Methods and compositions for preparing bacteriocins and antimicrobial peptides

Abstract

Methods and compositions for the preparation of bacteriocins are described in some embodiments herein. In some embodiments, a pro-polypeptide may be produced by a microbial cell comprising a nucleic acid encoding the pro-polypeptide comprising a desired proportion of bacteriocins that are cis-form and separated by cleavage sites. In some embodiments, microfluidic devices and methods for preparing specified mixtures of antimicrobial peptides and / or bacteriocins are described.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201880060807.0. The application date of Chinese Patent Application No. 201880060807.0 is August 30, 2018, and all the contents are incorporated herein.

[0002] Related Applications

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 720,804, filed on August 21, 2018, and U.S. Provisional Application No. 62 / 552,835, filed on August 31, 2017. The contents of the above applications are expressly incorporated herein by reference in their entirety.

[0004] Concerning a sequence listing, table or computer program listing

[0005] This application is submitted with a sequence listing in electronic format. The sequence listing is provided as a file named SYNG003WOSEQUENCE.TXT, which was created and last saved on August 29, 2018, and is 402,916 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. background

[0007] Microbial organism-mediated processes can be used in a variety of industrial processes to produce target products, such as fermentation in feedstocks. In addition, microbial organisms can be used to manufacture products in a sterile environment, such as manufacturing drugs, biologics, and cosmetics. In addition, populations of microbial organisms participate in maintaining the health and metabolic function of multicellular organisms, such as cultures of microbial flora in the human digestive tract and skin or in plant roots. The efficiency and efficacy of these processes may be affected by the culture conditions and the phenotypic characteristics of the microbial organisms present in the culture.

[0008] Modulating a population of microbial organisms, for example to reduce or eliminate undesirable microbial organisms, can be used to maintain industrial processes and to maintain the health of tissues containing microbial organisms.

[0009] field

[0010] Embodiments herein generally relate to gene products produced for controlling microbial growth. More specifically, some embodiments relate to methods, reagents and microfluidic devices for the preparation of bacteriocin. Bacteriocin can be produced in compositions, such as the specified mixture of bacteriocin, which can be used to control the growth of microbial organism colonies. In some embodiments, antimicrobial peptides and / or bacteriocin are produced in compositions with the ratio of expectation. Summary of the invention

