embodiments of the present invention.

[0017]

[0064] Terms and Concepts Certain terms and concepts are described below. These, in conjunction with the remainder of this document and the accompanying drawings, are intended to facilitate understanding of various embodiments of the present invention. These terms and concepts may be further clarified and understood based on accepted practices in the field of the present invention and the descriptions provided throughout this document and / or the accompanying drawings. Some other terms may be explicitly or implicitly defined in other sections of this specification and the accompanying drawings and may be used and understood based on accepted practices in the field of the present invention and the descriptions provided throughout this specification and / or the accompanying drawings. Terms that are not explicitly defined may also be defined and understood based on accepted practices in the field of the present invention and may be interpreted in the context of this document and / or the accompanying drawings.

[0018]

[0065] Unless otherwise indicated by the context, singular terms shall include the plural, and plural terms shall include the singular. Generally, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, and the techniques thereof, are well known and commonly used. Known methods and techniques are generally carried out according to well-known conventional methods and as described in various general and more specific references, unless otherwise indicated. The nomenclature used in connection with the experimental procedures and techniques described in this disclosure is well known and commonly used.

[0019]

[0066] Unless otherwise specified and / or dictated by context, the nomenclatures used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein, and the techniques thereof, are well known and commonly used in the art. Known methods and techniques are generally performed according to conventional methods well known in the art, unless otherwise indicated, and as described in the various general and more specific references discussed throughout this specification. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with the experimental procedures and techniques described herein are well known and commonly used in the art.

[0020]

[0067] As used in this disclosure, the terms "a," "an," and "the" can refer to one or more, unless otherwise specified.

[0021]

[0068] Use of the term "or" is used to mean "and / or" unless expressly indicated to refer to alternatives only or unless the alternatives are mutually exclusive, however, this disclosure supports a definition that refers to alternatives only and "and / or." As used in this disclosure, "another" can mean at least a second or more.

[0022]

[0069] As used in this disclosure, unless otherwise indicated, the terms "include," "including," and possibly similar terms (such as "have" or "having") mean "comprising."

[0023]

[0070] Where numerical ranges are provided in this disclosure, the numerical ranges include the endpoints of the range unless otherwise indicated. Unless otherwise indicated, the numerical ranges include all values ​​and subranges in this disclosure as if explicitly written out.

[0024]

[0071] As used in this disclosure, the terms "about" and "approximately" generally refer to an acceptable degree of error in the measured quantity, given the nature or precision of the measurement. Exemplary degrees of error are within 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of a given value or range of values. For example, any reference to "about X" or "approximately X" specifically indicates at least the values ​​X, 0.9X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, and 1.10X. In another example, the term "about" or "approximately" in reference to a reference numerical value can include values ​​within a range of plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of that value. Thus, the phrase "about X" or "approximately X" is intended to describe a claim limitation of, for example, "0.98X." Numerical values ​​provided in this disclosure are approximate unless otherwise specified, and the term "about" or "approximately" means that a value can be inferred when not explicitly stated. When the term "about" or "approximately" is applied to the beginning of a numerical range, it also applies to both ends of the range. When the term "about" or "approximately" appears before a series of values, it is intended to modify each value in the series.

[0025]

[0072] The term "administer" or "administration," when used in the context of administering a composition described in this disclosure to a subject (and related terms and phrases), refers to the act of physically delivering a substance present outside the body (e.g., a composition comprising one or more synthetic phages described in this disclosure) to a subject. Administration can be by mucosal, intradermal, intravenous, intramuscular, subcutaneous delivery, and / or any other known physical delivery method. Administration encompasses direct administration, such as administration to a subject by a healthcare professional or self-administration, or indirect administration, which can be the act of prescribing a composition described in this disclosure.

[0026]

[0073] As used herein, "analyte" refers to a molecule, compound, or cell being measured. The analyte of interest may, in certain embodiments, interact with a binding agent. As described herein, the term "analyte" may refer to a protein or peptide of interest. An analyte may be an agonist, antagonist, or modulator. Alternatively, an analyte may have no biological effect. Analytes may include small molecules, sugars, oligosaccharides, lipids, peptides, peptidomimetics, organic compounds, etc.

[0027]

[0074] As used herein, the term "chimera" and related terms and expressions refer to a nucleic acid sequence, amino acid sequence, polypeptide, or protein that is artificially constructed by combining or joining together nucleic acid sequences, amino acid sequences, or polypeptide or protein regions or domains, respectively, from two or more different sources (such as phages). The resulting combination is not found in nature. For example, the N-terminal region of a TSP from one phage is combined with the C-terminal region of a TSP from a different phage. In other cases, different locations may refer to different proteins within the same phage. For example, the C-terminal region of TSP2 is artificially duplicated by replacing the C-terminal region of TSP1 in CBA120 with the C-terminal region of TSP2 in CBA120.

[0028]

[0075] The terms "detectable moiety" or "detectable biomolecule" or "reporter" or "indicator agent" or "indicator moiety" refer to a molecule or compound produced by a molecule (such as an enzyme) that can be measured in a quantitative assay. For example, an indicator agent or indicator moiety can include an enzyme that can be used to convert a substrate into a measurable product. An indicator agent or indicator moiety can be an enzyme that catalyzes a reaction that produces bioluminescence (e.g., luciferase). Alternatively, an indicator agent or indicator moiety can be a radioisotope that can be quantified. Alternatively, an indicator moiety can be a fluorophore. Alternatively, other detectable molecules may be used. The term "indicator gene" is used to refer to a gene that encodes an indicator agent, such as a protein, e.g., an enzyme.

[0029]

[0076] The term "host" and related terms and phrases are used in this disclosure with respect to phages to refer to microorganisms that the phage can infect. Phages can lyse host cells, release new virions upon lysis, transfer genes between hosts, and form lysogens, thereby altering host function. As used herein, the phrase "host range" and related terms and phrases refer to the range or number of hosts infected by a phage. In other words, "host range" refers to the breadth of organisms (genus, species, strain, or other taxon) that a phage can infect, with the limitations of the host range resulting from phage, host, or environmental characteristics. Some phages have a fairly narrow host range, capable of infecting only a few strains within the same species. Other phages can infect many species of hosts, sometimes across different genera. The breadth of a particular phage's host range can be attributed in part to the specificity of the phage's host-binding proteins, biochemical interactions during infection, the presence of associated prophage or specific plasmids, and host-phage resistance mechanisms. The terms "customized host range," "modified host range," "altered host range," "altered tropism," and related terms are used herein to refer to artificially altered phage host ranges. Such altered host ranges can be achieved by at least some of the methods described in this disclosure, as well as other methods.

[0030]

[0077] The terms "nucleic acid," "nucleic acid sequence," "nucleotide sequence," "oligonucleotide," "polynucleotide," and related terms and phrases refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof. As discussed in this disclosure, nucleic acid sequences encompass all forms of nucleic acids, including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-loop structures, and the like. When an RNA sequence is described, its corresponding DNA sequence is also described, and uridine is represented as thymidine. When a DNA sequence is described, its corresponding RNA sequence is also described, and thymidine is represented as uridine. Unless otherwise specified, the term "nucleic acid" and related terms and phrases encompass nucleic acids containing known analogs of natural nucleotides that have similar properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. A nucleic acid sequence can include a combination of deoxyribonucleic acid and ribonucleic acid. Such deoxyribonucleic and ribonucleic acids include both naturally occurring molecules and synthetic analogs. Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses degenerate codon substitutions, alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly indicated sequence. Degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues.

[0031]

[0078] The terms "oligonucleotide," "polynucleotide," or "nucleic acid" encompass DNA or RNA molecules, including molecules produced synthetically or by recombinant technology. An oligonucleotide, polynucleotide, or nucleic acid may be single-stranded or double-stranded.

[0032]

[0079] As used herein, "phage" includes one or more of several viruses capable of invading living bacteria, fungi, mycoplasma, protozoa, yeast, and other microscopic living organisms. In this disclosure, the term "phage" and related terms include viruses such as bacteriophages capable of invading bacteria, viruses that infect archaea, which may be referred to as archaeal phages or bacteriophages, mycobacteriophages capable of invading mycobacteria (a family of bacteria that includes mycobacteria of the Mycobacterium tuberculosis complex, which contains the causative agent of tuberculosis, and mycobacteria of the Mycobacterium avis complex, which contains the causative agent of tuberculosis), mycoviruses capable of invading fungi, mycoplasma phages, and viruses capable of infecting protozoa, yeast, and other microscopic organisms. Here, "microscopic" means having a maximum dimension of one millimeter or less. Phages are viruses that have evolved in nature to use microscopic organisms as a means of replicating themselves. In nature, a phage can attach itself to a microorganism, inject its DNA (or RNA) into the microorganism, and then induce the microorganism to replicate the phage hundreds or thousands of times. This is called phage amplification.

[0033]

[0080] The terms "peptide," "polypeptide," or "protein" are used to refer to a polymer of amino acids linked by native and / or non-native amide bonds. A peptide, polypeptide, or protein may contain moieties other than amino acids (e.g., lipids or sugars). A peptide, polypeptide, or protein may be produced synthetically or by recombinant technology. Amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). In the broadest sense, naturally occurring amino acids can be divided into groups based on the chemical characteristics of their side chains. "Hydrophobic" amino acids refer to any of His, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys, or Pro. "Hydrophilic" amino acids refer to any of Gly, Asn, Gln, Ser, Thr, Asp, Glu, Lys, Arg, or His. This grouping of amino acids can be further subclassified as follows: "Uncharged hydrophilic" amino acids refer to any of Ser, Thr, Asn, or Gln. "Acidic" amino acids refer to any of Glu or Asp. "Basic" amino acids refer to any of Lys, Arg, or His. Non-natural (non-naturally occurring) amino acids include, but are not limited to, amino acid analogs, amino acid mimetics, synthetic amino acids, N-substituted glycines, and N-methyl amino acids in the L- or D-configuration that function in a manner similar to naturally occurring amino acids.For example, an "amino acid analog" may be a non-natural amino acid that has the same basic chemical structure as a naturally occurring amino acid (carbon bonded to a hydrogen, a carboxyl group, and an amino group), but has a modified side group or a modified peptide backbone, such as homoserine, norleucine, methionine sulfoxide, or methionine methylsulfonium. An "amino acid mimetic" refers to a chemical compound that has a structure different from the general chemical structure of an amino acid, but functions similarly to a naturally occurring amino acid. Amino acids may be referred to by either their commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. A "domain" of a protein or polypeptide refers to a region of the protein or polypeptide that is defined by structural and / or functional properties. Exemplary functional properties include enzymatic activity and / or the ability to bind to or be bound by another protein or non-protein entity.

[0034]

[0081] As used herein, the term "recombinant" and related terms and phrases refer to genetic (i.e., nucleic acid) modifications typically performed in a laboratory to bring together genetic material not otherwise found. The term can be used interchangeably with terms such as "modified," "synthetic," "engineered," "constructed," etc.

[0035]

[0082] The term "solid support" or "support" refers to a structure that provides a substrate and / or surface to which biomolecules, cells, etc. can be attached. For example, a solid support can be an assay well (i.e., a microtiter plate or multiwell plate, etc.), or the solid support can be a filter, an array, or a location on a movable support such as a bead or membrane (e.g., a filter plate or lateral flow strip).

[0036]

[0083] The terms "individual," "subject," and "patient" can be used interchangeably in this disclosure to refer to non-human animals or humans. Examples of subjects include, but are not limited to, humans and other primates, such as non-human primates, e.g., chimpanzees and other ape and monkey species; livestock, e.g., cattle, sheep, pigs, goats, and horses; domestic mammals, e.g., dogs and cats; laboratory animals, such as rodents, e.g., mice, rats, and guinea pigs; chickens, turkeys, and other poultry; domestic fowl, e.g., ducks, geese; and birds, e.g., wild birds and game birds. The terms "individual," "subject," and "patient" do not themselves denote a particular age, sex, race, or clinical condition. Thus, subjects of any age, regardless of sex, are intended to be encompassed by this disclosure, including, but not limited to, elderly people, adults, children, infants, babies, and young children. Similarly, the methods of the present invention are applicable to people of all races, for example, Caucasians (whites), African Americans (blacks), Native Americans, Native Hawaiians, Hispanics, Latin Americans, Asians, and Europeans. An infected subject is a subject known to be infected with an infectious organism, such as a bacterium.

[0037]

[0084] The term "sample" and related terms and phrases include, but are not limited to, environmental samples, food samples, water samples, medical samples, clinical samples, or veterinary samples. A sample can be liquid, solid, or semi-solid. A sample can be a swab of a solid surface. A sample can include environmental materials such as a water sample, or a filter from an air sample, or an aerosol sample from a cyclone collector. A sample can be a sample of fish, meat (e.g., beef, pork, or lamb), poultry, processed foods, peanut butter, powdered infant formula, milk powder, tea, starch, eggs, milk, cheese, or other dairy products. Medical, clinical, or veterinary samples include, but are not limited to, blood, sputum, cerebrospinal fluid, urine, and fecal samples. In some embodiments, a sample can be a different type of swab. The term "sample" encompasses a variety of appropriate control samples. For example, in the context of a microbial detection control sample that does not contain the microorganism of interest, it can be assayed to control for background signal levels.

[0038]

[0085] The term "variant," as used in this disclosure with reference to a reference amino acid or nucleic acid sequence, encompasses homologs, variants, isoforms, fragments, mutant forms, modified forms, and other variations of the amino acid or nucleic acid sequence described herein. The terms "homologue" and other related terms used herein with respect to various amino acids are intended to describe the degree of sequence similarity between amino acid sequences, calculated in accordance with accepted procedures. Homologous or similar sequences (which may also be described as "variants") may be at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous (or also described as having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% "sequence identity" or "sequence similarity"). As used herein, the "percent homology" (or "sequence identity" or "sequence similarity") of two amino acid sequences is determined using the Karlin and Altschul algorithm, which is incorporated into the Basic Local Alignment Search Tool (BLAST®, National Library of Medicine, Bethesda, Maryland) program available for public use through the National Institutes of Health (USA) website. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with words of the same length in a database sequence, match or meet some positive threshold score T. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using the parameters M (reward score for a pair of matching residues, always >0) and N (penalty score for mismatched residues, always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score falls by an amount X from its maximum achieved value, when the cumulative score falls below 0 due to the accumulation of one or more negative-scoring residue alignments, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, 1989). The BLAST algorithm also performs statistical analysis of the similarity between two sequences (Karlin and Altschul, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which indicates the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in comparing the test nucleic acid with the reference nucleic acid is less than about 0.01, more preferably less than about 10-5, and most preferably less than about 10-20. Variants or isoforms can be used instead of or in addition to homology. For comparison purposes, Gapped BLAST is used to obtain gapped alignments. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. Percent "homology" or "sequence similarity" can be used in the present disclosure to describe fragments, variants, or isoforms of amino acid or nucleic acid sequences, although other methods of describing fragments can also be used.Percent "homology" or "sequence similarity" can also be used in the present disclosure to describe nucleic acid and amino acid sequences that are "derived from" and "based on" other sequences. For example, when the amino acid sequence of a recombinant TSP, a region of a TSP according to the present disclosure (e.g., the N-terminal region, the C-terminal region, or any other region) is said to be "derived from" or "based on" another TSP amino acid sequence (which may be described as a "parent amino acid sequence" or "source amino acid sequence"), the amino acid sequence of the recombinant TSP can have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% "sequence identity" or "sequence similarity" with the source amino acid sequence.

[0039]

[0086] The term "conservatively modified variants" and related expressions can be applied to amino acid sequences and nucleic acid sequences encoding amino acid sequences. Substitutions, deletions, or additions to nucleic acid, peptide, polypeptide, or protein sequences that modify, add, or delete a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants," resulting in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the present invention. The following eight groups each contain amino acids that are conservative substitutions for each other: 1) Alanine (A), Glycine (G), 2) Aspartic acid (D), glutamic acid (E), 3) Asparagine (N), Glutamine (Q), 4) Arginine (R), Lysine (K), 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V), 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W), 7) serine (S), threonine (T), and 8) Cysteine ​​(C), methionine (M).

[0040]

[0087] "Virus" and related terms and phrases are used in both the plural and singular sense. A "virion" refers to a single virus. For example, the phrase "phage virion" refers to a phage particle.

[0041]

[0088] phage Phages possessing tails (molecular machines that specifically recognize and attach to host cells, penetrate the cell envelope, and deliver phage nucleic acid into the cell) can be called "tailed phages." Among naturally occurring bacteriophages, tailed phages are typically classified in the Caudoviricetes class. In addition to taxonomic separation by genomic relatedness, tailed phages historically can and have been distinguished by morphology. Myoviruses are characterized by long contractile tails (phages in the Straboviridae, Kyanoviridae, Herelleviridae, Chaseviridae, and Ackermannviridae families). Siphoviruses are characterized by long, non-contractile tails (e.g., phages of the Demerecviridae and Drexlerviridae families). Podoviruses are characterized by short, non-contractile tails (e.g., phages of the Autographiviridae families). Myoviruses of the Ackermannviridae family have a non-enveloped head, a collarless neck, a small baseplate, and a contractile tail. The host range of tailed phages is determined by the structures they use to bind to host cells. These structures include the tail fiber, tail spike, and tail tip. The host cell-binding structure of tailed phages binds to surface receptors on host cells. Infection of host cells by tailed phage virions begins with reversible recognition of host cell surface receptors by the binding structures, followed by irreversible binding and delivery of phage nucleic acid (DNA in the case of Caudoviricetes) to the host cell. The tail fibers, tail spikes, and tail tips act as receptor-binding proteins (RBPs) that specifically recognize host cell surface receptors.Host cell surface polysaccharides, such as lipopolysaccharides, often serve as surface receptors for phages. Other known cell surface receptors recognized by phages are teichoic acids, porins, and cellular appendages such as flagella and pili. More than one cell surface receptor may be involved in the recognition of a host cell by a phage virion. Tailed phages use their tails and associated RBPs to interact with host cells and create a channel for the delivery of phage nucleic acid into the host cell. The high genetic plasticity of tailed phage RBPs allows tailed phages to adapt to their host range. Phage taxonomy, the structure of tailed phages, including those of the family Ackermannviridae, and the host cell recognition mechanisms used by tailed phages are discussed in, for example, one or more of Nobrega et al. (2018), Prokhorov et al. (2017), Sorensen et al. (2021), Greenfield et al. (2020), Plattner et al. (2019), Bertozzi Silva et al. (2016), and Ackermann (2011). It should be understood that phage taxonomy and classification systems are a dynamic field. For example, current official phage classifications include many so-called "floating" members, which may not be assigned to an "order" and / or "family." Many current phage nomenclatures are based on morphology, but not necessarily molecular taxonomy.