[0011] Some embodiments include methods for preparing bacteriocins. The methods may include expressing a nucleic acid comprising a bacteriocin coding sequence and a second polypeptide coding sequence in a single reading frame, wherein the second polypeptide comprises, consists essentially of, or consists of a bacteriocin or a signal molecule. The nucleic acid may also include a cleavage site coding sequence disposed between the bacteriocin coding sequence and the second polypeptide coding sequence in a single reading frame. Thus, a propolypeptide comprising a bacteriocin, a second polypeptide, and a cleavage site disposed between the bacteriocin and the second polypeptide may be produced. In some embodiments, the methods further include cleaving the cleavage site, thereby separating the bacteriocin and the second polypeptide from each other. Thus, the methods may produce a composition comprising a bacteriocin and a second polypeptide. In some embodiments, the second polypeptide comprises, consists essentially of, or consists of a bacteriocin. In some embodiments, the second polypeptide comprises, consists essentially of, or consists of an antimicrobial peptide. In some embodiments, the second polypeptide comprises, consists essentially of, or consists of a signal molecule. In some embodiments, the expression is performed by a microbial cell that does not produce a functional immunomodulator of at least one bacteriocin. In some embodiments, the microbial cell does not produce any functional immunomodulator of a bacteriocin. In some embodiments, the expression is performed in vitro. In some embodiments, at least one bacteriocin is inactive when it is part of a propolypeptide. In some embodiments, the method further comprises separating the propolypeptide before cutting. In some embodiments, separation comprises affinity purification of the propolypeptide, wherein affinity purification comprises binding to an affinity tag encoded by a nucleic acid. In some embodiments, the nucleic acid comprises three bacteriocin coding sequences in a single reading frame. In some embodiments, at least two bacteriocins are different from each other. In some embodiments, the composition comprises a desired ratio of bacteriocins, or a desired ratio of signal molecules and bacteriocins. In some embodiments, at least a portion of the desired ratio is achieved by the ratio of bacteriocin coding sequences in a single reading frame of the nucleic acid or the ratio of bacteriocin and signal molecule coding sequences. In some embodiments, the desired ratio is further achieved by a second nucleic acid comprising a certain ratio of bacteriocin coding sequences and also comprising a cleavage site between bacteriocin coding sequences. In some embodiments, the desired ratio is achieved by the ratio of bacteriocin coding sequences in a single reading frame of the nucleic acid. In some embodiments, the desired bacteriocin ratio is selected to target an undesirable microbial organism or a population of undesirable microbial organisms, and / or the desired bacteriocin ratio is selected to balance the microbiome population in an animal, human organ, plant, plant root, and / or soil (e.g., in the case of plant roots and soil). In some embodiments, the ratio of the desired bacteriocin and the signal molecule is selected to control the genetic drift of the target microbial cell and stimulate the growth or production of the producing cell.In some embodiments, the desired ratio comprises a ratio of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 2:3, 2:5, 2:7, 2:9, 3:4, 3:5, 3:7, 3:8, 3:10, 4:5, 4:7, 4:9, 5:6, 5:7, 5:8, 5:9, 6:7, 7:8, 7:9, 7:10, 8:9 or 9:10 of the first bacteriocin to the second bacteriocin, wherein the first bacteriocin is different from the second bacteriocin. In some embodiments, the cleavage site is for a wild-type cleavage enzyme, a variant or a synthetic cleavage enzyme, such as an endopeptidase. In some embodiments, the cleavage site is for a cleavage enzyme selected from the group consisting of Arg-C protease, Asp-N endopeptidase, BNPS-skatole, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, chymotrypsin-high specificity, clostripain (clostridial peptidase B), CNBr, enterokinase, factor Xa, formic acid, glutamyl endopeptidase, granzyme B, hydroxylamine, iodosylbenzoic acid, LysC, neutrophil elastase, NTCB (2-nitro-5-thiocyanatobenzoic acid), pepsin (pH 1.3), pepsin (pH > 2), proline endopeptidase, proteinase K, staphylococcal peptidase I, thermolysin, thrombin, or trypsin. In some embodiments, the cleavage site is for a single cleavage enzyme, and wherein the cleavage enzyme does not cleave within the bacteriocin. In some embodiments, at least one cleavage site is for a first cleavage enzyme, another cleavage site is for a second cleavage enzyme, and neither the first cleavage enzyme nor the second cleavage enzyme cleaves within the bacteriocin. In some embodiments, the composition produced by the method further comprises a signal molecule (wherein the nucleotide further comprises a coding sequence for the signal molecule in a single reading frame) and a cleavage site sequence disposed between the signal molecule and the bacteriocin coding sequence. In some embodiments, the signal molecule is selected from: a quorum sensing molecule, a signal transduction receptor ligand, a growth factor, a hormone, and a cytokine, and wherein the signal molecule may be wild type, mutant, or synthetic. In some embodiments, the signal molecule comprises, consists of, or consists essentially of a quorum sensing peptide. In some embodiments, the length of the propolypeptide is no more than about 2000 amino acids. In some embodiments, the method further comprises expressing a second nucleic acid encoding a second propolypeptide, the second propolypeptide comprising two bacteriocins and a cleavage site disposed therebetween, wherein the second propolypeptide is different from the first propolypeptide. In some embodiments, cleavage of the propolypeptide comprises physical treatment of a peptide linker comprised by the cleavage site, wherein the peptide linker is chemically sensitive or pH sensitive. In some embodiments, the method further comprises chemically modifying the bacteriocin. In some embodiments, the bacteriocin is chemically co-translationally modified.In some embodiments, the bacteriocin is chemically modified after cutting. In some embodiments, the propolypeptide as described herein comprises two or more antimicrobial peptides instead of bacteriocin, and after cutting, produces a composition comprising the antimicrobial peptides. In some embodiments, the propolypeptide as described herein comprises one or more antimicrobial peptides and one or more bacteriocins, and after cutting, produces a composition comprising a mixture of antimicrobial peptides and bacteriocin.

[0012] Some embodiments include an isolated nucleic acid comprising a bacteriocin coding sequence and a second polypeptide coding sequence in a single reading frame, wherein the second polypeptide comprises a bacteriocin or a signal molecule, consists essentially of it or consists of it. The isolated nucleic acid may comprise a cleavage site coding sequence disposed between the bacteriocin coding sequences and in a single reading frame. In some embodiments, the second polypeptide comprises a bacteriocin, consists essentially of it or consists of it. In some embodiments, the second polypeptide comprises an antimicrobial peptide, consists essentially of it or consists of it. In some embodiments, the second polypeptide comprises a signal molecule, consists essentially of it or consists of it. In some embodiments, the cleavage site coding sequence encodes a cleavage site for a cleavage enzyme, and wherein the bacteriocin coding sequence does not comprise a cleavage site for a cleavage enzyme. In some embodiments, the nucleic acid comprises three bacteriocin coding sequences in a single reading frame. In some embodiments, the nucleic acid comprises at least 5, 10, 15 or 20 bacteriocin sequences in a single reading frame. In some embodiments, the cleavage site coding sequence is disposed in a frame between any two adjacent bacteriocin and / or signal molecule coding sequences. In some embodiments, at least two bacteriocin sequences encode bacteriocins that are different from each other. In some embodiments, the three bacteriocin sequences are present in a desired ratio or a portion of a desired ratio. In some embodiments, the desired ratio is selected to target an undesirable microbial organism or a population of undesirable microbial organisms. In some embodiments, the desired bacteriocin ratio is selected to balance the microbial community population in animals, human organs, plants, plant roots and / or soil (e.g., in the case of plant roots and soil). In some embodiments, the cleavage site is for a wild-type cleavage enzyme, a variant, or a synthetic cleavage enzyme, such as an endopeptidase. In some embodiments, the cleavage site is for a cleavage enzyme selected from the group consisting of Arg-C protease, Asp-N endopeptidase, BNPS-skatole, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, chymotrypsin-high specificity, clostripain (clostridial peptidase B), CNBr, enterokinase, factor Xa, formic acid, glutamyl endopeptidase, granzyme B, hydroxylamine, iodosylbenzoic acid, LysC, neutrophil elastase, NTCB (2-nitro-5-thiocyanatobenzoic acid), pepsin (pH 1.3), pepsin (pH > 2), proline endopeptidase, proteinase K, staphylococcal peptidase I, thermolysin, thrombin, or trypsin. In some embodiments, the first cleavage site encoding sequence encodes a first cleavage site for a first cleavage enzyme, and wherein the second cleavage site encoding sequence encodes a cleavage site for a second cleavage enzyme different from the first cleavage enzyme, and wherein the bacteriocin does not comprise a cleavage site for either the first cleavage enzyme or the second cleavage enzyme.In some embodiments, the cleavage site comprises a pH- or chemically sensitive linker. In some embodiments, the isolated nucleic acid further comprises a coding sequence of a signal molecule in a single reading frame, wherein the cleavage site coding sequence is arranged between the coding sequence of the signal molecule and an adjacent bacteriocin coding sequence. In some embodiments, the signal molecule is selected from: a quorum sensing molecule, a signal transduction receptor ligand, a growth factor, a hormone and a cytokine, and wherein the signal molecule can be wild type, mutant or synthetic. In some embodiments, the isolated nucleic acid as described herein comprises two or more antimicrobial peptide coding sequences instead of a bacteriocin coding sequence, and encodes a propolypeptide comprising an antimicrobial peptide. In some embodiments, the isolated nucleic acid as described herein comprises one or more antimicrobial peptide coding sequences and one or more bacteriocin coding sequences, and encodes a propolypeptide comprising one or more antimicrobial peptides and one or more bacteriocins.