[0042]

[0089] The tails of phages in the Ackermannviridae family contain up to four tail spike proteins (TSPs), each of which recognizes a specific host. Some phages in the Ackermannviridae family express four different TSPs, while some contain three TSP genes. Several recent studies of TSP sequence diversity have classified Ackermannviridae TSPs into distinct subtypes, TSP1, TSP2, TSP3, and TSP4, and found that each TSP subtype is specifically associated with a genera within the Ackermannviridae family (e.g., Kuttervirus, Agtrevirus, Limestonevirus, and Taipeivirus). See, for example, Sorensen et al. (2021). Although the amino acid sequences of Ackermannviridae TSPs are diverse, the overall TSP structure is well conserved. Ackermannviridae TSPs are stable homotrimers, with each monomer displaying a right-handed beta helix. The assembly of all TSPs on an Ackermannviridae phage virion forms a branched structure attached to a baseplate. Figure 1 shows a schematic representation of the four TSPs of CBA120, a member of the Ackermannviridae family. Each of TSPs 1-4 recognizes a unique bacterial serotype (TSP1 - Salmonella enterica subsp. enterica serovar Minnesota; TSP2 - E. coli O157; TSP3 - E. coli O77; TSP4 - E. coli O78). Figure 2 is a schematic diagram of the TSPs of an exemplary phage of the Ackermannviridae family, which has four TSPs.Each TSP is capable of recognizing a unique host cell. The TSPs form a branched complex attached to the baseplate of the phage virion. Described in this disclosure and included within embodiments of the invention are recombinant TSPs, some of which are derived from TSPs derived from Ackermannviridae phages. However, it should be understood that recombinant TSPs according to embodiments of the invention are not limited to those derived from Ackermannviridae phages. Recombinant TSPs according to embodiments of the invention may be derived, at least in part, from TSPs or homologous structures often identified as short-tailed or simple-tailed fibers found in other phage families, including, but not limited to, Autographiviridae, Straboviridae, Herelleviridae, Chaseviridae, Demerecvridae, and Drexlerviridae. The short-tailed fibers of many tailed phages may contain amino acid sequences useful as source sequences for the C-terminal region sequences of recombinant TSPs according to the invention. The amino acid sequences of recombinant TSPs according to embodiments of the invention may be derived, at least in part, from amino acid sequences of various tailed phages (class Cauodovriricetes). Recombinant TSPs according to embodiments of the invention may also be designed and constructed de novo.

[0043]

[0090] The TSP homotrimer comprises an N-terminal region or domain, an elongated C-terminal region or domain, and a helical "neck" or "hinge" region connecting the N-terminal and C-terminal regions or domains. The structure of the TSP trimer is shown in Figure 9, which schematically illustrates a recombinant TSP according to an embodiment described in this disclosure. Prior to the characterization of TSPs, many genes likely encoding TSPs were instead identified as tail fiber and short-tail fiber proteins. Thus, many Ackermannviridae viruses have erroneously labeled their TSPs as tail fiber proteins. The N-terminal domain or region of the TSP (which may be referred to as a "head-binding domain," "head domain," and other related terms and expressions) is structurally important for promoting either binding of the phage virion to the baseplate or binding to other TSPs to form the branched tailspike complex. The TSP acts as an RBP, enabling the initial, reversible contact between the phage virion and the host cell. The C-terminal domain or region of a TSP (which may be referred to as a "receptor-binding domain," "receptor-binding region," "body domain," and other related terms and expressions) contains a receptor-binding / catalytic module that facilitates the binding and degradation of the host cell receptor. After binding to a surface receptor on a host cell, the enzymatic activity of the TSP results in irreversible binding of the phage virion to that host cell. For example, the C-terminal domain of a TSP can exhibit lyase, glycosidase, or esterase activity against host cell lipopolysaccharide (LPS), resulting in irreversible binding of the phage virion to that host cell. It is believed that TSPs do not lyse host cells, but rather facilitate subsequent cell wall penetration by cleaving or modifying host cell surface receptors.

[0044]

[0091] Described in this disclosure and included among embodiments of the invention are synthetic phages, some of which are derived from naturally occurring Ackermannviridae phages (however, synthetic phages according to embodiments of the invention are not limited to synthetic Ackermannviridae-derived phages, but may be derived from or based on other tailed phages, or may be designed and synthesized de novo). Known Ackemannviridae taxa include: Agtreviruses (e.g., Agtrevirus AG3, Agtrevirus MK13, Agtrevirus P46FS4, Agtrevirus SKML39, Limestonevirus limestone, Limestonevirus RC2014, unclassified Aglimvirinae (e.g., Dickeya phage phiDP10.3, Dickeya phage phiDP23.1, Enterobacter phage fGh-Ecl02, Escherichia phage PC3, Escherichia phage PH4, Escherichia phage vB_EcoM-RPN242, Escherichia phage vB_EcoM-ZQ1; Campanileviruses (e.g., Vibriophage YC, Vibriophage vB_VcorM_GR28A, Cvivirinae), Kutterviruses (e.g., Kuttervirus aagejoakim, Kuttervirus allotria, Kuttervirus barely, Kuttervirus bering,Kuttervirus BSP101, Kuttervirus CBA120, Kuttervirus Det7, Kuttervirus dinky, Kuttervirus ECML4, Kuttervirus EP75, Kuttervirus FEC14, Kuttervirus GG32, Kuttervirus heyday, Kuttervirus kv38, Kuttervirus maane, Kuttervirus marshall, Kuttervirus maynard, Kuttervirus moki, Kuttervirus mutine, Kuttervirus pertusa pertopsoe), Kuttervirus PhaxI, Kuttervirus PM10, Kuttervirus rabagast, Kuttervirus S118, Kuttervirus SE14, Kuttervirus SeB, Kuttervirus SeG, Kuttervirus SeJ, Kuttervirus SenASZ3, Kuttervirus SeSz1, Kuttervirus SFP10, Kuttervirus SH19, Kuttervirus SJ2, Kuttervirus SJ3, Kuttervirus SP1, Kuttervirus SS9, Kuttervirus STML131, Kuttervirus STW77,and Kuttervirus ViI; Kujavirus, e.g., Kujavirus kuja (exemplified by vibriophage vB_VchM_Kuja); Miltonvirus, Miltonvirus 3M (Serratia phage vB_SmaA_3M), Miltonvirus MAM1 (exemplified by Serratia phage 2050H1 and Serratia phage phiMAM1), and unclassified Miltonvirus (exemplified by Serratia phage KNP4); Nezavisimistyvirus, e.g., Nezavisimistyvirus V1 bue1) (exemplified by Erwinia phage vB_EamM-Bue1) and Nezavisimistyvirus Ea2809 (exemplified by Erwinia phage phiEa2809); Taipeivirus, e.g., Taipeivirus 0507KN21, Taipeivirus IME250 (exemplified by Serratia phage vB-Sru-IME250), Taipeivirus KpS110 (exemplified by Klebsiella siella vB_KpnM_KpS110), Taipeiivirus KWBSE436 (exemplified by Escherichia phage vB_EcoM_KWBSE43-6), Taipeiivirus magnus (exemplified by Klebsiella magnus), Taipeiivirus Mei (exemplified by Klebsiella phage Mei), Taipeiivirus Menlo (exemplified by Klebsiella phage Menlo),Taipei virus UPM2146 (exemplified by Klebsiella phage UPM2146), unclassified Taipei virus (e.g., Klebsiella phage K751, Klebsiella phage PWKp5, Klebsiella phage T751, Klebsiella phage T765, Klebsiella phage vB_KqM-Bilbo, Klebsiella phage vB_KqM-LilBean, Klebsiella phage vB_KqM-Westerburg, Klebsiella virus UPM2146; Tedavirus, such as Tedavirus A829 (exemplified by Aeromonas phage phiA8-29); Vapseptimavirus, such as Vapseptimavirus VAP7 (exemplified by Vibriophage BX-1, Vibriophage VAP7, and Vibriophage VP-1). Furthermore, examples of the Ackermannviridae include unclassified Ackermannviridae, such as Acinetobacter phage SH-Ab15599, Agrobacterium phage Atu_ph04, Agrobacterium phage OLIVR5, Agrobacterium phage OLIVR6, and Lars Ralstonia phage RSP15, Rhizobium phage AF3, Rhizobium phage P9VFCI, Rhizobium phage RHph_I1_18, Rhizobium phage RHph_I1_9, Rhizobium phage RHph_I34, Rhizobium phage RHph_I46, Rhizobium phage RHph_I9,Rhizobium phage RHph_N34, Rhizobium phage RHph_Y68, Rhizobium phage RL2RES, Rhizobium phage RL38J1, Rhizobium phage vB_RleM_P10VF, Salmonella phage BRM13312, Salmonella phage BRM1331 4, Sinorhizobium phage phiM9, Stenotrophomonas phage vB_SmaS-DLP_6, Vibriophage 1.244.A._10N.261.54.C3, Vibriophage 1.255.O._10N.286.45.F1, Vibriophage 144E46.1, Vibriophage 207E48.1, Ackermannviridae species sp. ctaCq7, Ackermannviridae sp. ctClB2, Ackermannviridae sp. ctFRM8, Ackermannviridae sp. ctjwt21, Ackermannviridae sp. ctkHJ36, Ackermannviridae sp. ctQad106, and Ackermannviridae sp. ctUml7.

[0045]

[0092] Synthetic phage and recombinant tail spike protein (TSP) As described in more detail in this disclosure, compositions, methods, systems, and kits according to embodiments of the present invention can include one or more synthetic phages with customized host ranges. In certain embodiments, the synthetic phages include at least one recombinant or engineered tail spike protein (TSP) that confers its host recognition characteristics (also referred to as "host range") to the synthetic phages. Engineered or recombinant TSPs according to the present disclosure are included in embodiments of the present invention. The C-terminal region of the TSP is responsible for phage host recognition. Thus, engineered or recombinant TSPs according to the present disclosure are constructed with a C-terminal region that has the desired recognition ability (the ability or lack of ability to recognize a particular host, or an altered ability or lack of ability to recognize a particular host (such as low affinity or high affinity)). It should be understood that a TSP amino acid sequence described as being located in the C-terminal region, or a region that affects host recognition, is not necessarily located at the C-terminus of the TSP amino acid sequence; it can be located anywhere in the C-terminal region of the TSP sequence (e.g., found within the C-terminal region). As currently understood, the N-terminal region or domain (which may be referred to as the "head" domain) of naturally occurring phage TSPs is responsible for binding to the phage baseplate or other TSPs. The N-terminal and C-terminal regions of a phage TSP are involved in phage structural assembly, such as the N-terminal region ("neck"), whereas regions or domains C-terminal to the regions involved in phage assembly may be involved in host recognition. The N-terminal and C-terminal regions of a phage TSP are connected by a so-called "neck" or "hinge" region. Exemplary structures of naturally occurring phage TSPs are shown and can be understood with reference to Figures 21, 29, and 35. Exemplary structures of recombinant TSPs according to embodiments of the invention are shown and can be understood with reference to Figures 9, 22, and 36. The N-terminal region of the amino acid sequence of a TSP is located N-terminal ("upstream") of the "neck" or "hinge" region. The C-terminal region of the amino acid sequence of a TSP is located C-terminal ("downstream") of the "neck" or "hinge" region.

[0046]

[0093] An example of an engineered or recombinant TSP is a recombinant TSP that contains a combination of N- and C-terminal regions that have been engineered in the laboratory (i.e., do not occur in nature). Both the N- and C-terminal regions of such a recombinant TSP can (but do not necessarily) contain amino acid sequences that naturally occur in the N- and C-terminal regions, respectively, of different phage TSPs. For example, in a recombinant TSP, N- and C-terminal regions occurring in two different TSPs (which may be referred to as "parent" TSPs) are artificially combined to create a recombinant TSP with desired host recognition properties. Even if both the N- and C-terminal amino acid sequences exist separately in nature, the resulting combination (which may be described as a "hybrid" or "chimeric" amino acid sequence) is artificial. Such a recombinant TSP may be referred to as a "chimera." A chimeric TSP may contain amino acid sequences from three or more (3, 4, 5, etc.) parent TSPs.

[0047]

[0094] Another example of an engineered or recombinant TSP is a TSP containing at least one non-naturally occurring amino acid sequence in its C-terminal region. For example, such a non-naturally occurring amino acid sequence can be artificially modified from the naturally occurring C-terminal amino acid sequence of a naturally occurring phage TSP. For example, an engineered or recombinant TSP can be constructed from a parent TSP, which can be an engineered or recombinant TSP, by replacing at least a portion of the C-terminal region of the naturally occurring or parent (first) TSP with at least a portion of the C-terminal region of a different (second) TSP. At least a portion of the C-terminal region from the second TSP can be naturally occurring, engineered, or recombinant. In this exemplary case, the first TSP has a different recognition ability from the second TSP, and the resulting engineered or recombinant TSP has the recognition ability of the second TSP. In another example, an engineered or recombinant TSP can be constructed from a parent TSP by modifying one or more amino acids in the C-terminal region of the parent TSP to modify the recognition ability of the parent TSP. In another example, an engineered or recombinant TSP contains one or more engineered sequences within its C-terminal region (in some cases, the entire amino acid sequence of the C-terminal region can be engineered de novo) that confer a desired recognition ability on the engineered TSP.

[0048]

[0095] In some embodiments according to the present disclosure, the amino acid sequence of the N-terminal region of a recombinant or engineered TSP, such as a chimeric TSP, is derived from the amino acid sequence of the N-terminal region of a TSP found in a phage used as a "parent" for a synthetic phage incorporating the chimeric TSP. Engineering the C-terminal domain of the chimeric TSP allows for tailoring (customizing) its host recognition ability while retaining the N-terminal TSP region found in the parent phage, maintaining the ability of the chimeric TSP to be incorporated into the phage structure during assembly.

[0049]

[0096] In some embodiments, inclusion of a recombinant or engineered TSP in a synthetic phage with a C-terminal region capable of recognizing a target host confers the synthetic phage the ability to recognize the target host, meaning that the synthetic phage acquires an ability to recognize a target host that was not present in the parent phage from which the synthetic phage was constructed. In some embodiments, inclusion of a recombinant TSP in a synthetic phage with a C-terminal region capable of recognizing a target host improves the ability of the synthetic phage to recognize the target host compared to the parent phage from which the synthetic phage was constructed. For example, the recombinant TSP may have a higher affinity for the target host than the TSP in the parent phage. In another example, the recombinant TSP (and therefore the synthetic phage) may have a higher specificity for the target host than the parent phage. In another example, a synthetic bacteriophage may be constructed to contain two or more TSPs that recognize the same target host, thereby improving the ability of the synthetic bacteriophage to recognize the target host. In this case, the two or more TSPs that recognize the same target host can be identical TSPs, non-identical TSPs that recognize the same surface receptor on the target host, or non-identical TSPs that recognize different surface receptors on the same target host. In some embodiments, including a recombinant or engineered TSP in a synthetic phage deprives the synthetic phage of the ability to recognize at least one host recognized by the parent phage from which the synthetic phage was constructed (meaning that the synthetic phage loses the ability to recognize at least one host that recognized the parent phage). In some embodiments, including a recombinant TSP in a synthetic phage impairs the synthetic phage's ability to recognize a target host compared to the parent phage from which the synthetic phage was constructed. For example, the recombinant TSP (and thus the synthetic phage) can have a lower affinity for the target host than the TSP in the parent phage. In another example, the recombinant TSP (and thus the synthetic phage) can have a lower specificity for the target host than the parent phage. In the latter case, the synthetic phage can recognize a wider range of related hosts than the parent phage.In some embodiments, one or more native TSPs of a parent phage may be replaced with truncated versions of the TSP genes, thereby eliminating the bacterial specificity of the truncated TSPs while ensuring proper TSP complex formation and protein folding. For example, CBA120 TSP1 may be truncated to only the N-terminal region upstream of the "neck" connecting the C-terminal catalytic and receptor-binding domains that confers specificity to Salmonella Minnesota. If the presence of the N-terminus of TSP1 binding to TSP4 stabilizes the TSP complex, including a truncated TSP1 with only the N-terminus, this may result in a more viable phage than simply deleting TSP1 or providing it in trans with a replicator or a TSP-deleted TSP deletion construct.

[0050]

[0097] Engineered or recombinant TSPs are described in this disclosure and are included within the embodiments of the present invention. The amino acid sequences of engineered or recombinant TSPs according to embodiments of the present invention, as well as nucleotide sequences encoding such amino acid sequences, are included within the embodiments of the present invention. As discussed throughout this disclosure, TSPs mediate recognition and adhesion between phage and the surface of their host. Phage TSPs are a subtype of phage receptor-binding protein (RBP). The TSP designation is reserved for RBPs that often possess enzymatic and active penetrating "spike"-like attributes, as opposed to the phage "tail fiber," which can refer to RBPs that only facilitate binding. In other words, TSPs can be described as phage RBPs that facilitate the binding and degradation of target host receptors. TSPs are often characterized by distinct regions or domains: an N-terminal ("head") region or domain that serves to bind to the phage baseplate or other TSPs, and a C-terminal ("tail") region or domain that serves to bind to and degrade target host receptors. In naturally occurring phages, such as Ackermannviridae phages, the amino acid sequences of the N-terminal region are highly conserved, but the C-terminal region amino acid sequences are variable. The C-terminal region of phage TSPs has the same or similar C-terminal amino acid sequences appearing in the TSPs of multiple Ackermannviridae and even Autographiviridae phages, demonstrating horizontal gene transfer. For example, phages SPTD1, Det7, and P22 share the same or very similar TSP C-terminal amino acid sequences. In another example, phage STML-13-1 shares the C-terminal amino acid sequence of TSP1 with phage SPTD1, the C-terminal amino acid sequence of TSP2 with CBA120, the C-terminal amino acid sequence of TSP3 with Salmonella phage Matapan, and the C-terminal amino acid sequence of TSP4 with Citrobacter phage Sajours1.Many naturally occurring TSPs are incorrectly labeled as tail fiber proteins (or tail fibers) in existing literature; however, TSPs have distinct morphologies that differ from those of other RBPs, such as tail fiber proteins. For example, in electron microscopy (EM) images of phage, TSPs appear as clusters on the baseplate of phage virions (see, e.g., Figure 5), whereas tail fiber proteins appear as fibers. Each TSP is encoded by a single gene and conveys specificity to a particular host. TSPs in phage virions form "TSP complexes" that combine the activities of multiple TSP proteins. For example, in the Ackermannviridae family, TSP complexes confer multiple receptor-binding properties to phage virions (different from the typical tail fiber proteins, which are typically identical in phage virions).