[0013] Some embodiments include a microbial cell comprising a promoter operably linked to an isolated nucleic acid in any embodiment described herein, wherein the isolated microbial cell does not produce a functional immunomodulator of a bacteriocin encoded by the isolated nucleic acid. In some embodiments, the cell does not produce a functional immunomodulator of any bacteriocin encoded by the isolated nucleic acid.

[0014] Some embodiments include separated propolypeptides, which include two bacteriocins, and / or bacteriocins and signal molecules, a cleavage site disposed between the bacteriocin and / or bacteriocin and the signal molecule; and an affinity tag. In some embodiments, the propolypeptide includes two bacteriocins. In some embodiments, the propolypeptide includes a bacteriocin and a signal molecule. In some embodiments, the propolypeptide includes three bacteriocins. In some embodiments, the propolypeptide includes at least 5, 10, 15 or 20 bacteriocins. In some embodiments, the propolypeptide includes a signal molecule. In some embodiments, the cleavage site is for a cleavage enzyme, and wherein the bacteriocin encoding sequence does not include a cleavage site for a cleavage enzyme. In some embodiments, the cleavage site is for a first cleavage enzyme, wherein another cleavage site is for a second cleavage enzyme different from the first cleavage enzyme, and wherein the bacteriocin does not include a cleavage site for any of the first or second cleavage enzymes. In some embodiments, the separated propolypeptide also includes a co-translational or post-translational modification. In some embodiments, the separated propolypeptide described herein includes two or more antimicrobial peptides instead of bacteriocins. In some embodiments, the separated propolypeptide described herein includes one or more antimicrobial peptides and one or more bacteriocins.

[0015] In some embodiments, a composition comprising two or more bacteriocins selected to target a microbial cell or a microbial cell population is described. Each bacteriocin in the composition can include a portion of a cleavage sequence that has been cut at its N-terminus, C-terminus, or N-terminus and C-terminus, wherein the portion of the cleavage sequence at the N-, C-, or N- and C-terminus of the bacteriocin is a cleavage site for the same or different cleavage enzymes. In some embodiments, at least some bacteriocins also include a tag. In some embodiments, the tag is selected from an affinity tag, a signal sequence, or a stability tag. In some embodiments, the composition also includes a signal molecule in a desired ratio with the bacteriocin, wherein the signal molecule includes a portion of a cleavage sequence that has been cut at its N-terminus, C-terminus, or N-terminus and C-terminus, wherein the portion of the cleavage sequence at the N-, C-, or N- and C-terminus of the signal molecule is used for the cleavage site of the same or different cleavage enzymes. In some embodiments, the ratio of the bacteriocin is selected to target an undesirable microbial organism or an undesirable microbial organism population. In some embodiments, the ratio of bacteriocin is selected to balance the microbial community population in animals, human organs, plants, plant roots and / or soil (e.g., in the case of plant roots and soil). In some embodiments, the composition is formulated for topical or oral administration to human subjects. In some embodiments, the composition is used to balance the microbial community population in, for example, animals, human organs (such as intestinal tract or skin), plants, plant roots and / or soil. In some embodiments, the isolated propolypeptide described herein comprises one or more antimicrobial peptides. The propolypeptide can be cleaved, and the composition can comprise one or more antimicrobial peptides, and the antimicrobial peptide comprises a part of a cleavage sequence at the N-, C-, or N- and C-termini.