[0051]

[0098] An engineered or recombinant TSP according to the present invention is a protein that can be incorporated into a tailed phage virion during virion assembly and contains a C-terminal binding domain responsible for phage host recognition. The term "responsible for recognition" encompasses the ability of the engineered recombinant TSP's C-terminal region or domain to recognize the target host, the degree to which the engineered or recombinant TSP's C-terminal region domain recognizes the target host (which may be characterized by binding affinity or specificity), and the lack of ability of the engineered recombinant TSP's C-terminal region domain to recognize a particular host. Embodiments of engineered or recombinant TSPs according to the present invention may be characterized by their three-dimensional structure. For example, in some embodiments, an engineered or recombinant TSP may be described as a homotrimeric protein containing at least two distinct regions or domains: an N-terminal region domain capable of binding to the phage tail (in some embodiments, the baseplate of the phage tail) and / or other TSPs to form a TSP complex, and a C-terminal region or domain responsible for phage host recognition. The N-terminal region or domain and the C-terminal region domain may be connected by a "neck" or "hinge" region or domain. The N-terminal region or domain of an engineered or recombinant TSP according to embodiments of the invention may comprise or consist of about 100 to about 600 amino acids and may comprise or consist of two to six subregions or subdomains variously involved in binding to phage baseplates or other TSPs. The C-terminal region or domain of an engineered or recombinant TSP according to embodiments of the invention may comprise or consist of about 400 to about 1200 amino acids, optionally including at least one possible catalytic subregion or subdomain and at least one receptor-binding subregion or subdomain. The "neck" or "hinge" domain or region of an engineered or recombinant TSP according to some embodiments of the invention may comprise or consist of about 5 to about 20 amino acids forming at least one alpha helix and may include a D3' domain as described in Chao et al., 2022.The N-terminal domain, the C-terminal domain, or both of an engineered or recombinant TSP can be derived from the same Ackermannviridae phage or different Ackermannviridae phages. For example, the N-terminal and C-terminal domains of an engineered or recombinant TSP can both be derived from the same Ackermannviridae phage, with at least the C-terminal domain comprising one or more amino acid sequences modified from the amino acid sequence of the C-terminal domain of an Ackermannviridae phage TSP found in nature. In another example, the N-terminal and C-terminal domains of an engineered or recombinant TSP each have or are composed of amino acid sequences that are naturally occurring Ackermannviridae phage amino acid sequences, but the combination of the amino acid sequences of the C-terminal and N-terminal domains does not occur in nature because they are derived from two different Ackermannviridae phages. In another example, the N-terminal domain of an engineered or recombinant TSP is, or comprises or consists of, a naturally occurring Ackermannviridae phage amino acid sequence or an amino acid sequence derived (by artificial modification) from such a sequence, while the C-terminal domain comprises a sequence derived from a phage classified as a different family of tailed phage (i.e., a non-Ackermannviridae phage), some of which are discussed elsewhere in this disclosure. In yet another example, the C-terminal domain of an engineered or recombinant TSP is, or comprises an amino acid sequence derived (by artificial modification) from a naturally occurring Ackermannviridae phage amino acid sequence or an N-terminal domain comprises a sequence derived from a phage classified as a different family of tailed phage (i.e., a non-Ackermannviridae phage), some of which are discussed elsewhere in this disclosure.For example, the TSP from the podovirus phiAB6 of the Autographiviridae family is homologous to the TSP from the myovirus Acinetobacter SH-Ab15599 of the Ackermannviridae family, demonstrating horizontal gene transfer. Therefore, the amino acid sequence of a TSP from a phage related to phiAB6 can be engineered to be in the Ackermannviridae family. In another example, the amino acid sequence of a TSP from the podovirus TR2 can be engineered to be in the Ackermannviridae family. The above examples are not intended to be limiting.

[0052]

[0099] With reference to exemplary sequences of several naturally occurring TSPs of Ackermannviridae phages, the N-terminal domain is located at or includes approximately amino acids 1-165 of the TSP1 amino acid sequence of CBA120 (SEQ ID NO:15), amino acids 1-257 of the TSP2 amino acid sequence of CBA120 (SEQ ID NO:9), amino acids 1-167 of the TSP3 amino acid sequence of CBA120 (SEQ ID NO:11), amino acids 1-479 of the TSP4 amino acid sequence of CBA120 (SEQ ID NO:13), amino acids 1-162 of the TSP1 amino acid sequence of SPTD1 (SEQ ID NO:2), amino acids 1-257 of the TSP2 amino acid sequence of SPTD1 (SEQ ID NO:3), amino acids 1-167 of the TSP3 amino acid sequence of SPTD1 (SEQ ID NO:4), amino acids 1-479 of the TSP4 amino acid sequence of SPTD1 (SEQ ID NO:5), or amino acids 1-251 of the Det7 TSP2 sequence (SEQ ID NO:16). With reference to exemplary sequences of naturally occurring TSPs of Ackermannviridae phages, the C-terminal domain may be approximately amino acids 166 to 770 of the TSP1 amino acid sequence of CBA120 (SEQ ID NO: 15), amino acids 258 to 921 of the TSP2 amino acid sequence of CBA120 (SEQ ID NO: 9), amino acids 168 to 627 of the TSP3 amino acid sequence of CBA120 (SEQ ID NO: 11), amino acids 490 to 1036 of the TSP4 amino acid sequence of CBA120 (SEQ ID NO: 13), amino acids 163 to 615 of the TSP1 amino acid sequence of SPTD1 (SEQ ID NO: 2), amino acids 257 to 724 of the TSP2 amino acid sequence of SPTD1 (SEQ ID NO: 3), amino acids 168 to 708 of the TSP3 amino acid sequence of SPTD1 (SEQ ID NO: 4), amino acids 490 to 1013 of the TSP4 amino acid sequence of SPTD1 (SEQ ID NO: 5), or Det7. Located at or including amino acids 261 to 791 of the TSP2 sequence (SEQ ID NO: 16).With reference to exemplary sequences of naturally occurring TSPs of Ackermannviridae phages, the "neck" or "hinge" domain is approximately amino acids 155-165 of the TSP1 amino acid sequence of CBA120 (SEQ ID NO: 15), amino acids 245-257 of the TSP2 amino acid sequence of CBA120 (SEQ ID NO: 9), amino acids 155-168 of the TSP3 amino acid sequence of CBA120 (SEQ ID NO: 11), and amino acids 245-257 of the TSP4 amino acid sequence of CBA120 (SEQ ID NO: 12). It is located at or contains amino acids 480 to 488 or 480 to 490 of the TSP4 amino acid sequence (SEQ ID NO: 13), amino acids 152 to 162 of the TSP1 amino acid sequence of SPTD1 (SEQ ID NO: 2) based on structural prediction, amino acids 247 to 257 of the TSP2 amino acid sequence of SPTD1 (SEQ ID NO: 3), amino acids 155 to 168 of the TSP3 amino acid sequence of SPTD1 (SEQ ID NO: 4), amino acids 480 to 490 of the TSP4 amino acid sequence of SPTD1 (SEQ ID NO: 5), or amino acids 252 to 260 of the Det7 TSP2 sequence (SEQ ID NO: 16). For exemplary sequences of several naturally occurring TSPs of non-Ackermannviridae phages, the N-terminal domain is located at or includes approximately amino acids 1-362 of the TSP amino acid sequence of TR2 (SEQ ID NO:7), the C-terminal domain is located at or includes approximately amino acids 376-875 of the TSP amino acid sequence of TR2 (SEQ ID NO:7), and the "neck" or "hinge" domain is located at or includes approximately amino acids 363-375 of the TSP amino acid sequence of TR2 (SEQ ID NO:7).

[0053]

[0100] Some examples of amino acid sequences of recombinant or engineered TSPs according to embodiments of the invention include the amino acid sequence of the N-terminal domain of TSP1 of SPTD1 (SEQ ID NO: 1), an amino acid sequence or variant of said amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity to said amino acid sequence; the amino acid sequence of the N-terminal domain of TSP2 of SPTD1 (SEQ ID NO: 3), an amino acid sequence or variant of said amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity to said amino acid sequence; a variant of the above amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity to the above amino acid sequence; the amino acid sequence of the N-terminal domain of TSP3 of SPTD1 (SEQ ID NO: 4); a variant of the above amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity to the above amino acid sequence; the amino acid sequence of the N-terminal domain of TSP4 of SPTD1 (SEQ ID NO: 5); a variant of the above amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity to the amino acid sequence of the N-terminal domain of TSP1 of CBA120 (SEQ ID NO: 15); a variant of the above amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity to the amino acid sequence of the N-terminal domain of TSP2 of CBA120 (SEQ ID NO: 16); an amino acid sequence (SEQ ID NO: 9); a variant of the amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity to the amino acid sequence; an amino acid sequence of the N-terminal domain of TSP3 of CBA120 (SEQ ID NO: 11); a variant of the amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity to the amino acid sequence;the amino acid sequence of the N-terminal domain of TSP4 of CBA120 (SEQ ID NO: 13), a variant of the above amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity to the above amino acid sequence; the C-terminal domain of TSP1 of SPTD1 (SEQ ID NO: 1), an amino acid sequence or a variant of the above amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity to the above amino acid sequence; a variant of the amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity to the amino acid sequence of the C-terminal domain of TSP2 of SPTD1 (SEQ ID NO: 3); a variant of the amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity to the amino acid sequence of the C-terminal domain of TSP3 of SPTD1 (SEQ ID NO: 4); or variants of the above amino acid sequences having at least 99% sequence similarity; the amino acid sequence of the C-terminal domain of TSP4 of SPTD1 (SEQ ID NO: 5); variants of the above amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity to the above amino acid sequence; the amino acid sequence of the C-terminal domain of TSP1 of CBA120 (SEQ ID NO: 15); variants of the above amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85% sequence similarity to the above amino acid sequence; Variants of the above amino acid sequences having at least 90%, at least 95%, or at least 99% sequence similarity, the amino acid sequence of the C-terminal domain of TSP2 of CBA120 (SEQ ID NO: 9), variants of the above amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity with the above amino acid sequence, the amino acid sequence of the C-terminal domain of TSP3 of CBA120 (SEQ ID NO: 11), variants of the above amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity with the above amino acid sequence,a variant of the above amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity to the above amino acid sequence; the amino acid sequence of the C-terminal domain of TSP4 of CBA120 (SEQ ID NO: 13); a variant of the above amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity to the above amino acid sequence; the amino acid sequence of the C-terminal domain of Det7 (SEQ ID NO: 16); Examples of amino acid sequences of recombinant or engineered TSPs according to embodiments of the invention include amino acid sequences that share at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity with the amino acid sequence of the C-terminal domain of TSP of TR2 (SEQ ID NO: 17), or variants of the above amino acid sequences that share at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity with the amino acid sequence of SEQ ID NO: 6, 7, 8, 10, 12, 14, or 18. Examples of amino acid sequences of recombinant or engineered TSPs according to embodiments of the invention include amino acid sequences that share at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity with SEQ ID NO: 6, 7, 8, 10, 12, 14, or 18. Examples of amino acid sequences of recombinant or engineered TSPs according to embodiments of the invention include or consist of SEQ ID NO: 6, 7, 8, 10, 12, 14, or 18. Recombinant TSPs or nucleic acid molecules encoding TSPs according to the present disclosure are included within embodiments of the present invention. Such nucleic acid molecules may be contained in vectors, such as bacteriophages, plasmids, phagemids, viruses, and other suitable vectors.

[0054]

[0101] In some examples, synthetic phages according to the present disclosure are lytic. Lytic phages take over the cell's machinery and make phage components. They then lyse the cell, releasing new phage particles. Synthetic phages according to embodiments of the present invention can be constructed from natural or recombinant (genetically modified, synthetic, or engineered) phages, referred to as "parent" phages. In some examples, such parent phages can be naturally occurring Ackermannviridae phages or genetically modified Ackermannviridae phages. Some non-limiting examples of natural Ackermannviridae phages that can serve as parent phages for the synthetic phages described in this disclosure include CBA120, SPTD1, STML-13-1, KOPDS1, Salmonella phage Det7, Salmonella phage Chennai, Acinetobacter phage SH-Ab-15599, Salmonella phage Di Examples of engineered TSPs include Dinky, Escherichia phage PhaxI, Citrobacter phage Sajous1, Escherichia phage ECML-4, Escherichia phage Matapan, Salmonella phage Maynard, Salmonella phage ST-W77, and Salmonella phage SKML-39. Synthetic phage can have a different number of TSPs than its parent phage. Synthetic phage can contain one engineered or recombinant TSP or multiple (at least two, three, or four) engineered or recombinant TSPs according to the present disclosure. In some examples, the synthetic phage contains one engineered or recombinant TSP that recognizes a target host not recognized by other TSPs of the synthetic phage.In some examples, a synthetic phage contains two engineered or recombinant TSPs that recognize two different target hosts that are not recognized by other TSPs on the synthetic phage. In some examples, a synthetic phage contains three engineered or recombinant TSPs that recognize three different target hosts. In some examples, a synthetic phage contains four engineered or recombinant TSPs that recognize four different target hosts. In some examples, a synthetic phage contains two or more recombinant TSPs that recognize the same target host. Nucleic acid sequences encoding synthetic phages according to the present disclosure are included in embodiments of the present invention.

[0055]

[0102] Synthetic phages according to the present disclosure may include a reporter gene or indicator gene. In certain embodiments, the indicator gene does not encode a fusion protein. For example, in certain embodiments, expression of the indicator gene after infection of a host microorganism, such as a bacterium, results in a soluble indicator protein product. In certain embodiments, the indicator gene may be inserted into the late gene region of the synthetic phage, i.e., a region of the viral genome that is transcribed late in the viral life cycle. Late gene regions typically contain the most abundantly expressed genes (e.g., structural proteins assembled into bacteriophage particles). Phage late genes may be referred to as "class III genes" and include genes with structural and assembly functions. The indicator gene may be inserted into the phage genome so that it is under the control of a phage promoter. The indicator gene may be inserted to replace at least a portion of the sequence of a late phage gene. Inclusion of stop codons in all three reading frames of the indicator gene may help increase expression by reducing read-through (also known as leaky expression). This strategy also avoids the possibility of low-level production of the fusion protein, which would appear as a background signal that cannot be separated from the phage. Thus, in some embodiments, the indicator gene is not part of the fusion protein. That is, in some embodiments, the genetic modification can be configured so that the indicator protein product does not contain the phage polypeptide. In some embodiments, the non-native indicator gene is under the control of a late promoter. Use of a viral late gene promoter ensures that the reporter gene (e.g., luciferase) is not only expressed at high levels like the viral capsid protein, but also does not shut down as similar endogenous bacterial or early bacteriophage genes. In some embodiments, the late promoter is a T4, T7, or ViI-like promoter, or another phage promoter similar to those found in naturally occurring phages.

[0056]

[0103] The indicator gene may encode a variety of biomolecules or may itself be a detectable biomolecule. For example, the indicator gene may encode a detectable polypeptide or protein. In another example, the indicator gene may be a gene that expresses a detectable product or an enzyme that produces a detectable product. In another example, the indicator gene may encode or include a detectable nucleic acid. For example, the indicator gene may encode a detectable aptamer such as RNA Mango, or the indicator gene may include a nucleic acid sequence that is detectable by real-time polymerase chain reaction (RT-PCR). In some embodiments, the product of the indicator gene may be a detectable enzyme. The indicator gene product may produce light and / or be detectable by a color change. A variety of suitable enzymes are commercially available, such as alkaline phosphatase (AP), horseradish peroxidase (HRP), or luciferase (Luc). In some embodiments, these enzymes may serve as indicator moieties. For example, in some embodiments, the indicator gene encodes a luciferase enzyme. Various types of luciferases can be used. The luciferase can be one of Oplophorus luciferase, firefly luciferase, Lucia luciferase, Renilla luciferase, or an engineered luciferase. In some embodiments, firefly luciferase is the indicator moiety. In some embodiments, the luciferase gene is derived from Oplophorus. Other engineered luciferases or other enzymes that generate a detectable signal can also be suitable indicator moieties.

[0057]

[0104] The choice of indicator gene to include in a synthetic phage can be guided by various considerations. For example, most phages can package DNA several percent larger than their native genome. Due to this consideration, a smaller indicator gene may be a more appropriate choice for engineering bacteriophage, especially those with smaller genomes. OpLuc and NANOLUC® (Promega Corporation, Madison, Wisconsin) proteins are only approximately 20 kDa (encoding approximately 500-600 bp), while FLuc is approximately 62 kDa (encoding approximately 1,700 bp). For comparison, the T7 genome is approximately 40 kbp, while the T4 genome is approximately 170 kbp. Furthermore, the reporter gene should not be endogenously expressed by the synthetic phage's host(s), generate a high signal-to-background ratio, and be easily detectable in a timely manner. NANOLUC® is a modified Oplophorus gracilirostris (deep-sea shrimp) luciferase. In some embodiments, NANOLUC® in combination with NANO-GLO® (Promega Corporation, Madison, Wisconsin), an imidazopyrazinone substrate (furimazine), can provide a robust signal with low background. In some embodiments, two or more indicator genes can be inserted into the synthetic phage. For example, two or more copies (e.g., two copies) of the same indicator gene can be inserted, which can improve the signal strength and / or signal-to-noise ratio of assays using the synthetic phage. In another example, different indicator genes, such as two different indicator genes, can be inserted, which can enable bimodal signal detection. For example, the NANOLUC® gene can be inserted together with a gene encoding green fluorescent protein (GFP), or the NANOLUC® gene can be inserted together with a gene encoding a different luciferase, such as firefly luciferase.

[0058]

[0105] Recombinant TSPs according to the present disclosure can include a reporter or indicator moiety, such as a detectable polypeptide or protein. The detectable moiety can be an enzyme that produces a detectable product, such as, but not limited to, alkaline phosphatase (AP), horseradish peroxidase (HRP), or luciferase (Luc). The detectable product can produce light and / or be detectable by a color change.

[0059]

[0106] Compositions comprising synthetic phage or recombinant TSPs according to the present disclosure may contain one or more types of synthetic phage and / or recombinant TSPs. A composition comprising two or more types of synthetic phage or recombinant TSPs (e.g., two, three, four, five, six, etc.) may be referred to as a "cocktail." In some embodiments, a composition may contain a cocktail of different TSPs specific to different hosts of interest. In some embodiments, a composition contains a cocktail of different synthetic phages with recombinant TSPs specific to different hosts of interest. Such cocktails can be used for simultaneous recognition of multiple microorganisms of interest, e.g., to simultaneously detect the presence or absence of each of the microorganisms of interest in a sample. In another example, a cocktail of synthetic phages can be administered to a subject for therapeutic purposes to treat potential infections caused by multiple microorganisms. In some instances, the nature of the subject's infection may be unknown, making it beneficial to administer a broad-spectrum synthetic phage cocktail. In some instances, it may be beneficial to administer a broad-spectrum synthetic phage cocktail prophylactically.