[0016] Some embodiments include methods for producing a specified mixture of, for example, bacteriocin and / or antimicrobial protein. The method may include selecting a mixture comprising two or more different bacteriocins and / or antimicrobial proteins. The method may include, in a microfluidic device comprising a discrete coding substrate encoding an antimicrobial peptide or bacteriocin separately: placing a discrete coding substrate encoding an antimicrobial peptide and / or bacteriocin in a specified mixture in communication with an in vitro transcription / translation solution fluid; incubating the discrete coding substrate with an in vitro transcription / translation solution, thereby producing an antimicrobial peptide and / or bacteriocin encoded by the discrete coding substrate; and mixing the antimicrobial peptide and / or bacteriocin in a microfluidic device, thereby producing a specified mixture of an antimicrobial peptide and / or bacteriocin. In some embodiments, the method may also include generating two or more submixtures of a subset of a specified mixture comprising an antimicrobial peptide and / or bacteriocin separately, and combining the submixtures to produce a specified mixture of an antimicrobial peptide and / or bacteriocin. In some embodiments, selecting may also include selecting the stoichiometry of two or more different antimicrobial peptides and / or bacteriocins in a specified mixture. In some embodiments, the designated mixture of antimicrobial peptides and / or bacteriocin comprises a designated stoichiometry, and the submixture is combined to produce a designated stoichiometry. In some embodiments, discrete coding substrates are included in separate chambers. In some embodiments, discrete coding substrates include nucleic acids fixed thereon. In some embodiments, discrete coding substrates (but not other discrete coding substrates) encoding antimicrobial peptides and / or bacteriocins in a designated mixture are placed in communication with in vitro transcription / translation solution fluid. In some embodiments, discrete coding substrates are incubated together with in vitro transcription / translation solution and include in vitro transcription / translation solution flowing into each chamber. In some embodiments, in vitro transcription / translation solution comprises in vitro transcription reagents and / or in vitro translation reagents. In some embodiments, discrete coding substrates encoding antimicrobial peptides and / or bacteriocins in a designated mixture are placed in communication with in vitro transcription / translation solution fluids and include (i) opening a valve so that the source (source) of in vitro transcription / translation solution is placed in communication with discrete coding substrate fluids; (ii) closing a valve so as to suppress the fluid communication between the source of in vitro transcription / translation solution and other discrete coding substrates, or a combination of (i) and (ii). In some embodiments, the antimicrobial peptide and / or bacteriocin in the mixing microfluidic device comprises opening a valve to place a discrete coding substrate in communication with a fluid reservoir fluid, wherein the antimicrobial peptide and / or bacteriocin are mixed in the fluid reservoir. In some embodiments, the method further comprises in situ screening of a mixture of antimicrobial peptides and / or bacteriocin for a desired effect.In some embodiments, screening is for suppressing the growth or reproduction of pathogenic microorganisms in the microbial community in the object (such as skin, intestine, gastrointestinal tract, mammary gland, placenta, tissue, biological fluid, semen, uterus, vagina, follicle, lung, saliva, oral cavity, mucosa, conjunctiva or bile duct). In some embodiments, screening is for the mixture of antimicrobial peptides and / or bacteriocin to the microbial community in the object (such as skin, intestine, gastrointestinal tract, mammary gland, placenta, tissue, biological fluid, semen, uterus, vagina, follicle, lung, saliva, oral