[0060]

[0107] The synthetic phage and recombinant TSPs of the present disclosure can recognize a variety of microorganisms, including, but not limited to, bacteria. As used herein, the term "bacteria" encompasses all variants of bacteria, including non-pathogenic and pathogenic bacteria. Examples of bacteria that may be recognized by the synthetic phage and / or recombinant TSPs according to the present disclosure include, but are not limited to, Yersinia spp., Escherichia spp., Klebsiella spp., Bordetella spp., Neisseria spp., Aeromonas spp., Francisella spp., Corynebacterium spp., Citrobacter spp., Chlamydia spp., Hemophilus spp., Brucella spp., Mycobacterium spp., Legionella spp., and the like. spp.), Rhodococcus spp., Pseudomonas spp., Helicobacter spp., Salmonella spp., Vibrio spp., Bacillus spp., Erysipelothrix spp., Streptomyces spp., Bacteroides spp., Prevotella spp., Clostridium spp., Bifidobacterium spp., Lactobacillus spp., Pseudomonas spp., Cronobacter spp., Shigella spp., Campylobacter spp. In some embodiments, the bacterial cell isBacteroides thetaiotaomicron, Bacteroides fragilis, Bacteroides distasonis, Bacteroides vulgatus, Clostridium leptum, Clostridium coccoides, Staphylococcus aureus, Bacillus subtilis, Clostridium butyricum, Brevibacterium lactofermentum, Streptococcus agalactiae, Lactococcus lactis lactis, Leuconostoc lactis, Actinobacillus actinomycetemcomitans, Cyanobacteria, Escherichia coli, Helicobacter pylori, Selenomonas ruminantium, Shigella sonnei, Zymomonas mobilis, Mycoplasma mycoides, Treponema denticola, Bacillus thuringiensis, Staphylococcus lugdunensis lugdunensis, Leuconostoc oenos, Corynebacterium xerosis, Lactobacillus plantarum,Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus acidophilus, Streptococcus Enterococcus faecalis, Bacillus coagulans, Bacillus cereus, Bacillus popillae, Synechocystis strain PCC6803, Bacillus liquefaciens, Pyrococcus abyssi, Selenomonas nominantium nominantium, Lactobacillus hilgardii, Streptococcus ferus, Lactobacillus pentosus, Bacteroides fragilis, Staphylococcus epidermidis, Zymomonas mobilis, Streptomyces phaechromogenes, or Streptomyces ghanaensis. In some embodiments, synthetic phages according to the present disclosure can recognize the following non-limiting examples of bacteria: Actinobacteria, Aquificae, Armatimonadetes, Bacteroidetes, Caldiserica, Chlamydiae, Chloroflexi, Chrysiogenetes, Cyanobacteria,Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Elusimicrobia, Fibrobacteres, Firmicutes, Bacillus, Listeria, Staphylococcus, Fusobacteria, Gemmatimo Gemmatimonadetes, Nitrospirae, Planctomycetes, Proteobacteria, Acidobacillus, Aeromonas, Burkholderia, Neisseria, Shewanella, Citrobacter, Enterobacter, Erwinia, Escherichia, Klebsiella, Kluyvera, Morganella, Salmonella, Shigella, Yersinia, Coxiella, Rickettsia, Legionella, Avibacterium, Haemophilus, Pasteurella Pasteurella, Acinetobacter, Moraxella, Pseudomonas, Vibrio, Xanthomonas, Spirochaetes, Synergistets, Tenericutes, (e.g., Mycoplasma, Spiroplasma, Ureaplasma,Thermodesulfobacteria and Thermotogae. Synthetic phage and recombinant TSPs according to the present disclosure can also act as archaea (viruses that infect archaea, such as viruses of the Thaliviridae family). Thus, in some embodiments, a synthetic phage or recombinant TSP according to the present disclosure can recognize archaea.

[0061]

[0108] In some embodiments, synthetic phages or recombinant TSPs are designed to optimize desired traits for use in various applications, such as methods for detecting and / or controlling microorganisms, some of which are described elsewhere in this disclosure. In some embodiments, bioinformatics and prior analysis of genetic modifications are used to optimize desired traits. For example, in some embodiments, the gene encoding the TSP can be optimized to recognize and bind to microorganisms of a specific taxonomic group, or to not recognize and bind to microorganisms of a specific taxonomic group. In other embodiments, the gene encoding the TSP can be optimized to recognize and bind to entire categories of microorganisms or specific groups within a category. In this manner, recombinant TSPs and synthetic phages containing them can be optimized to detect broader or narrower groups of microorganisms.

[0062]

[0109] In some exemplary embodiments, the Ackermannviridae phage CBA120 is modified to generate a parent phage for producing synthetic phage by generating an engineered CBA120-based phage that can infect only Escherichia coli. Such a CBA120-based phage can be engineered by including one native TSP (TSP2) from CBA120 in the CBA120-based phage. This allows infection and propagation of the CBA120-based parent phage in Escherichia coli. Synthetic phage can be constructed from such a CBA120-based parent phage, also using E. coli as a phage host. One or more of the remaining three TSPs (TSP1, TSP3, and TSP4) of the parent phage can be replaced with one or more recombinant TSPs capable of recognizing one or more highly pathogenic hosts of interest (e.g., B. pseudomallei or Y. pestis) without the need for laboratory work with such hosts. In some exemplary embodiments, synthetic phages with higher specificity for a target bacterial host are constructed by replacing the off-target TSP with a second, third, or fourth copy of the on-target TSP, e.g., by replacing the C-terminal regions of TSP1, TSP3, and TSP4 of CBA120 with the O157:H7-specific TSP2 C-terminal region. The presence of four customizable TSPs allows for the generation of phage cocktails with reduced complexity.

[0063]

[0110] Methods for constructing synthetic phages and recombinant tailspike proteins Methods for constructing synthetic phages and recombinant TSPs according to the present disclosure are included within the embodiments of the present invention. An embodiment of a method for constructing a synthetic phage can begin with the selection of a parent phage for constructing the synthetic phage. The parent phage can be a wild-type phage found in any environment or an engineered phage. In some embodiments, it may be preferable to use a parent phage isolated from the environment to produce a synthetic phage so that the parent phage can recognize naturally occurring microorganisms. Any suitable method can be used to isolate the parent phage from the environment. In some embodiments, the parent phage can be specific to at least some desired target hosts and can be further modified according to the methods of the present disclosure to customize its host range. In some examples, the parent phage can be modified to recognize additional hosts, i.e., to modify and expand the host range of the parent phage, thereby resulting in a synthetic phage with a broader host range than the parent phage. In some examples, the parent phage can be modified to recognize fewer hosts, i.e., to modify and narrow the host range of the parent phage, thereby resulting in a synthetic phage with a narrower host range than the parent phage. In some examples, a parent phage can be modified to recognize a set of target hosts that differs from some or all of the hosts of the parent phage, thereby resulting in a synthetic phage with a modified or customized host range. The above examples are non-limiting, and other examples are contemplated (e.g., broadening the host range of the parent phage for one host category and narrowing the host range of the parent phage for another host category). Methods for modifying phage host range are included among embodiments of the present invention. The use of phage in various applications, such as microbial detection or control (some related methods are described elsewhere in this disclosure), is influenced by the phage host range. In many applications, it is desirable to use phage with high specificity and minimal off-target recognition. Synthetic phage according to the present disclosure can be used to enhance the specificity of the parent phage and / or reduce off-target recognition.

[0064]

[0111] Some embodiments of methods for constructing synthetic phage or modifying phage host range according to embodiments of the present invention include incorporating at least one (e.g., 1, 2, 3, 4) recombinant TSP according to the present disclosure into a parent phage. In some embodiments, the incorporation of at least one recombinant TSP is achieved by modifying only the C-terminal region or domain of an existing TSP of the parent phage (rather than incorporating the entire gene for the recombinant TSP into the synthetic phage genome). Modifying only the C-terminal region or domain of an existing TSP of the parent phage (e.g., by exchanging it with the C-terminal region or domain of a TSP from a different phage, thus creating a chimeric TSP as discussed elsewhere in this disclosure) has the advantage of likely preserving the structure of the TSP complex (because the N-terminal TSP region, which is responsible for assembly of the TSP complex, remains unmodified). Also, modifying only the C-terminal region of an existing TSP of the parent phage reduces the time and cost of the genetic engineering involved. Furthermore, due to the presence of multiple homologous sequences in the TSPN terminal region, HRs incorporating only the C-terminal region into phage TSP are less error-prone than HRs incorporating the entire recombinant TSP gene.

[0065]

[0112] For example, a method for modifying phage host range can include modifying at least one tail spike protein (TSP) of a parent phage and thereby generating a synthetic phage with an altered host range. In some embodiments, a method for modifying phage host range can include incorporating at least one recombinant TSP described elsewhere in this disclosure into a parent phage using applicable molecular biology techniques, some of which are discussed herein. It should be understood that the construction of synthetic phage or modification of phage host range is not limited by the techniques described in this disclosure and can employ any applicable technique from the fields of molecular biology, virology, biotechnology, and related fields. For example, phage with an altered host range can be constructed de novo based on genome fragments or sequences derived from one or more parent phages. The engineered genome or synthetic phage genome can be "rebooted" to generate viable phage virions. The source can be assembled DNA fragments, cosmid inserts, or yeast artificial chromosomes. A fully functional synthetic phage constructed de novo may require the ability to control a host to produce the phage, an origin of replication and associated replication functions, a complete set of genes enabling capsid assembly, a complete set of genes enabling tail assembly, a TSP, and packaging functions. In some embodiments, the host range modified synthetic phage is capable of recognizing at least one host different from the host recognized by the parent phage. In some embodiments, the host range modified synthetic phage is incapable of recognizing at least one host recognized by the parent phage. In some embodiments, the host range modified synthetic phage is capable of recognizing at least one host different from the host recognized by the parent phage and is incapable of recognizing at least one host recognized by the parent phage.

[0066]

[0113] Some embodiments of a method for constructing a synthetic phage according to the present disclosure may include selecting a parent phage capable of infecting a microorganism such as a bacterium, preparing a homologous recombination plasmid / vector containing a nucleic acid sequence encoding a recombinant TSP according to the present disclosure, transforming the homologous recombination plasmid / vector into a microorganism, infecting the transformed target pathogenic bacterium with the parent phage to cause homologous recombination between the plasmid / vector and the phage genome, and isolating a synthetic phage clone containing the recombinant TSP. Various methods can be used to design and prepare the homologous recombination plasmid. Various methods can be used to transform a microorganism with the plasmid, such as heat shock, F pilus-mediated bacterial conjugation, electroporation, and other methods. Various methods can be used to isolate specific clones after homologous recombination.

[0067]

[0114] Some embodiments of the method for constructing a synthetic phage may include determining the nucleic acid sequence within a region of the genome of a parental phage that encodes a TSP, annotating the genome to identify at least one TSP gene in the parental phage, and designing sequences for homologous recombination adjacent to the identified TSP gene. Some embodiments of the method for constructing a synthetic phage may include incorporating the sequences designed for homologous recombination into a plasmid / vector. Some embodiments of the method for constructing a synthetic phage may include transforming the plasmid / vector into a microorganism and then selecting a transformed microorganism, such as a bacterium. After infecting the transformed microorganism with the parental phage and allowing homologous recombination to occur between the plasmid and the phage genome, some embodiments of the method for constructing a synthetic phage may include determining the titer of a synthetic bacteriophage lysate. Some embodiments of the method for constructing a synthetic phage may include performing a limiting dilution assay to concentrate and isolate the synthetic phage. Some embodiments may include repeating the limiting dilution and titering steps, as necessary, after the first limiting dilution assay until the synthetic bacteriophage represent a detectable fraction of the mixture. Large-scale production can be carried out to obtain high-titer stocks of synthetic phage. Phage particles can be separated using a variety of methods, including but not limited to, cesium chloride isopycnic gradient centrifugation.

[0068]

[0115] Methods for constructing recombinant TSPs described in this disclosure are included within the embodiments of the present invention. Any suitable molecular biology technique or techniques can be used to generate the nucleic acid sequence of a recombinant TSP. Such nucleic acid sequences can be derived from naturally occurring TSPs or artificially constructed TSPs. Naturally occurring TSP sequences can be based on (or isolated from and synthesized based on) TSP sequences of naturally occurring phages in various environments. In some embodiments, it can be advantageous to use TSP sequences from naturally occurring phages in various environments to generate recombinant TSPs capable of specifically binding to naturally occurring microorganisms. Any suitable method can be used to generate TSP nucleic acid sequences based on phages from the environment. For example, TSP nucleic acid sequences can be generated from the isolated genome of a naturally occurring phage. The isolated phage nucleic acid can be used for further manipulation. If the phage genome sequence is unknown, various sequencing methods can be used. For example, next-generation sequencing techniques can be used to generate large amounts of data (contigs) that can be used to assemble contiguous phage sequence pieces, while PCR-based techniques can be used to fill in the gaps. Primers designed to anneal to the ends of contigs can be used on the phage nucleic acid, and the resulting PCR products can be sequenced by conventional Sanger sequencing to close gaps between contigs. Modified Sanger sequencing can also be used to sequence the phage nucleic acid.

[0069]

[0116] The specificity of each TSP can be experimentally determined by comparing the infectivity of a panel of bacteria between the TSP swap recombinant and its parent phage. For example, performing plaque assays on multiple bacterial strains carrying SPTD1 using chimeric TSPs derived from the CBA120 TSP may result in successful infection of additional strains that the parent SPTD1 phage was previously unable to infect. Thus, the original CBA120 TSP can be determined to be specific to these new strains. Sequence homology to TSPs with known targets may also indicate likely bacterial targets. For example, Salmonella phage STML-13-1 TSP2 shares sequence homology with CBA120 TSP2, which is known to target Escherichia coli (E. coli) O157:H7. Therefore, it was hypothesized that STML-13-1 could infect E. coli (E. coli) O157:H7, which was subsequently confirmed experimentally by plaque assay. Sequence homology between various TSPs can be determined using alignment software such as Clustal Omega. A combination of these and other techniques can be used to determine the specificity of a particular TSP. For example, if a phage is known to infect a particular host, and three of the four TSPs on the phage are determined experimentally and / or through homology with known TSP targets to not infect that host, it can be concluded that the fourth TSP on the phage is responsible for host targeting.

[0070]

[0117] TSP nucleic acid sequences from naturally occurring phages can be analyzed to identify nucleic acid sequences encoding the C-terminal domain of a TSP with the desired host recognition ability. The nucleic acid sequence encoding the C-terminal domain of a TSP (the "donor phage" sequence) can then be combined ("spliced") with a nucleic acid sequence encoding the N-terminal domain sequence of a TSP from a parent phage, thereby generating a nucleic acid sequence encoding a recombinant TSP. The junction (or "splice") site between the sequences can be selected based on the analyzed amino acid sequences of the two proteins, the TSP from the donor phage and the TSP from the parent phage. For example, the two sequences can be joined at the "neck" or "hinge" regions of both TSPs. The location of the "neck" or "hinge" regions on the amino acid sequence can be determined by several methods. One such method is alignment of the amino acid sequence to well-characterized TSPs using Needle Pairwise Alignment, Clustal, or BLAST. Because the "neck" or "hinge" region is an alpha helix, it is possible to sequence the amino acids using a secondary structure prediction tool such as PHYRE2 and compare it to known or typical N-terminal TSP regions to align domains and secondary structures such as alpha helices and beta sheets. Structural matching tools such as Swiss-Model can also be used to find homologous proteins with known three-dimensional structures, often allowing the neck or adjacent domains to be visually determined. Artificial intelligence-based fold prediction software such as DeepMind's AlphaFold2 or its derivatives can also be used to determine the three-dimensional structure of TSPs using either the entire amino acid sequence or a subset of the sequence to minimize computational effort. Figures 22 and 36 are examples of three-dimensional protein structures generated using AlphaFold2. Three-dimensional structures can also be determined experimentally using established structural biology techniques such as X-ray crystallography, nuclear magnetic resonance (NMR) structure determination, or cryo-EM (electron microscopy).Domains are typically spliced ​​between the N-terminal region and the "neck" from the donor TSP, but splice sites can also be created between other domains to preserve binding to other TSPs or to preserve predicted interactions with the donor TSP. For example, two constructs were designed to replace TSP2 in a phage of interest with TSP2 from Salmonella phage Det7. One construct had a splice at the "neck," while the other had a splice between the XD3 and TD1 domains in the N-terminal region, replacing the TD1 domain with the C-terminal region. Structural prediction software showed that this construct had a conformation closer to that of native Det7 TSP2. Nucleic acid molecules with sequences encoding recombinant TSPs can be synthesized in the laboratory.

[0071]

[0118] Methods for constructing synthetic PAGEs and recombinant TSPs according to the present disclosure can include preparing various types of plasmids, including, but not limited to, preparing plasmids for homologous recombination. Various methods and commercial products for preparing plasmids can be used. For example, plasmids can be prepared using a combination of PCR, site-directed mutagenesis, restriction digestion, ligation, cloning, and other techniques. Synthetic plasmids can also be ordered commercially (e.g., GeneWiz). Cosmids can also be used, or the CRISPR / CAS9 system can be used to selectively edit phage genomes. Some embodiments of methods for constructing synthetic PAGEs according to the present disclosure include designing a plasmid that can easily recombine with a parent bacteriophage genome to generate a recombinant genome. In designing the plasmid, some embodiments include adding a codon-optimized reporter gene, such as a luciferase gene. Some embodiments for constructing synthetic PAGEs according to the present disclosure can include adding elements to an upstream untranslated region. The untranslated region may include a promoter such as a T4, T4-like, T7, T7-like, Salmonella- or Staphylococcus-specific bacteriophage, ViI, or ViI-like promoter. The untranslated region may also include a ribosome entry / binding site (RBS), also known as the "Shine-Dalgarno sequence" with bacterial systems. Either or both of these or other untranslated elements can be embedded within a short upstream untranslated region consisting of random sequences containing approximately the same GC content as the rest of the phage genome. The random region should not contain an ATG sequence, as this will act as an initiation codon.

[0072]

[0119] As an example of how to construct a synthetic PAGE according to the present disclosure, Figures 3 and 4 schematically illustrate the use of Ackermannviridae phage as a customizable, modular platform for constructing synthetic phage for detecting Gram-negative bacteria. As shown in Figure 3, the entire genomic region encoding TSP is deleted in an Ackermannviridae phage, such as CBA120, to generate a recombinant Ackermannviridae phage lacking the ability to produce TSP. This is achieved using a homologous recombination strategy. Desired recombinants can be selected based on the expression of marker genes, such as fluorescent marker genes (e.g., luciferase), antibiotic resistance markers, colorimetric marker genes, and other appropriate markers, but must be propagated on a complementation strain that provides TSP in trans via a transformation plasmid to produce infectious progeny. Figure 4 is a schematic diagram of a complementing bacterial host that expresses TSP in trans to complement the recombinant phage deficient in TSP production, resulting in the production of viable, infectious recombinants that can then undergo a single round of infection in wild-type bacteria. However, wild-type bacteria cannot support the production of infectious progeny. Progeny phage produced during infection of wild-type bacteria lack the TSP gene in their genome and therefore lack the TSP, which is essential for binding to the bacterial host. Phage derived from bacteria harboring a complementary TSP-expressing plasmid possess the TSP on the virion but lack the TSP-encoding gene. Therefore, while the original virions produced in bacteria harboring a complementary TSP-expressing plasmid can infect wild-type bacteria, they are unable to produce the TSP due to the lack of the TSP gene, meaning that their progeny phage cannot infect bacteria. Figure 4 shows a schematic diagram of customizing the host range of Ackermannviridae phages using various complementary bacterial strains. Different plasmids encoding unique TSPs enable the assembly of recombinants with customizable host ranges.

[0073]

[0120] Methods for using synthetic phage and recombinant TSP Methods using synthetic phage and / or recombinant TSPs according to the present disclosure are included among the embodiments of the present invention. Method embodiments according to the present disclosure can be applied to the detection, including qualitative and quantitative detection (quantification), of various microorganisms in a variety of contexts, including, but not limited to, the detection of microorganisms in clinical, food, water, and commercial samples. Such methods may be referred to as "detection methods." The methods of the present disclosure provide high detection sensitivity and specificity and rapid detection. Method embodiments according to the present disclosure can be applied to methods for controlling one or more microorganisms (including, but not limited to, reducing, inhibiting, or maintaining levels, including concentration and / or amount, of microorganisms, reducing, inhibiting, or maintaining the growth and / or growth rate of microorganisms, etc.) in a variety of contexts. For example, methods according to the present disclosure include therapeutic methods comprising administering a synthetic phage to a subject to control one or more microorganisms in the subject. In another example, a method according to the present disclosure comprises contacting a subject, material, or device with a synthetic phage to control a microorganism or microorganisms on or in the subject or device. Microbial control methods

[0074]

[0121] Methods for controlling microorganisms using synthetic phages include therapeutic methods. Such methods include methods for treating infections caused by one or more microorganisms, including, but not limited to, bacterial infections. The methods involve administering one or more synthetic phages according to the present disclosure to a subject with a bacterial infection in need of treatment. It should be understood that methods for controlling microorganisms involving administering one or more synthetic phages according to the present disclosure are not limited to therapeutic methods. Such methods may also be cosmetic methods, methods for improving the well-being of a subject, and the like. In this case, the subject may not be suffering from a disease or condition caused by a microorganism. Synthetic phages according to embodiments of the present disclosure may be administered to a subject as part of a pharmaceutical composition or formulation. Examples of pharmaceutical compositions or formulations are described elsewhere in this disclosure.

[0075]

[0122] Diseases or conditions caused by microorganisms include, but are not limited to, bacterial infections, such as, but not limited to, soft diarrhea, chlamydia infection, Crohn's disease, conjunctivitis, cholecystitis, colon cancer, polyposis, dysbiosis, Lyme disease, diarrhea, diphtheria, duodenal ulcer, endocarditis, typhoid fever, fever, glomerulonephritis, gastroenteritis, gastric ulcer, Guillain-Barré syndrome, tetanus, gonorrhea, gingivitis, inflammatory bowel disease, irritable bowel syndrome, leptospirosis, leprosy, listeriosis, tuberculosis, Lady Wing syndrome, and the like. These include Myrrh syndrome, Legionnaires' disease, meningitis, mucopurulent conjunctivitis, multidrug-resistant bacterial infections, multidrug-resistant bacterial colonization, myonecrotizing gas gangrene, nontuberculous mycobacterial disease (Mycobacterium avium complex), neonatal necrotizing enterocolitis, nocardiosis, hospital-acquired infections, otitis media (or otitis media), periodontitis, pharyngitis, pneumonia, peritonitis, purpuric fever, Rocky Mountain spotted fever, dysentery, syphilis, sinusitis, sigmoiditis, sepsis, subcutaneous abscess, tularemia, bronchitis, tonsillitis, typhoid fever, ulcerative colitis, urinary tract infection, and whooping cough. The disease or condition caused by bacteria may be a skin infection, such as acne; an intestinal infection, such as esophagitis, gastritis, enteritis, colitis, sigmoiditis, proctitis, peritonitis; a urinary tract infection; a vaginal infection; an upper female reproductive tract infection, such as salpingitis, endometritis, oophoritis, metritis, parametritis, an infection of the pelvic peritoneum; a respiratory infection, such as pneumonia, an intra-amniotic cavity infection, an odontogenic infection, an endodontic infection, fibrosis, meningitis, a bloodstream infection; a hospital-acquired infection, such as a catheter-associated infection, a hospital-acquired pneumonia, a postpartum infection, a hospital-acquired gastroenteritis, a hospital-acquired urinary tract infection, or a combination thereof. In some cases, the bacterial infection is caused by antibiotic-resistant bacteria. The disease or condition caused by bacteria may also be a metabolic disorder, such as obesity and diabetes. The disease or condition caused by bacteria may also be a pathology involving bacteria from an animal microbiota, including, but not limited to, inflammatory and autoimmune diseases, cancer, infectious diseases, or brain disorders. For example, some bacteria in an animal's microbiome may secrete molecules that induce and / or enhance the development of inflammatory or autoimmune diseases or cancer without causing any infection. In another example, some bacteria in the microbiome may secrete molecules that affect the brain. Therefore, a method for regulating an animal's microbiome is included in an embodiment of the present invention.For example, synthetic phage according to the present disclosure may be administered to a subject to improve the efficacy of immunotherapies based on, for example, chimeric antigen receptor T (CAR-T) cells, tumor infiltrating lymphocytes (TILs), regulatory T cells (Tregs), also known as suppressor T cells, immune checkpoint inhibitors, including but not limited to, programmed cell death protein 1 (PD-1) inhibitors, programmed death-ligand 1 (PD-L1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

[0076]

[0123] Methods for using synthetic phages according to the present disclosure include methods for personalized treatment of subjects in need of treatment for microbial infections, including bacterial infections. An exemplary method may include obtaining a biological sample from a subject and determining the nucleic acid (e.g., DNA) sequence of a group of microorganisms (e.g., bacteria) from the sample. Based on the sequencing, one or more pathogenic microorganisms (e.g., bacteria) found in the sample can be identified, and the subject can be administered one or more synthetic phages according to the present disclosure that can recognize one or more pathogenic microorganisms identified in the sample. Methods for using synthetic phages according to the present disclosure also include methods that involve administering one or more synthetic phages to a subject to improve the effectiveness of a drug or other therapeutic agent. It is known that some microorganisms in the microbiome, such as bacteria, are not themselves pathogenic but can metabolize drugs and modify them into ineffective or harmful molecules.

[0077]

[0124] Methods of controlling microorganisms by using synthetic phages according to the present disclosure include methods of controlling microorganisms in or on various objects, devices, and materials. Such objects or devices include veterinary and medical devices (including, but not limited to, medical and veterinary instruments and artificial implants such as endoscopes) and non-medical equipment and devices such as sewers, food preparation, and storage devices. It should be understood that the devices and devices may be found in a variety of situations and environments. For example, artificial implants found inside or outside of a subject may be subjected to methods of controlling microorganisms according to embodiments of the present disclosure. Methods of controlling microorganisms by using synthetic phages according to the present disclosure also include methods of controlling microorganisms in or on various biological samples and materials such as blood, plasma, cultured cells, and transplants. It should be understood that the biological samples and materials may have natural origins, be artificially produced, or be found in a variety of situations and environments. For example, transplants found inside or outside of a subject may be subjected to methods of controlling microorganisms according to embodiments of the present disclosure. The methods of controlling microorganisms by using synthetic phages according to the present disclosure are not limited by any particular situation or environment. For example, such methods may include controlling microorganisms in water to control bacterial contamination of crops in various contexts, such as during food processing and packaging, and in the agricultural industry for bioremediation and / or water purification. Methods for controlling microorganisms in or on various objects, devices, materials (biological and non-biological), samples, etc., include contacting one or more synthetic phages with the object, device, material, or sample for a period of time sufficient for the one or more synthetic phages to infect and lyse one or more microorganisms found on the object, device, material, or sample. For example, a method may be a method for inhibiting or controlling one or more microorganisms that form biofilms, by contacting the object or device with one or more synthetic phages capable of infecting one or more microorganisms that form biofilms.In another example, the method may be a method for controlling one or more organisms in environments where the growth of one or more microorganisms is considered undesirable or harmful, such as medical and veterinary environments (including operating room facilities), or food or food preparation surfaces or areas (including those where raw meat or fish is handled or discarded). Microbial control methods may also be used to sterilize heat-sensitive objects, medical devices, and tissue implants, such as grafts. In some examples, only specific species or groups of microorganisms are undesirable or dangerous, and synthetic phages can be designed to target these groups. For example, it may be desirable to control specific strains of Escherichia coli (E. coli), such as O157:H7, while leaving other naturally occurring, harmless strains of E. coli unaffected. Thus, selective or complete control of microorganisms using methods according to the present disclosure is contemplated. Detection Method

[0078]

[0125] Some embodiments of the present invention include methods for detecting a microorganism of interest. The methods may use one or more synthetic phages and / or one or more recombinant TSPs according to the present disclosure for the detection of the microorganism of interest. The methods may include detecting the microorganism of interest in a sample by incubating the sample with one or more synthetic phages that infect the one or more microorganisms of interest. In some embodiments, the synthetic phage includes an indicator gene encoding an indicator gene product, as discussed elsewhere in this disclosure. The method may then include detecting the indicator gene product, where positive detection of the indicator gene product indicates the presence of the microorganism of interest in the sample. In some embodiments, the indicator gene product is a protein. In some embodiments, the indicator gene product is a soluble protein.

[0079]

[0126] An exemplary detection method according to one embodiment of the present invention can be a method for detecting a microorganism of interest, e.g., bacteria, in a sample, the method comprising incubating the sample with a synthetic bacteriophage that infects the microorganism of interest. The synthetic phage can include an indicator gene such that expression of the indicator gene during synthetic phage replication following infection of the microorganism of interest results in the production and detection of a soluble indicator protein product, with positive detection of the indicator protein product indicating the presence of the microorganism of interest in the sample. In some embodiments, the amount of indicator moiety detected corresponds to the amount of the microorganism of interest present in the sample. In some variations of the detection method, the synthetic phage need not include an indicator gene and can be detected by a variety of other suitable methods, e.g., immunochemical methods and assays utilizing appropriate antibodies that specifically bind to synthetic phage proteins, such as capsid proteins.

[0080]

[0127] Some embodiments of the detection method may include capturing at least one target microorganism before incubating with the synthetic phage according to the present disclosure. For example, in some embodiments, the target microorganism may be captured by binding to a solid support, such as, but not limited to, the surface of a plate or a filter (e.g., a bacteriological filter having a pore size of 0.45 μm, such as a spin filter or plate filter). The indicator phage is then contacted with the solid support. The solid support may then be washed one or more times to remove excess unbound synthetic phage. In one embodiment, culture medium (e.g., Luria-Bertani broth (LB broth), or tryptic soy broth or tryptone soy broth (TSB), or buffered peptone water (BPTW) may be added for an additional incubation period to allow replication of the synthetic phage and high-level expression of the gene encoding the indicator moiety. The incubation step may be long enough for a single phage life cycle. One replication cycle of the synthetic phage may be sufficient for sensitive and rapid detection of the target microorganism. The soluble indicator released into the surrounding liquid upon lysis of the bacteria can then be measured and quantified. Alternatively, the indicator signal may be measured directly on the solid support. For example, if the indicator moiety is an enzyme such as luciferase or HRP, an indicator substrate may be incubated with the portion of the sample that remains on the solid support, such as bound to a filter or plate surface. Thus, in some embodiments, the enzymatic reaction can be detected by placing the plate, such as a 96-well plate, directly into an appropriate detection device.

[0081]

[0128] Lysis of cells of the target microorganism, such as bacteria, can occur before, during, or after the detection step. In some embodiments, unenumerated infected cells may be detectable upon addition of the indicator substrate, for example, if the indicator exits the cells and / or the substrate enters the cells without complete cell lysis. Thus, in embodiments utilizing a spin filter system in which only the indicator released in the lysate is analyzed, lysis is required for detection. However, in embodiments utilizing filter plates or 96-well plates containing samples in solution or suspension, where the original plate filled with intact and lysed cells is directly assayed, lysis is not required for detection. In some embodiments, the reaction between the indicator moiety and the substrate may continue for 30 minutes or more, and detection at various time points may be desirable to optimize sensitivity. For example, in embodiments using a 96-well plate as the solid support, detector readings can be taken initially at 10- or 15-minute intervals until the reaction is complete.

[0082]

[0129] Detection methods may be implemented to utilize a general concept that can be modified to accommodate different sample types or sizes and assay formats. Embodiments of the present methods may have a total detection run time (which may be referred to as "assay time") of less than 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, or 26.0 hours, depending on the sample type, sample size, cell concentration in the sample, and assay format. For example, the amount of time required may be somewhat shorter or longer depending on the assay, type and size of the sample being tested, the conditions required for target viability, the complexity of the physical / chemical environment, and the concentration of "endogenous" non-target contaminants in the synthetic phage and microorganisms, e.g., bacteria, to be detected.

[0083]

[0130] In an exemplary method, synthetic phage are engineered to encode a soluble luciferase and express it during phage replication. Luciferase expression is driven by a viral capsid promoter (e.g., the bacteriophage T5 or T4 late promoter), resulting in high expression. Because the parent phage is substantially purified from luciferase, the luciferase detected in the assay is most likely derived from progeny phage replication during infection of cells of the microbial organism being detected. Therefore, separation of the parent phage from the progeny phage is not necessary. At least a portion of a sample that may contain the microorganism of interest is placed in a spin column filter, centrifuged to remove the liquid, and an appropriate number of synthetic phage is added. The infected cells can be incubated for a sufficient time (e.g., 30-120 minutes at 37°C) to allow progeny phage replication and cell lysis to occur. The parent and progeny phage plus free luciferase in the lysate can then be collected, for example, by centrifugation, and the level of luciferase in the filtrate can be quantified using a luminometer. Alternatively, a high-throughput method may be used in which samples are applied to a 96-well filter plate and, after the manipulations listed above, assayed for luciferase directly in the original 96-well filter plate without the final centrifugation step.

[0084]

[0131] Another exemplary method involves a filter plate assay using synthetic phage to detect target microorganisms, such as bacteria. Briefly, a sample containing the target microorganism is added to the wells of a multi-well filter plate, which can be spun to concentrate the sample by removing liquid from the sample. Synthetic phage is added to the wells and incubated with additional medium for a time sufficient for adsorption, subsequent infection of the target microorganism, and progression of the phage life cycle (e.g., about 45 minutes). Finally, a luciferase substrate is added and reacts with any luciferase present. The resulting luminescence is measured using a luminometer that detects luciferase activity.

[0085]

[0132] In some instances, detection can occur without concentrating the microorganisms on or near the capture surface. In such instances, the sample is not concentrated but is directly incubated with the synthetic phage for a period of time and then assayed for indicator activity. Aliquots of the synthetic phage are dispensed into individual wells of a multi-well plate, and then a test sample aliquot, which may contain the microorganism of interest, is added and incubated for a period of time sufficient for the synthetic phage to replicate and produce soluble indicator protein. The plate wells containing the soluble indicator and phage can then be assayed by an appropriate method to measure the indicator activity on the plate.

[0086]

[0133] In some embodiments, the sample may be enriched prior to testing by incubation in conditions conducive to growth. In such embodiments, the enrichment period may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours or longer, depending on the type and size of the sample. In some embodiments, the synthetic phage includes a detectable indicator moiety, and infection of a single cell of a target microorganism, such as a bacterial cell, can be detected by an amplified signal generated via the indicator moiety. In some embodiments, the synthetic phage is added to the sample at a concentration sufficient to rapidly locate, bind, and infect a target microorganism present in very low numbers in the sample, e.g., a single cell. In some embodiments, the synthetic phage concentration may be sufficient to locate, bind, and infect a target microorganism in less than one hour. In other embodiments, these events may occur in less than two or three hours after adding the synthetic phage to the sample. For example, in certain embodiments, the synthetic phage concentration in the incubation step is greater than 1×10 5 PFU / mL, greater than 1×10 6 PFU / mL, or greater than 1×10 7 PFU / mL.

[0087]

[0134] The synthetic phage can be purified to be substantially free of residual indicator protein that may be generated during production of the infectious agent stock. Thus, in certain embodiments of the detection method, the synthetic phage can be purified using cesium chloride isopycnic gradient centrifugation or other suitable methods before incubation with the sample. In some embodiments of the detection method, microorganisms can be detected without isolating or purifying any of the microorganisms from the sample. For example, in certain embodiments, a sample containing one or a few microorganisms of interest can be applied directly to an assay vessel, such as a spin column, microtiter well, or filter, and the assay is performed in that assay vessel. Aliquots of the sample can be dispensed directly into wells of a multiwell plate, and the synthetic phage can be added. After a sufficient time for infection, a lysis buffer and a substrate for the indicator protein can be added and assayed for detection of the indicator signal. Some embodiments of the method can be performed on a filter plate. Some embodiments of the detection method can be performed with or without concentrating the sample before infection with the synthetic phage. The methods of the present invention can include various other steps to enhance sensitivity. For example, as discussed in more detail herein, the method may include washing the captured, infected bacteria after adding the bacteriophage but before incubation to remove excess parent bacteriophage and / or luciferase or other reporter protein that may contaminate the bacteriophage preparation.

[0088]

[0135] In some embodiments of the detection method, detection of the microorganism of interest can be completed without the need to culture the sample as a way to increase the population of the microorganism. For example, in certain embodiments, the total time required for detection is less than 26.0 hours, 25.0 hours, 24.0 hours, 23.0 hours, 22.0 hours, 21.0 hours, 20.0 hours, 19.0 hours, 18.0 hours, 17.0 hours, 16.0 hours, 15.0 hours, 14.0 hours, 13.0 hours, 12.0 hours, 11.0 hours, 10.0 hours, 9.0 hours, 8.0 hours, 7.0 hours, 6.0 hours, 5.0 hours, 4.0 hours, 3.0 hours, 2.5 hours, 2.0 hours, 1.5 hours, 1.0 hour, 45 minutes, or 30 minutes. Some embodiments of the detection method may detect 10 or fewer cells of a microorganism (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 microorganisms) present in a sample. In certain embodiments, the detection method detects on the order of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 cells of a microorganism in a sample.

[0089]

[0136] Some detection methods according to embodiments of the present invention, in addition to detection with synthetic phage or recombinant TSPs according to the present disclosure, are the use of binding agents (e.g., antibodies) to purify and / or concentrate the microorganism of interest from a sample. For example, in certain embodiments, the present invention includes a method for detecting a microorganism of interest in a sample, the method comprising the steps of incubating the sample with a synthetic phage that infects the microorganism of interest, capturing the microorganism from the sample on a solid support using a capture antibody specific for the microorganism of interest, the synthetic phage comprising an indicator gene inserted into a late gene region of the synthetic phage, whereby expression of the indicator gene during synthetic phage replication following infection of the microorganism of interest results in a soluble indicator protein product, and detecting the indicator protein product, wherein positive detection of the indicator protein product indicates the presence of the microorganism of interest in the sample.

[0090]

[0137] Various applications of the detection methods according to the present disclosure are envisioned. For example, the detection methods can be used to test an initial patient sample for the presence of a particular pathogen, such as a particular bacterial genus or species. In some embodiments, the methods can be used to detect a particular pathogen in a clinical sample. The methods can be used to evaluate the potential effectiveness of a treatment in the context of an infectious disease or other pathogenic condition in a given patient. In another example, the detection methods can be applied to non-clinical applications. For example, the detection methods can be used as food safety detection methods to identify the presence of a particular bacterium in food. In another example, the detection methods can be used to detect pathogens in a patient sample after the initiation of some type of treatment, such as antibiotic treatment or some other type of drug or therapy. Detection can be used to monitor the progress or effectiveness of any type of treatment or therapy.