cavity, mucosa, conjunctiva or bile duct) without harmful effects. In some embodiments, screening is carried out in real time. For example, screening can be carried out at 120 minutes, 60 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes or 1 minute produced by the specified mixture of antimicrobial peptides and / or bacteriocin. In some embodiments, screening is for the stability of antimicrobial peptides and / or bacteriocin or for the destruction of microbial biofilm. In some embodiments, one or more discrete coding substrates encode the auxiliary protein with antiprotease activity. In some embodiments, the screening is for enhancing the growth or reproduction of non-pathogenic microbial organisms in the microbial community in the object (such as skin, intestine, gastrointestinal tract, breast, placenta, tissue, biological fluid, semen, uterus, vagina, follicle, lung, saliva, oral cavity, mucosa, conjunctiva or bile duct). In some embodiments, one or more discrete encoding substrates encode auxiliary proteins that attract non-pathogenic microbial organisms or enhance the growth or reproduction of non-pathogenic microbial organisms in the microbial community in the object. In some embodiments, the method also includes delivering a specified mixture of antimicrobial peptides and / or bacteriocins to the wound via a tube or membrane, thereby cleaning or dressing the wound. In some embodiments, the desired effect includes antimicrobial activity. In some embodiments, the method also includes screening 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 different mixtures for microbial infection. In some embodiments, the method also includes delivering a mixture of specified antimicrobial peptides and / or bacteriocins in combination with chemical antibiotics and / or phage antibiotics to the object. In some embodiments, the in vitro transcription / translation solution is lyophilized, and further comprises adding water to the in vitro transcription / translation solution. In some embodiments, the method comprises preparing a specified mixture of bacteriocins. Thus, the method may comprise, in a microfluidic device comprising discrete coding substrates each encoding a bacteriocin: placing a discrete coding substrate encoding a bacteriocin in a specified mixture in fluid communication with the in vitro transcription / translation solution; incubating the discrete coding substrate with the in vitro transcription / translation solution to produce a bacteriocin encoded by the discrete coding substrate; and mixing the bacteriocins in the microfluidic device to produce a specified mixture of bacteriocins. In some embodiments, the method comprises preparing a specified mixture that does not contain an antimicrobial bacteriocin.In some embodiments, described method comprises the specified mixture of the preparation antimicrobial peptide.In some embodiments, described method comprises the specified mixture of the preparation antimicrobial peptide that does not comprise bacteriocin.In some embodiments, described method comprises the specified mixture of the preparation bacteriocin and antimicrobial peptide.Should be understood that if method as herein described, microfluidic device or system are used to prepare the specified mixture of the bacteriocin that does not comprise antimicrobial peptide, then this method, microfluidic device or system do not need to comprise the part of selecting antimicrobial peptide as the specified mixture, do not need to comprise the discrete coding substrate of encoding antimicrobial peptide, and do not need to produce or flow any antimicrobial peptide.Should be understood that if method as herein described, microfluidic device or system are used to prepare the specified mixture of the antimicrobial peptide that does not comprise bacteriocin, then this method, microfluidic device or system do not need to comprise the part of selecting bacteriocin as the specified mixture, do not need to comprise the discrete coding substrate of encoding bacteriocin, and do not need to produce or flow any bacteriocin.