[0091]

[0138] Systems and Kits Some embodiments of the present invention include systems and kits. In some embodiments, a system (e.g., an automated system) or kit according to the present disclosure includes components, such as reagents, materials, or devices, for performing a method according to the present disclosure. The term "component" is broadly defined and includes any suitable device or collection of devices, reagent, or reagents suitable for performing a method according to the present disclosure. Components need not be integrally connected or arranged with each other in any particular manner. The present invention encompasses any suitable arrangement of components relative to each other. For example, components need not be in the same room. However, in some embodiments, components are connected to each other within an integrated unit. In some embodiments, the same component may perform multiple functions. Some embodiments of a system or kit include at least one synthetic phage according to the present disclosure and / or at least one recombinant TSP according to the present disclosure. Some embodiments of systems and kits are particularly suitable for automating and / or performing a method according to the present disclosure in a high-throughput manner. In certain embodiments, a system or kit may include a self-contained unit deliverable from a first site to a second site.

[0092]

[0139] In some embodiments, the present invention includes systems or kits for detecting a microorganism of interest in a sample. Such systems and / or kits may be referred to as diagnostic systems and / or kits or detection systems and / or kits. The systems or kits, in certain embodiments, may include components for incubating a sample with one or more synthetic phages specific for one or more microorganisms of interest. The synthetic phages may include an indicator gene, and the systems or kits may include one or more components for detecting the product of the indicator gene (e.g., an enzyme substrate, if the product of the indicator gene is an enzyme). In some embodiments of both the systems and kits, the synthetic phage is capable of specifically infecting a microorganism of interest and includes an indicator gene inserted into a late gene region, such that expression of the indicator gene during infection of the microorganism results in a soluble indicator protein product. Some systems and kits may include components for capturing the microorganism of interest on a solid support. In certain embodiments, the systems or kits may include a device including a solid support containing a cell-binding component. The systems or kits, in certain embodiments, may include a signal detection component capable of detecting the indicator gene product produced by infecting a microorganism in a sample with the synthetic phage. In some examples, the signal detection component is a luminometer, which can be a handheld device. The system or kit may further include a vessel or container containing a substrate and / or medium. In certain embodiments, the system and / or kit may include components for washing the captured microorganism sample. In some embodiments, the system or kit may include components for isolating the microorganism of interest from other components in the sample. In some detection systems and / or kits according to embodiments of the present invention, the steps performed by the system or kit are automated or controlled by a user via computer input. A liquid-handling robot performs at least one step.In a computerized system, the system may be fully automated, semi-automated, or directed by a user via a computer (or some combination thereof).

[0093]

[0140] In exemplary embodiments, a system or kit includes components for detecting a microorganism of interest in a sample. Such a system or kit may include, for example, components for isolating at least one microorganism from other components in the sample, components for infecting at least one microorganism with one or more synthetic phages, components for lysing at least one infected microorganism to release the synthetic phages present in the microorganism, and components for detecting the synthetic phages, including soluble proteins encoded and expressed by indicator genes contained in the synthetic phages. When such exemplary systems or kits are used to perform detection methods according to the present disclosure, detection of the soluble protein product of the synthetic phages indicates the presence of the microorganism in the sample.

[0094]

[0141] In some embodiments, the present invention includes systems or kits involved in implementing methods for controlling microorganisms or target microorganisms in various contexts, some of which are described in this disclosure. For example, a kit can include one or more synthetic phages or compositions containing one or more synthetic phages according to the present disclosure and a container for their storage, such as a bag or vial. Such a container can have a sterile access port, e.g., a bag or vial with a stopper pierceable by a hypodermic injection needle. In another example, a kit can include one or more synthetic phages or compositions containing one or more synthetic phages according to the present disclosure in lyophilized or concentrated form and a diluent. In such kits, the diluent can be a pharmaceutically acceptable carrier or excipient, as described elsewhere in this disclosure. Non-limiting examples of diluents that can be included in such kits are saline, buffered saline, water, or sucrose. In another example, a kit can include one or more synthetic phages or compositions comprising one or more synthetic phages according to the present disclosure, and a device for administering one or more synthetic phages or compositions comprising one or more synthetic phages according to the present disclosure, or a device for contacting one or more synthetic phages or compositions comprising one or more synthetic phages according to the present disclosure with a subject or device. The device can be a syringe for injection or oral administration (e.g., the kit can be a syringe pre-filled with a liquid composition), a microneedle device such as a microneedle patch, an inhaler, nebulizer, sprayer, or pump. The kit can include multiple devices.

[0095]

[0142] composition Compositions comprising at least one synthetic phage according to the present disclosure are included among the embodiments of the present invention. In some examples, the composition is a pharmaceutical composition comprising one or more synthetic phage according to the present disclosure. For pharmaceutical use, one or more synthetic phage according to the present disclosure can be formulated as a pharmaceutical preparation or composition comprising at least one pharmaceutically acceptable carrier, diluent, or excipient (the foregoing terms may be used interchangeably). Pharmaceutical compositions include compositions suitable for therapeutic use as well as compositions suitable for non-therapeutic use, such as, but not limited to, cosmetic use or use to improve the well-being of a subject. Pharmaceutical compositions may further comprise one or more pharmaceutically active compounds. Pharmaceutical compositions can be administered orally, parenterally (e.g., by intravenous, intramuscular, or subcutaneous injection). The dosage form (which may also be referred to as a "dosage form") may be in a form suitable for administration by intravenous injection (by injection or intravenous infusion), topical administration, administration by inhalation, skin patch, implant, suppository, etc. The dosage form (which may also be referred to as a "dosage form") may be solid, semi-solid, or liquid, depending on the mode and route of administration. For example, a formulation for oral administration may be provided with an enteric coating that allows one or more synthetic phages in the formulation to resist the gastric environment and pass into the intestine. More generally, a synthetic formulation for oral administration may be appropriately formulated for delivery to any desired part of the gastrointestinal tract. Suppositories may be used for delivery to the gastrointestinal tract.

[0096]

[0143] The compositions of the present disclosure can include carriers or vehicles such as pharmaceutically acceptable carrier vehicles.Solid pharmaceutically acceptable vehicles can include one or more substances that can also act as flavoring agents, lubricants, solubilizers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet disintegrating agents.Suitable solid vehicles include, for example, calcium phosphate, magnesium stearate, talc, sugar, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidone, low-melting waxes, and ion exchange resins.The pharmaceutical compositions can be prepared as sterile solid compositions that can be suspended in sterile water, saline, or other suitable sterile injectable medium at the time of administration. Pharmaceutical compositions can be administered in the form of a sterile solution or suspension containing other solutes or suspending agents (e.g., sufficient saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monooleate, polysorbates, oleic acid esters of sorbitol and its anhydrides copolymerized with ethylene oxide, and the like. Pharmaceutical compositions can be administered orally in either liquid or solid composition form. Compositions suitable for oral administration include solid forms such as pills, capsules, granules, tablets, and powders, as well as liquid forms such as solutions, syrups, elixirs, and suspensions. Forms useful for enteral administration include sterile solutions, emulsions, and suspensions.

[0097]

[0144] One or more synthetic phages according to the present disclosure can be dissolved or suspended in a liquid vehicle such as water, an organic solvent, a mixture of both, or a pharmaceutically acceptable oil or fat. The liquid vehicle can contain other suitable additives, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavorings, suspending agents, thickeners, colorants, viscosity adjusters, stabilizers, or osmolality adjusters. Suitable examples of liquid vehicles include water (which may contain various additives, for example, cellulose derivatives such as sodium carboxymethylcellulose solution), alcohols (such as monohydric and polyhydric alcohols) and their derivatives, and oils. The liquid carrier or vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate. The liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant. For external administration to a subject (e.g., wound, skin, or transdermal), the composition can be formulated into an ointment, cream, or gel form, and permeation can be facilitated using an appropriate penetrant or detergent, such as dimethyl sulfoxide, dimethylacetamide, and dimethylformamide. For transmucosal administration, nasal sprays, rectal, or vaginal suppositories can be used. The composition can include, but is not limited to, various suppository bases, cocoa butter, polyethylene glycol (carbowax), polyethylene sorbitan monostearate, and mixtures thereof with other suitable materials to modify the melting point or dissolution rate.

[0098]

[0145] In some embodiments, the composition can be prepared in a dry form (e.g., a dehydrated form), such as a lyophilized form. Such a formulation can be referred to as "lyophilized" or "lyophilized." Lyophilization is a freeze-drying process, or a process of freezing and drying, during which the solvent is removed from a liquid formulation. The freeze-drying process can include one or more simultaneous or sequential steps of freezing and drying. The composition is freeze-dried in a non-volatile buffer or an aqueous solution containing a volatile buffer. Non-limiting examples of suitable non-volatile buffers are PBS, Tris-HCl, HEPES, or L-histidine buffer. Non-limiting examples of suitable volatile buffers are ammonium bicarbonate, ammonia / acetic acid, or N-ethylmorpholine / acetic acid buffer. The lyophilized composition can include a suitable carrier or excipient. Such suitable excipients can include, but are not limited to, cryopreservatives, bulking agents, surfactants, or combinations thereof. Exemplary excipients include one or more of polyols, disaccharides, or polysaccharides, such as mannitol, sorbitol, sucrose, trehalose, and / or dextran 40. In some examples, the cryopreservative may be sucrose and / or trehalose. In some examples, the bulking agent may be glycine or mannitol. In one example, the surfactant may be a polysorbate, such as polysorbate-20 and / or polysorbate-80. The lyophilized composition may be, for example, in cake or powder form. The lyophilized composition can be rehydrated / solubilized / reconstituted in a carrier or excipient (such as water or a buffer) prior to use.

[0099]

[0146] Computer System and Computer-Readable Medium The systems or kits described in this disclosure, or any of their components, may be embodied in the form of a computer system, typical examples of which include general purpose computers, programmed microprocessors, microcontrollers, peripheral integrated circuit devices, and other devices or arrangements of devices capable of performing the steps constituting the methods of the present technology.

[0100]

[0147] The computer system may include a computer, an input device, a display unit, and / or the Internet. The computer may further include a microprocessor. The microprocessor may be connected to a communication bus. The computer may also include memory. The memory may include random access memory (RAM) and read-only memory (ROM). The computer system may further include a storage device. The storage device may be a hard disk drive or a removable storage drive, such as a floppy disk drive or an optical disk drive. The storage device may also be other similar means for loading computer programs or other instructions into the computer system. The computer system may also include a communication unit. The communication unit allows the computer to connect to other databases and the Internet via an I / O interface. The communication unit allows data to be transferred to and received from other databases. The communication unit may include a modem, an Ethernet card, or any similar device that allows the computer system to connect to databases and networks, such as a LAN, a MAN, a WAN, and the Internet. Thus, the computer system may facilitate input from a user via an input device accessible to the system via the I / O interface.

[0101]

[0148] A computing device typically includes an operating system that provides executable program instructions for the general management and operation of the computing device, and typically includes a computer-readable storage medium (e.g., a hard disk, random access memory, read-only memory, etc.) that stores instructions that, when executed by a processor of a server, enable the computing device to perform its intended functions. Suitable implementations for operating systems and the general functions of computing devices are known or commercially available and will be readily implemented by those skilled in the art, especially in light of the disclosure herein.

[0102]

[0149] A computer system executes a set of instructions stored in one or more storage elements to process input data. The storage elements may also hold data or other information as needed. The storage elements may be in the form of information sources or physical memory elements present within a processing machine.

[0103]

[0150] The environment may include various data stores and other memory and storage media, as described above. These may reside in a variety of locations, such as on storage media local to (and / or resident on) one or more computers, or on storage media remote from any or all of the computers via a network. In a particular set of embodiments, information may reside within a storage area network ("SAN") familiar to those skilled in the art. Similarly, any files necessary to perform functions attributed to a computer, server, or other network device may be stored locally and / or remotely, as appropriate. Where a system includes computing devices, each such device may include hardware elements that may be electrically coupled via a bus, including, for example, at least one central processing unit (CPU), at least one input device (e.g., a mouse, keyboard, controller, touchscreen, or keypad), and at least one output device (e.g., a display device, printer, or speaker). Such systems may also include one or more storage devices, such as disk drives, optical storage devices, and solid-state storage devices, such as random access memory ("RAM") or read-only memory ("ROM"), as well as removable media devices, memory cards, flash cards, etc.

[0104]

[0151] Such devices may also include computer-readable storage medium readers, communication devices (e.g., modems, network cards (wireless or wired), infrared communication devices, etc.), and working memory, as described above. The computer-readable storage medium readers may be configured to connect to or receive computer-readable storage media representing remote, local, fixed, and / or removable storage devices, as well as storage media for temporarily and / or more permanently storing, storing, transmitting, and retrieving computer-readable information. Systems and various devices also typically include several software applications, modules, services, or other elements located within at least one working memory device, including an operating system and application programs such as client applications or web browsers. It should be understood that alternative embodiments may have numerous variations from those described above. For example, customized hardware may be used, and / or particular elements may be implemented in hardware, software (including portable software such as applets), or both. Additionally, connections to other computing devices, such as network input / output devices, may be employed.

[0105]

[0152] Non-transitory storage media and computer-readable media for storing code or portions of code can include any suitable media known or used in the art, including, but not limited to, storage media and communication media such as volatile and nonvolatile, removable and non-removable media implemented in any method or technology for storing and / or transmitting information, such as computer-readable instructions, data structures, program modules, or other data, including RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disk (DVD) or other optical storage, magnetic cassette, magnetic tape, magnetic disk storage or other magnetic storage device, or any other medium that can be used to store the required information and that can be accessed by a system device. Based on the disclosure and teachings provided herein, one skilled in the art will appreciate other manners and / or methods for implementing the various embodiments.

[0106]

[0153] A computer-readable medium may include, but is not limited to, electronic, optical, magnetic, or other storage devices capable of providing computer-readable instructions to a processor. Other examples include, but are not limited to, floppy disks, CD-ROMs, DVDs, magnetic disks, memory chips, ROMs, RAMs, SRAMs, DRAMs, content addressable memories ("CAMs"), flash memory such as DDR, NAND flash, or NOR flash, ASICs, configured processors, optical storage devices, magnetic tape, or other magnetic storage devices, or any other medium from which a computer processor can read instructions. In one embodiment, a computing device may include a single type of computer-readable medium, such as random access memory (RAM). In other embodiments, a computing device may include two or more types of computer-readable media, such as random access memory (RAM), a disk drive, and a cache. A computing device may communicate with one or more external computer-readable media, such as an external hard disk drive or an external DVD or Blu-Ray drive.

[0107]

[0154] As described above, embodiments include a processor configured to execute computer-executable program instructions and / or access information stored in memory. The instructions may include processor-specific instructions generated by a compiler and / or interpreter from code written in any suitable computer programming language, such as, for example, C, C++, C#, Visual Basic, Java, Python, Perl, JavaScript, and ActionScript (Adobe Systems, Mountain View, Calif.). In one embodiment, a computing device includes a single processor. In other embodiments, the device includes two or more processors. Such processors may include microprocessors, digital signal processors (DSPs), application-specific integrated circuits (ASICs), field-programmable gate arrays (FPGAs), and state machines. Such processors may further include programmable electronic devices, such as programmable logic devices (PLCs), programmable interrupt controllers (PICs), programmable logic devices (PLDs), programmable read-only memories (PROMs), electronically programmable read-only memories (EPROMs or EEPROMs), or other similar devices.

[0108]

[0155] The computing device comprises a network interface. In some embodiments, the network interface is configured to communicate via a wired or wireless communication link. For example, the network interface may enable communication over a network via Ethernet, IEEE 802.11 (Wi-Fi), 802.16 (Wi-Max), Bluetooth, infrared, etc. As another example, the network interface may enable communication over a network such as a CDMA, GSM, UMTS, or other cellular communication network. In some embodiments, the network interface may enable a point-to-point connection with another device, such as via a Universal Serial Bus (USB), 1394 FireWire, serial or parallel connection, or similar interface. Some embodiments of a suitable computing device may comprise two or more network interfaces for communicating over one or more networks. In some embodiments, the computing device may include a data store in addition to or instead of a network interface.

[0109]

[0156] A suitable computing device embodiment may include or communicate with several external or internal devices, such as a mouse, external memory, a CD-ROM, a DVD, a keyboard, a display, audio speakers, one or more microphones, or any other input or output device. For example, a computing device may communicate with various user interface devices and displays. Displays may use any suitable technology, including, but not limited to, LCD, LED, CRT, etc.

[0110]

[0157] A set of instructions for execution by a computer system may include various commands that instruct a processing machine to perform particular tasks, such as steps constituting the methods of the present technology. The set of instructions may be in the form of a software program. Further, the software may be in the form of a collection of separate programs, a program module with a larger program, or a portion of a program module, as in the present technology. The software may also include modular programming in the form of object-oriented programming. The processing of input data by a processing machine may be in response to user commands, results of previous processing, or a request made by another processing machine.

[0111]

[0158] While the present invention has been disclosed with reference to particular embodiments, many modifications, alterations, and variations to the described embodiments are possible without departing from the scope and spirit of the invention as defined in the appended claims. Accordingly, it is intended that the present invention not be limited to the described embodiments, but rather have the full scope defined by the language of the following claims and equivalents thereof. [Example]

[0112]

[0159] The following examples are offered to illustrate, but not to limit, the claimed invention.

[0113]

[0160] Example 1: Construction and testing of synthetic phage using recombinant TSP3 An exemplary synthetic phage was constructed from the naturally occurring parent phage CBA120 by replacing the TSP3 of CBA120 with a recombinant TSP ("CBA120-SPTD1 TSP3") containing the N-terminal domain from CBA120 TSP3 and the C-terminal domain from phage SPTD1 TSP3. Phage SPTD1 (SEQ ID NO: 1; GenBank accession number OP991882) was isolated from a sewage sample and sequenced as described in U.S. Patent Application No. 16 / 247,486, filed January 14, 2019, where SPTD1 is designated as TSP1. The sequence of the CBA120 phage is available at NCBI GenBank under accession number NC_016570. The CBA120 and SPTD1 phages each have four TSPs: TSP1, TSP2, TSP3, and TSP4 (six copies each). Examples of electron microscopy images of the SPTD1 and CBA120 phages are shown in Figure 6. Figure 7 is a schematic diagram of the structure of the CBA120 virion with TSP1, TSP2, TSP3, and TSP4 proteins. The CBA120 TSPs recognize the following bacteria: TSP1 - Salmonella Minnesota; TSP2 - E. coli O157; TSP3 - E. coli O77; TSP4 - E. coli O78. SPTD1 TSP2 recognizes Citrobacter sedlakii, and SPTD1 TSP3 recognizes Salmonella.

[0114]

[0161] To design a nucleic acid sequence encoding CBA120-SPTD1 TSP3, the amino acid sequences of CBA120 TSP3 and SPTD1 TSP3 were analyzed. Figure 14 is a schematic diagram of the results of a pairwise alignment of the amino acid sequences of CBA120 TSP3 and SPTD1 TSP3 performed using EMBOSS Needle. The "neck" regions of both TSPs are shown in boxes. CBA120 TSP3 showed N-terminal homology with SPTD1 TSP3. Additional amino acid sequence analysis revealed that the Swiss-fold region of CBA120 TSP3 matches the N-terminal region of SPTD1 TSP3 and the C-terminal region of Salmonella phage Det7 TSP3 (which recognizes Salmonella Typhimurium (LT2) ATCC 19585), indicating that the α-helix is ​​shared between the two proteins. The Det7 genome sequence has GenBank accession numbers NC_27119.1 and / or KP797973.1. The Det7 TSP3 and SPTD1 TSP3 amino acid sequences showed 95% identity over 90% coverage, serving as potential evidence of horizontal gene transfer. After analyzing the amino acid sequences of Det7 CBA120 TSP3 and SPTD1 TSP3, the site between amino acids 158 and 159 within the "neck" region of each TSP was selected as the junction ("splice") site between the N-terminal domain of CBA120 TSP3 and the C-terminal domain of SPTD1 TSP3. Figure 15 shows a pairwise alignment of the amino acid sequences of CBA120 TSP3 and the recombinant CBA120-SPTD1 TSP3, performed using Emboss Needle. The junction between amino acids 158 and 159 in the "neck" region (boxed) of recombinant CBA120-SPTD1 TSP3 is indicated by an arrow. Figure 16 shows a pairwise alignment of the amino acid sequences of SPTD1 TSP3 and recombinant CBA120-SPTD1 TSP3, performed with Emboss Needle. The "neck" region is boxed.

[0115]

[0162] The nucleic acid sequence encoding CBA120-SPTD1 TSP3 was generated by determining the location of potential "neck" regions in SPTD1 TSP3 based on homology to CBA120 TSP3 and structural predictions using the Swiss-Model and Protein Homology / analogY Recognition Engine V 2.0 (Phyre 2, available online from the Structural Bioinformatics Group, Imperial College, London). The corresponding regions in the nucleotide sequence were then determined from the amino acid sequence. The nucleic acid sequence encoding the SPTD1 TSP3 "neck" regions was then copied to the ends of the gene, and the corresponding "neck" regions were then replaced with the ends of the CBA120 TSP3 gene to generate a chimeric open reading frame (ORF) called "CBA120-SP-TD1 chimeric TSP3." The copied section, "SPTD1 TSP3 catalytic & binding domain," was considered the donor DNA sequence. 500 bp of flanking CBA120 genomic sequence flanking the sequence to be replaced, at each end of the donor sequence, were synthesized by Integrated DNA Technologies (IDT) in the pUCIDT plasmid used for subsequent HR. Figure 18 is a schematic diagram of the HR.CBA120-TSP1.chimeric TSP3HR cassette.pUCIDT.HR.CBA120-SPTD1.chimeric TSP3 plasmid, which contains the nucleic acid sequence encoding the CBA120 TSP3 N-terminal domain, followed by the codon encoding amino acid 158, followed by the codon encoding amino acid 159 of SPTD1 TSP3, and the nucleic acid sequence encoding the C-terminal domain of SPTD1 TSP3.

[0116]

[0163] HR was performed as follows to generate a synthetic phage from SPTD1 phage by exchanging SPTD1 TSP3 with recombinant CBA120-SPTD1 TSP3, which contains the N-terminal domain from SPTD1 TSP3 and the C-terminal domain from CBA120 TSP3. The HR plasmid containing pUCIDT.HR.CBA120-SPTD1.chimeric TSP3 was transformed into Escherichia coli (E. coli) O157:H7 ATCC 43888 by electroporation using a MicroPulser (Bio-Rad® Laboratories, Inc., Hercules, California, USA) according to the manufacturer's instructions. Five colonies were grown in 2 ml of TSB containing 100 μg / ml carbenicillin at 37°C with shaking at 250 rpm until they reached 10 cells / ml. Transformed bacteria, along with untransformed controls, were infected with CBA120.NL at an MOI of 0.1, a recombinant form of CBA120 expressing NanoLuc® (Promega Corporation, Madison, Wisconsin, USA), and incubated at 37°C for 3 hours with shaking at 250 rpm, then collected and centrifuged at 6800 g for 2 minutes. The supernatant was filtered through a 0.45 μm pore size filter and stored at 4°C. The lysates were serially diluted and spotted onto Petri dishes seeded with either E. coli O157:H7 43888 as a control or a panel of Salmonella strains (ATCC 19585, 7001, 14028, 27869, and 51158), some of which contain the O4 antigen of the known SPTD1 TSP3 target, Salmonella Typhimurium LT2 strain. Individual plaques distinguishable from the spot assays from Salmonella ATCC 19585 and 14028 were harvested and used to inoculate Salmonella LT2 strain ATCC 19585 cultures, which were subsequently prepared as large-scale stocks.Figure 13 is a schematic diagram of the HR process used to generate a synthetic phage from SPTD1 phage by replacing SPTD1 TSP3 with a recombinant TSP containing the C-terminal domain from SPTD1 TSP3 and the N-terminal domain from CBA120 TSP3 ("CBA120-SPTD1 TSP3"). The resulting synthetic phage was designated CBA120-SPTD1.chiTSP3 or RBP-CBA120-1.

[0117]

[0164] To test the properties of the synthetic CBA120-SPTD1.chiTSP3 (alternatively designated RBP-CBA120-1) phage, plating experiments were performed. The results of an exemplary plating experiment are shown in Figure 18 and also summarized in Tables 1 and 2. Compared to CBA120, the synthetic phage CBA120-SPTD1.chiTSP3 lost the ability to infect E. coli O77 and gained the ability to infect S. Typhimurium.

[0118]

[0165] Example 2: Construction and testing of synthetic phage using recombinant TSP2 An exemplary synthetic phage was constructed from the naturally occurring parent phage CBA120 by replacing CBA120 TSP2 with a recombinant TSP ("CBA120-SPTD1 TSP2") containing the N-terminal domain from CBA120 TSP2 and the C-terminal domain from phage SPTD1 TSP2. A nucleic acid sequence encoding CBA120-SPTD1 TSP2 was produced, and a synthetic phage designated CBA120-SPTD1.chiTSP2 (alternatively designated RBP-CBA120-2) was constructed using procedures substantially similar to those described in Example 1. Figure 11 is a schematic diagram of the HR.CBA120-SPTD1.chimeric TSP2 HR cassette. This HR cassette contained a nucleic acid sequence encoding the CBA120 TSP2 N-terminal domain via a codon encoding amino acid 244, followed by a codon encoding amino acid 245 of SPTD1 TSP2, and a nucleic acid sequence encoding the C-terminal domain of SPTD1 TSP2. Figure 7 is a schematic diagram of the HR process used to generate a synthetic phage from CBA120 phage by replacing SPTD1 TSP2 with a recombinant TSP containing the N-terminal domain from CBA120 TSP2 and the C-terminal domain from SPTD1 TSP2 ("CBA120-SPTD1 TSP2"). The resulting synthetic phage was designated CBA120-SPTD1.chiTSP2 or alternatively RBP-CBA-120-2.

[0119]

[0166] To design a nucleic acid sequence encoding CBA120-SPTD1 TSP2, the amino acid sequences of CBA120 TSP2 and SPTD1 TSP2 were analyzed. Figure 8 is a schematic representation of the results of a pairwise alignment of the amino acid sequences of CBA120 TSP2 and SPTD1 TSP2 performed with Emboss Needle. The "neck" regions of both TSPs are shown boxed. As shown in Figure 8, the amino acid sequences are identical up to two amino acids upstream of the neck (as indicated by the arrows). After analyzing the amino acid sequences of CBA120 TSP2 and SPTD1 TSP2, the site between amino acids 244 and 245 of each TSP was selected as the junction ("splice") site between the C-terminal domain of SPTD1 TSP2 and the N-terminal domain of CBA120 TSP2. Figure 9 shows a pairwise alignment of the amino acid sequences of CBA120 TSP2 and recombinant CBA120-SPTD1 TSP2, performed with an Emboss Needle, in the region of the junction between the two sequences. The junction between amino acids 244 and 245 of recombinant CBA120-SPTD1 TSP2 is indicated by an arrow. As shown in the right panel of Figure 9, the structure of the recombinant CBA120-SPTD1 TSP2 protein contains three parts: an N-terminal region that binds to the baseplate of bacteriophage virions via TSP4 (TSP2 binds to the XD2 domain in the N-terminal region of TSP4, while TSP4 binds to the baseplate via the XD1 domain), a "neck" ("hinge") region, and a C-terminal region that binds to bacterial receptors. Figure 10 shows a pairwise alignment of the amino acid sequences of SPTD1 TSP2 and recombinant CBA120-SPTD1 TSP2, performed with an Emboss Needle. The predicted "neck" region is indicated by a box. Recombinant CBA120-SPTD1 TSP2 contains the complete neck alpha helix from SPTD1 TSP2.

[0120]

[0167] Plating experiments were performed to test the properties of the synthetic CBA120-SPTD1.chiTSP2 (RBP-CBA-120-2) phage. The results of exemplary plating experiments are shown in Figures 12 and 18 and are also summarized in Tables 1 and 2. Compared to CBA120, the synthetic phage CBA120-SPTD1.chiTSP2 lost the ability to infect E. coli O157 and gained the ability to infect Citrobacter sedlakii.

[0121]

[0168] Example 3: Construction and testing of synthetic phage using recombinant TSP4 An exemplary synthetic phage was constructed from the naturally occurring parent phage SPTD1 by replacing SPTD1 TSP4 with a recombinant TSP containing the N-terminal domain from SPTD1 TSP4 and the C-terminal domain from CBA120 TSP4 ("SPTD1-CBA120 TSP4"). A nucleic acid sequence encoding SPTD1-CBA120 TSP4 was produced, and a synthetic phage designated SPTD1-CBA120.chiTSP4 (alternatively designated RBP-SPTD1-1) was constructed using procedures substantially similar to those described in Example 1. To design a nucleic acid sequence encoding SPTD1-CBA120 TSP4, the amino acid sequences of CBA120 TSP4 and SPTD1 TSP4 were analyzed. Figure 21 is a schematic diagram of the results of a pairwise alignment of the amino acid sequences of CBA120 TSP4 and SPTD1 TSP4 performed with an Emboss Needle. The "neck" region of SPTD1 TSP4 is indicated. After analyzing the amino acid sequences of CBA120 TSP4 and SPTD1 TSP4, the site between amino acids 479 and 480 of each TSP was selected as the junction ("splice") site between the N-terminal domain of SPTD1 TSP4 and the C-terminal domain of CBA120 TSP4. Figure 23 shows a pairwise alignment of the amino acid sequences of SPTD1 TSP4 and recombinant SPTD1-CBA120 TSP4 performed on an Emboss Needle. The predicted "neck" region is indicated by a box. Figure 21 is a schematic diagram of the structure of the CBA120 TSP4 protein. Figure 22 is a schematic diagram of the hypothetical structure of the recombinant SPTD1 CBA120 TSP4 protein. Figure 23 shows a pairwise alignment of the amino acid sequences of SPTD1 TSP4 and recombinant SPTD1-CBA120 TSP4 performed on an Emboss Needle. The junction site between amino acid 479 of SPTD1 TSP4 and amino acid 480 of CBA120 TSP4 is indicated by an arrow. The "neck" region (based on the published TSP structure) is indicated by a box. Figure 24 shows a pairwise alignment of the amino acid sequences of CBA120 TSP4 and recombinant SPTD1-CBA12 TSP4 performed on an Emboss Needle.The junction site between amino acid 479 of SPTD1 and amino acid 480 of recombinant SPTD1-CBA120-TSP4 is indicated by an arrow. The neck region (based on the published TSP structure) is indicated by a box. Figure 19 is a schematic diagram of the HR process used to generate the SPTD1-derived synthetic phage SPTD1-CBA120.chiTSP4 (alternatively designated RBP-SPTD1-1) by exchanging SPTD1 TSP4 with a recombinant TSP4 containing the N-terminal domain from SPTD1 TSP4 and the C-terminal domain from CBA120 TSP4 ("SPTD1-CBA120 TSP4"). This HR cassette contained a nucleic acid sequence encoding the SPTD1 TSP4 N-terminal domain via a codon encoding amino acid 479, followed by a codon encoding amino acid 480 of CBA120 TSP4, and a nucleic acid sequence encoding the C-terminal domain of CBA120 TSP4. Figure 25 is a schematic diagram of the HR.SPTD1-CBA120chiTSP4 plasmid insert.

[0122]

[0169] A plating experiment was performed to test the ability of the synthetic SPTD1-CBA120.chiTSP4(RBP-SPTD1-1) phage to infect E. coli O78. Figure 26 is a photographic image of a plate culture showing the results of plating synthetic bacteriophage SPTD1-CBA120.chiTSP4(RBP-SPTD1-1) onto E. coli O78.

[0123]

[0170] Example 4: Construction and testing of synthetic phage using recombinant TSP1 An exemplary synthetic phage was constructed from the naturally occurring parent phage SPTD1 by replacing SPTD1 TSP1 with a recombinant TSP1 comprising the N-terminal domain from SPTD1 TSP1 and the C-terminal domain from phage CBA120 TSP1 ("SPTD1-CBA120 TSP1"). A nucleic acid sequence encoding SPTD1-CBA120 TSP1 was produced, and a synthetic phage designated SPTD1-CBA120.chiTSP1 (alternatively designated RBP-SPTD1-2) was constructed using procedures substantially similar to those described in Example 1. Figure 27 is a schematic diagram of the homologous recombination process used to generate the SPTD1-derived synthetic phage SPTD1-CBA120.chiTSP1(RBP-SPTD1-2) by exchanging SPTD1 TSP1 with a recombinant TSP1 comprising the N-terminal domain from SPTD1 TSP1 and the C-terminal domain from CBA120 TSP1 ("SPTD1-CBA120 TSP1"). Figure 28 shows a pairwise alignment of the amino acid sequences of CBA120 TSP1 and SPTD1 TSP1 performed on an Emboss Needle. The arrow indicates the junction site between amino acid 149 of SPTD1 TSP1 and amino acid 152 of CBA120 TSP1. The "neck" region (based on the published TSP structure) is indicated by a box. Figure 29 is a schematic diagram of the structure of the CBA120 TSP1 protein. Figure 30 shows a pairwise alignment of the amino acid sequences of SPTD1 TSP1 and SPTD1-CBA120 TSP1 performed on an Emboss Needle. The junction site between amino acid SPTD1 TSP1 and amino acid 149 of CBA120 TSP1 is marked with an arrow. Figure 31 shows a pairwise alignment of the amino acid sequences of CBA120 TSP1 and recombinant SPTD1-CBA120 TSP1 performed on an Emboss Needle. The junction site between amino acid SPTD1 TSP1 and amino acid 149 of CBA120 TSP1 is marked with an arrow. Figure 32 is a schematic diagram of the HR.SPTD1-CBA120chiTSP1 plasmid insert. The upstream homologous recombination region is the N-terminal region of TSP1 of SPTD1, which contains the TD1 and TD2 domains.

[0124]

[0171] Example 5: Construction and testing of synthetic phages using recombinant TSP1 and TSP2 An exemplary synthetic phage was constructed from the synthetic parent phage SPTD1-CBA120.chiTSP1 (RBP-SPTD1-2) discussed in Example 4 by replacing its TSP2 with a recombinant TSP2 comprising the N-terminal domain from SPTD1 TSP2 and the C-terminal domain from Det7 TSP2 ("SPTD1-Det7 TSP2"). A nucleic acid sequence encoding SPTD1-Det7 TSP2 was produced, and a synthetic phage called SPTD1-CBA120.chiTSP1-Det7.chiTSP2 (alternatively designated RBP-SPTD1-3) was constructed using procedures substantially similar to those described in the previous examples. Figure 33 is a schematic diagram of the homologous recombination process used to generate the synthetic phage SPTD1-CBA120.chiTSP1-Det7.chiTSP2 (alternatively designated RBP-SPTD1-3) derived from SPTD1-CBA120.chiTSP1 (RBP-SPTD1-2) by replacing the TSP2 of SPTD1-CBA120.chiTSP1 (RBP-SPTD1-2) with a recombinant TSP2 containing the N-terminal domain from SPTD1 TSP2 and the C-terminal domain from Det7 TSP2 ("SPTD1-Det7 TSP2"). Figure 34 shows a pairwise alignment performed with Emboss Needle of the amino acid sequences of Det7 TSP2, CBA120 TSP2, and SPTD1 TSP2. The arrow indicates the junction site between amino acid 252 of SPTD1 TSP2 and amino acid 256 of Det7 TSP2. The "neck" region, based on published TSP structures, is indicated by a box. Figure 35 is a schematic diagram of the structure of the CBA120 TSP2 protein. Figure 36 is a schematic diagram of the predicted structure of a recombinant TSP containing the N-terminus of SPTD1 TSP2 and the C-terminus of Det7 TSP2. Figure 37 shows a pairwise alignment of the amino acid sequences of chimeric SPTD1-Det7 TSP2 (SPTD1.Chi.TSP2) and SPTD1 TSP2, performed with Emboss Needle. The arrow indicates the junction site between amino acid 252 of SPTD1 TSP2 and amino acid 256 of Det7 TSP2.Figure 38 shows a pairwise alignment of the amino acid sequences of recombinant SPTD1-Det7 TSP2 and Det7 TSP2 performed with an Emboss Needle. The arrow indicates the junction site between amino acid 252 of SPTD1 TSP2 and amino acid 256 of Det7 TSP2. Figure 39 is a schematic diagram of the HR.SPTD1-CBA120chiTSP2 plasmid insert. The upstream homologous recombination region is the N-terminal region of SPTD1 TSP2, which contains the attachment domain (AD), XD2, XD3, and TD1 domains.

[0125]

[0172] Example 6: Testing of synthetic phage with recombinant TSP The synthetic phages carrying the recombinant TSP were tested by spotting a suspension of each phage onto plated bacterial strains. The appearance of clear plaques indicated the ability of the phage to infect the plated bacterial strain (a positive result), while the absence of clear plaques indicated the phage's lack of ability to infect the plated bacterial strain (a negative result). These results are summarized in Tables 1 and 2. The results of the test are shown below.

[0126]

[0173] Compared with its parent phage CBA120, CBA120-SPTD1.chiTSP2 (RBP-CBA120-2) gained the ability to infect Citrobacter sedlakii but lost the ability to infect Escherichia coli O157:H7. TSP1, TSP3, and TSP4 of RBP-CBA120-2 maintained the phage's ability to infect Salmonella Minnesota, E. coli O77, and E. coli O78, respectively.

[0127]

[0174] Compared to its parent phage CBA120, CBA120-SPTD1.chiTSP3(RBP-CBA120-1) gained the ability to infect the same Salmonella strains that SPTD1 can infect, but lost the ability to infect Escherichia coli O77. TSP1, TSP2, and TSP4 of CBA120-SPTD1.chiTSP3(RBP-CBA120-1) maintained the phage's ability to infect Salmonella Minnesota, E. coli O157:H7, and E. coli O78, respectively.

[0128]

[0175] Compared to its parent phage SPTD1, SPTD1-CBA120.chiTSP4(RBP-SPTD1-1) gained the ability to infect Escherichia coli O78. TSP2 of SPTD1-CBA120.chiTSP4(RBP-SPTD1-1) maintained the phage's ability to infect Citrobacter sedlakii. TSP3 of SPTD1-CBA120.chiTSP4(RBP-SPTD1-1) maintained the phage's ability to infect the same Salmonella strains that SPTD1 can infect. [Table 1] JPEG2025538196000003.jpg164170 [Table 2]

[0129]

[0176] Example 7: Construction of synthetic phages based on the SPTD1 parent phage A synthetic phage based on the CBA120 parent phage was constructed by replacing TSP1, TSP3, and TSP4 of CBA120 with recombinant TSPs specific for Escherichia coli (E. coli) serotype O157, which is of major concern for food safety due to the serotype's ability to cause severe disease after human infection. The CBA120-based synthetic phage specific for E. coli serotype O157 was used to detect E. coli serotype O157 in food samples with improved specificity and a low false-positive detection rate.

[0130]

[0177] Example 8: Construction of synthetic phages based on the SPTD1 parent phage The SPTD1 phage can recognize both Salmonella and Citrobacter. A synthetic phage is constructed by replacing one or more TSPs that recognize Citrobacter with one or more TSPs that cannot recognize Citrobacter. The resulting synthetic phage can be used to detect Salmonella with reduced false-positive detection rates in samples containing background flora, including Citrobacter, thereby improving the specificity of Salmonella detection.

[0131]

[0178] The resulting synthetic phage can also be used to improve phage therapy in chickens to reduce Salmonella colonization and improve food safety. Citrobacter is often a member of the microbiota of healthy chickens and would be targeted by SPTD1 phage. Therefore, disruption of the native microbiota (dysbiosis) in chickens can result from phage therapy with SPTD1. Dysbiosis is associated with adverse outcomes, such as increased risk of infection. Synthetic phages based on SPTD1 but unable to recognize Citrobacter can be used for phage therapy in chickens, resulting in the killing of Citrobacter present in the commensal gut microbiota, thereby improving the outcome of phage therapy.

[0132]

[0179] Example 9: Construction of synthetic phages for Salmonella recognition The SPTD1 phage can infect Salmonella Typhimurium but not Salmonella Anatum or Salmonella Minnesota strains, and can also infect Citrobacter. Replacing the C-terminal region of SPTD1 TSP1 with that of CBA120 TSP1 conferred the ability to recognize Salmonella Minnesota to SPTD1, while replacing the C-terminal region of SPTD1 TSP2 with that of Salmonella phage Det7 conferred the ability to recognize Salmonella Anatum but eliminated the ability to recognize Citrobacter. Furthermore, replacing the SPTD1 TSP4 with the TR2 podovirus TSP conferred on SPTD1 the ability to infect Salmonella Kentucky, resulting in a Salmonella-specific phage that can infect a wider range of Salmonella species than the parent phage SPTD1.

[0133]

[0180] An exemplary synthetic phage was constructed from the synthetic parent phage RBP-SPTD1-3 discussed in Example 5 by replacing its TSP4 with a recombinant TSP4 ("SPTD1.TSP4-TR2.TSP") comprising the N-terminal domain from SPTD1 TSP4 from podovirus Salmonella phage vB_SalP_TR2 ("TR2"), GenBank accession number NC_055921, and the C-terminal domain of KVN79_gp66 ("TR2.TSP"). A nucleic acid sequence encoding SPTD1.TSP4-TR2.TSP was produced, and a synthetic phage designated RBP-SPTD1-5 was constructed using procedures substantially similar to those described in the previous example. Due to the substantial evolutionary distance between SPTD1 (a myovirus) and TR2 (a podovirus), the C-terminal region of the TR2 TSP was codon-optimized to match the late gene codon usage of SPTD1. Figure 40 is a schematic diagram of the homologous recombination process used to generate the synthetic phage RBP-SPTD1-5 derived from RBP-SPTD1-3 by exchanging TSP4 of RBP-SPTD1-3 with a recombinant TSP4 containing the N-terminal domain from SPTD1 TSP4 and the C-terminal domain from TR2 TSP ("SPTD1.TSP4-TR2.TSP"). Figure 41 shows a pairwise alignment of the amino acid sequences of SPTD1 TSP4 and TR2 TSP performed with an Emboss Needle. The arrow indicates the junction site between amino acid 479 of SPTD1 TSP2 and amino acid 363 of TR2 TSP. The "neck" region, based on disclosed TSP structural homology and AlphaFold2 prediction, is indicated by a box. Figure 42 is a schematic diagram of the structure of the SPTD1 TSP4 protein generated by AlphaFold2, containing amino acids 1-959. Figure 43 is a schematic diagram of the structure of the TR2 TSP protein generated by AlphaFold2. Figure 44 is a schematic diagram of the predicted structure of a recombinant TSP containing the N-terminus of SPTD1 TSP4 and the C-terminus of TR2 TSP.Figure 45 shows a pairwise alignment, performed with an Emboss Needle, of the amino acid sequences of chimeric SPTD1.TSP4-TR2.TSP ("chi.TSP4") and SPTD1 TSP4 ("SPTD1.TSP4"). The arrow indicates the junction site between amino acid 479 of SPTD1 TSP4 and amino acid 363 of TR2 TSP. Figure 46 shows a pairwise alignment, performed with an Emboss Needle, of the amino acid sequences of recombinant SPTD1.TSP4-TR2.TSP ("chi.TSP4") and TR2 TSP ("TR2.TSP"). The arrow indicates the junction site between amino acid 479 of SPTD1 TSP4 and amino acid 363 of TR2 TSP. Figure 47 is a schematic diagram of the HR.SPTD1.TSP4-TR2.TSP plasmid insert. The upstream homologous recombination region is 500 bp of the N-terminal region of SPTD1 TSP4.

[0134]

[0181] Plating experiments were performed to test the ability of synthetic phage RBP-SPTD1-5 to infect Salmonella Kentucky and other target strains. Figure 48 is a photographic image of a plate culture showing the results of plating synthetic bacteriophage RBP-SPTD1-5 on Salmonella Kentucky. To demonstrate the ability of synthetic phages employing chimeric TSP1, TSP2, and TSP4 to infect target Salmonella strains, including Salmonella Minnesota (TSP1), Salmonella anatum (TSP2), Salmonella enterica Typhimurium 19585 (TSP3), and Salmonella Kentucky (TSP4), limit of detection assays (LODs) were performed using RBP-SPTD1-5. The results are summarized in Table 3A–3E. [Table 3] [Table 4] [Table 5] [Table 6] [Table 7]

[0135]

[0182] Example 10: Construction of TSP-deleted synthetic phage for rapid modularity We use members of the Ackermannviridae family, such as CBA120, to create synthetic TSP-deficient (ΔTSP) phage for use as a modular platform capable of rapid generation of synthetic phages with customizable TSP complexes. One or more TSPs are provided in trans, for example, encoded on a bacterial plasmid, to complement the TSP deficiency. The generation of conditionally replicating TSP-deficient phage is illustrated in Figure 3 and involves the use of HR to replace the TSP gene cluster with an operable marker or reporter, such as luciferase. The synthetic TSP-deficient phage is expected to acquire its TSP from that encoded by a wild-type copy of the phage genome present in the same cell upon recombination. Following this step, recombinants must be isolated and maintained on bacterial strains transformed with a plasmid encoding the TSP or another mechanism for trans-delivery of the TSP. The TSP-deficient phage is conditionally replicative and produces infectious progeny only in the appropriate complementation strain. This dependency on trans-TSP is exploited as follows.

[0136]

[0183] For example, when CBA120.ΔTSP phage is prepared in a bacterial strain expressing TSP1, TSP2, TSP3, and TSP4 from CBA120, the resulting progeny phage recognize the same strain as the CBA120 parent. In another example, when CBA120.ΔTSP is prepared in a bacterial strain expressing TSP1, TSP2, TSP3, and TSP4 from SPTD1, the progeny phage recognize the same strain as the SPTD1 donor. This is illustrated in Figure 4, where a synthetic phage that does not encode its own TSP adopts the TSP (and therefore the host range) of the donor TSP provided in trans. In some cases, the trans TSP can also be a chimeric TSP, with the N-terminal region equivalent to the CBA120 TSP and the C-terminal region equivalent to any other known TSP. Thus, a modular platform is created from TSP-deficient Ackermannviridae phage that allows for the rapid generation of progeny phage with customized host ranges (capacities). Example 11: Phage Sequences [Table 8]

[0137]

[0184] This application contains a Sequence Listing that has been submitted electronically in xml format and is incorporated herein by reference in its entirety. The Sequence Listing does not exceed the disclosure in this international application as filed. The file containing the Sequence Listing, created on November 6, 2023, has the file name 057618-1413719 PhDx 2022-01-PCTWO.xml and is 348,160 bytes in size.

[0138]

[0185] It is understood that the examples and embodiments described in this disclosure are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art and are intended to be included within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited in this disclosure are incorporated herein by reference in their entirety for all purposes. References Nobrega et al.Targeting mechanisms of tailed bacteriophages.Nat Rev Microbiol 16,760-773(2018).doi:10.1038 / s41579-018-0070-8

[0186] Prokhorov et al.Function of bacteriophage G7C esterase tailspike in host cell adsorption.Mol Microbiol.105(3):385-398(2017).doi:10.1111 / mmi.13710 Leiman et al.Morphogenesis of the T4 tail and tail fibers.Virol J 7,355(2010).doi:10.1186 / 1743-422X-7-355 Sorensen et al.Subtypes of tail spike proteins predicts the host range of Ackermannviridae phages.Comput Struct Biotechnol J.Aug 21;19:4854-4867(2021).doi:10.1016 / j.csbj.2021.08.030 Greenfield et al.Structure and function of bacteriophage CBA120 ORF211(TSP2),the determinant of phage specificity towards E.coli O157:H7.Sci Rep.10(1):15402.(2020)doi:10.1038 / s41598-020-72373-0 Plattner et al.Structure and Function of the Branched Receptor-Binding Complex of Bacteriophage CBA120.J Mol Biol.431(19):3718-3739(2019).doi:10.1016 / j.jmb.2019.07.022 Walter et al.Structure of the receptor-binding protein of bacteriophage det7:a podoviral tail spike in a myovirus.J Virol.82(5):2265-73(2008).doi:10.1128 / JVI.01641-07 Bertozzi Silva et al.Host receptors for bacteriophage adsorption.FEMS Microbiol Lett.363(4):fnw002.(2016)doi:10.1093 / femsle / fnw00 Ackermann Bacteriophage taxonomy.Microbiology Australia 32(2):90-94(2011) Henikoff and Henikoff,1989,”Amino acid substitution matrices from protein blocks” Proc.Natl.Acad.Sci.USA 89:10915-10919. Karlin and Altschul,1993,”Applications and statistics for multiple high-scoring segments in molecular sequences.”Proc.Nat’l.Acad.Sci.USA 90:5873-5787. Chao et al.Structure of Escherichia coli O157:H7 bacteriophage CBA120 tailspike protein 4 baseplate anchor and tailspike assembly domains(TSP4-N).Sci Rep.12(1):2061(2022)doi:10.1038 / s41598-022-06073-2.PMID:35136138;PMCID:PMC8825819

Claims

1. A synthetic phage comprising at least one recombinant tail spike protein (TSP) comprising an N-terminal region and a C-terminal region, wherein (a) a combination of the N-terminal region and at least a portion of the C-terminal region is engineered in the laboratory, and / or (b) the C-terminal region comprises one or more engineered amino acid sequences, and wherein the synthetic phage is constructed from a parent phage that is an Ackermannviridae phage.

2. The synthetic phage of claim 1 , wherein the C-terminal region is capable of recognizing a target host.

3. 3. The synthetic phage of claim 1 or 2, wherein the at least one recombinant TSP confers to the synthetic phage the ability to recognize the target host, the ability to recognize the target host not being present in the parent phage.

4. 4. The synthetic phage of claim 3, wherein the C-terminal region of the at least one recombinant TSP comprises at least one amino acid sequence occurring within the C-terminal region of a TSP derived from a phage that is different from the parent phage and capable of recognizing the target host.

5. 5. The synthetic phage of claim 4, wherein the phage that is different from the parent phage is a non-Ackermannviridae phage.

6. The synthetic phage of claim 5, wherein the C-terminal region of the at least one recombinant TSP comprises at least one amino acid sequence that occurs within the C-terminal region of a TSP in a phage that is unable to infect the at least one host that is recognized by the parent phage.

7. The synthetic phage of any one of claims 1 to 6, wherein the parent phage is a recombinant phage.

8. The synthetic phage of any one of claims 1 to 7, wherein the at least one recombinant TSP is a plurality of recombinant TSPs.

9. 9. The synthetic phage of claim 8, comprising at least two recombinant TSPs capable of recognizing at least two different target hosts.

10. 10. The synthetic phage of any one of claims 1 to 9, wherein the at least one recombinant TSP has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity to SEQ ID NO: 6, 7, 8, 10, 12, 14, or 18.

11. A recombinant tail spike protein (TSP) comprising an N-terminal region and a C-terminal region comprising amino acid sequences derived from an Ackermannviridae phage, wherein (a) a combination of the N-terminal region and at least a portion of the C-terminal region is engineered in the laboratory, and / or (b) the C-terminal region comprises one or more engineered amino acid sequences.

12. The recombinant TSP of claim 11, wherein the C-terminal region is capable of recognizing a target host.

13. 13. The recombinant TSP of claim 11 or 12, wherein the amino acid sequences of the C-terminal region and the N-terminal region are derived from the same Ackermannviridae phage.

14. The recombinant TSP of claim 11 or 12, wherein the amino acid sequences of the C-terminal region and the N-terminal region are derived from different phages.

15. 15. The recombinant TSP of claim 14, wherein the amino acid sequence of the C-terminal region is derived from a non-Ackermannviridae phage.

16. The recombinant TSP of any one of claims 11 to 15, wherein the amino acid sequence of the C-terminal region comprises one or more amino acid sequences of TSPs of SPTD1 phage, CBA120 phage, Det7 phage, or TR2 phage.

17. 16. The recombinant TSP of any one of claims 11-15, wherein the recombinant TSP has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence similarity to SEQ ID NO: 6, 7, 8, 10, 12, 14, or 18.

18. A nucleic acid sequence encoding a recombinant TSP according to any one of claims 11 to 17.

19. 17. A method for modifying a phage host range, comprising modifying a parent phage that is an Ackermannviridae phage to include at least one recombinant tail spike protein (TSP), wherein the at least one recombinant TSP is a recombinant TSP of any one of claims 11-16, thereby generating a synthetic phage with an altered host range.

20. 20. The method of claim 19, wherein the parent phage is capable of recognizing at least one host and the synthetic phage having the modified host range is incapable of recognizing the at least one host.

21. 21. The method of claim 20, wherein the parent phage is capable of recognizing at least one host and the synthetic phage having the modified host range is capable of recognizing at least one target host that is different from the at least one host.

22. 20. The method of claim 19, wherein the synthetic phage is unable to recognize the at least one host.

23. 20. The method of claim 19, wherein the modified host range is broader or narrower than the host range of the parent phage.

24. 24. The method of any one of claims 19 to 23, wherein modifying the parent phage comprises modifying a C-terminal region of at least one tail spike protein (TSP) of the parent phage.

25. 25. The method of claim 24, wherein modifying the C-terminal region of at least the TSP of the parent phage comprises exchanging at least a portion of the C-terminal region of the TSP of the parent phage with the C-terminal region of the recombinant TSP.

26. 26. The method of claim 25, wherein the exchange is performed using homologous recombination.

27. A method for detecting said target host using the synthetic phage of any one of claims 2 to 10, comprising: contacting the sample with the synthetic phage for a period of time sufficient for the synthetic phage to infect the target host; detecting the synthetic phage or a progeny phage of the synthetic phage. wherein positive detection of said synthetic phage or said progeny phage or said synthetic phage indicates that said target host is present in said sample.

28. 28. The method of claim 27, wherein the synthetic phage comprises an indicator gene, and wherein said detecting comprises detecting an indicator protein product produced by the synthetic phage or the progeny phage or the synthetic phage, and wherein positive detection of the indicator protein product indicates that the target host is present in the sample.

29. 29. A kit or system for carrying out the method of claim 27 or 28, comprising said synthetic phage.

30. A method of controlling microorganisms using a synthetic phage according to any one of claims 2 to 10, comprising administering to a subject or contacting a sample, object, device or material with said synthetic phage, wherein said synthetic phage is lytic.

31. 31. The method of any one of claims 27, 28 or 30, wherein the sample is a food sample, an environmental sample, a water sample, a commercial sample, or a clinical sample.

32. 31. A kit or system for carrying out the method of claim 30, comprising said synthetic phage.

33. 1. A method for constructing a synthetic phage, comprising: selecting a parent phage that is capable of infecting a first microorganism and is incapable of infecting a second microorganism, wherein the parent phage is an Ackermannviridae phage; Transforming the first microorganism with a homologous recombination (HR) plasmid containing a nucleic acid sequence encoding a C-terminal region of a tail spike protein (TSP) capable of recognizing the second microorganism and a HR sequence flanking a nucleic acid sequence complementary to the corresponding sequence in the TSP of the parent phage, thereby generating a transformed microorganism; infecting the first microorganism with the parent phage to allow HR to occur between the HR plasmid and the genome of the parent phage, thereby generating a plurality of synthetic phage clones; isolating a clone of the synthetic phage containing the recombinant TSP, thereby constructing the synthetic phage capable of infecting the second microorganism; A method comprising:

34. 1. A method for constructing a synthetic phage with a desired host recognition ability, comprising: (a) providing a virion of a recombinant phage constructed from an Ackermannviridae phage, wherein the virion lacks a tail spike protein-encoding gene (TSP-encoding gene) and comprises a tail spike protein (TSP) capable of recognizing a bacterial host; (b) providing the bacterial host containing one or more plasmids encoding the TSPs with the desired host recognition ability; (c) infecting the bacterial host with the virions of the recombinant phage; (d) producing progeny phage in the bacterial host, wherein the progeny phage virions lack a TSP-encoding gene and comprise a TSP encoded by the one or more plasmids in the bacterial host, and the progeny phage are the synthetic phage having the desired host recognition ability; A method comprising:

35. Step (a) selecting a parent phage capable of infecting a suitable bacterial host to undergo homologous recombination (HR), wherein said parent phage is an Ackermannviridae phage or an engineered phage constructed from said Ackermannviridae phage; Transforming the bacterial host suitable for HR with an HR plasmid comprising a nucleic acid sequence encoding an operable marker and sequences flanking said nucleic acid sequence that are complementary to corresponding sequences flanking the TSP-encoding gene cluster of the parent phage; infecting the bacterial host suitable for HR with the parent phage, allowing HR to occur between the HR plasmid and the genome of the parent phage, thereby generating a plurality of phage clones; isolating a phage clone containing the operable marker from the plurality of phage clones, thereby constructing the recombinant phage; 35. The method of claim 34, comprising:

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