[0017] Some embodiments include a microfluidic device for producing a specified mixture of antimicrobial peptides and / or bacteriocins. The device may include: a discrete coding substrate, each of which encodes an antimicrobial peptide and / or bacteriocin; an in vitro transcription / translation solution; a fluid reservoir; and a valve, each valve being arranged on a fluid path between the discrete coding substrate and the fluid reservoir, each valve being configured to regulate the flow between the discrete coding substrate and the fluid reservoir. The device can be configured to be placed in data communication with a processor, which is configured to: based on a specified mixture of antimicrobial peptides and / or bacteriocins, configure a valve to place discrete coding substrates (but not other discrete coding substrates) encoding antimicrobial peptides and / or bacteriocins in the coding mixture in communication with a fluid reservoir; allow in vitro transcription / translation solution to be incubated with discrete coding substrates encoding antimicrobial peptides and / or bacteriocins in a specified mixture, thereby producing antimicrobial peptides and / or bacteriocins in a specified mixture; allow antimicrobial peptides and / or bacteriocins to flow into the fluid reservoir through a valve; and control the flow of fluid in the fluid reservoir, wherein the flow includes the movement of antimicrobial peptides and / or bacteriocins in the fluid reservoir, thereby producing a specified mixture of antimicrobial peptides and / or bacteriocins in the fluid reservoir. In some embodiments, the specified mixture of antimicrobial peptides and / or bacteriocins comprises two or more submixtures, each of which comprises a subset of antimicrobial peptides and / or bacteriocins, is substantially composed of or is composed of them; and the processor is configured to allow each submixture to flow into the fluid reservoir, thereby producing a specified mixture of antimicrobial peptides and / or bacteriocins. In some embodiments, the specified mixture of antimicrobial peptides and / or bacteriocin comprises the sum of the subsets of the antimicrobial peptides and / or bacteriocin of the specified stoichiometry, and wherein the combination of submixtures produces the specified stoichiometry. In some embodiments, discrete coding substrates are included in separate chambers. In some embodiments, discrete coding substrates include nucleic acids fixed thereon. In some embodiments, discrete coding substrates include materials or products selected from: chips, beads, nanoparticles, holes, films, substrates, plastics, metals, glass, polymers, polysaccharides and paramagnetic compounds. In some embodiments, in vitro transcription / translation solution includes in vitro transcription reagents and / or in vitro translation reagents. In some embodiments, the device is portable. In some embodiments, one or more discrete coding substrates encode auxiliary proteins, and the auxiliary proteins include the following, are essentially composed of or are composed of: proteins for stabilizing antimicrobial peptides and / or bacteriocins, proteins with antiprotease activity or proteins for destroying microbial biofilms.In some embodiments, one or more discrete coding substrates encode auxiliary proteins that attract non-pathogenic microbial organisms, or enhance the growth or reproduction of non-pathogenic microbial organisms in the microbial community in the object (such as skin, intestine, gastrointestinal tract, breast, placenta, tissue, biological fluid, semen, uterus, vagina, follicle, lung, saliva, oral cavity, mucosa, conjunctiva or bile duct). In some embodiments, the fluid reservoir is configured to be placed in communication with the tissue fluid of the object. In some embodiments, the tissue comprises a microbial community, and the tissue is such as skin, intestine, gastrointestinal tract, breast, placenta, tissue, biological fluid, semen, uterus, vagina, follicle, lung, saliva, oral cavity, mucosa, conjunctiva or bile duct. In some embodiments, the fluid reservoir is configured to be placed in communication with the wound fluid of the object. In some embodiments, the microfluidic device also includes a fluid channel such as a tube or a membrane, through which the fluid reservoir can be placed in communication with the microbial community or wound fluid, and through which the specified mixture of antimicrobial peptides and / or bacteriocins can be delivered to the microbial community or wound. In some embodiments, wherein each discrete coding substrate encodes different bacteriocins.In some embodiments, the discrete coding substrate comprises a nucleic acid of separation as described herein, and the nucleic acid of the separation comprises the following, is substantially composed of or is composed of: an antimicrobial peptide and / or a bacteriocin coding sequence and a second polypeptide coding sequence in a single reading frame.In some embodiments, the discrete coding substrate encodes a propolypeptide of separation as described herein.In embodiments, the microfluidic device also comprises a processor.In some embodiments, the microfluidic device also comprises a reservoir of chemistry or bacteriophage antibiotics, which is configured to mix with the mixture of specified antimicrobial peptides and / or bacteriocins.In some embodiments, the microfluidic device also comprises an antibiotic, and the antibiotic comprises a chemical antibiotic and / or a bacteriophage antibiotic, is substantially composed of or is composed of.In some embodiments, the in vitro transcription / translation solution is lyophilized.In some embodiments, the microfluidic device is used to produce a specified mixture of bacteriocins. Therefore, the device can include discrete coding substrates, which encode bacteriocins separately; in vitro transcription / translation solution; fluid reservoir; and valves, each valve is arranged on the fluid path between the discrete coding substrate and the fluid reservoir, and each valve is configured to regulate the flow between the discrete coding substrate and the fluid reservoir. In some embodiments, the microfluidic device is used to produce a specified mixture of the bacteriocin that does not comprise an antimicrobial peptide. In some embodiments, the microfluidic device is used to produce a specified mixture of the antimicrobial peptide that does not comprise a bacteriocin. In some embodiments, the microfluidic device is used to produce a specified mixture of a bacteriocin and an antimicrobial peptide.

[0018] Some embodiments include system. The system may include microfluidic device as described herein and processor, the processor being configured to: based on the specified mixture of antimicrobial peptides and / or bacteriocin (as described herein), configure valve to place discrete coding substrates (but not other discrete coding substrates) of antimicrobial peptides and / or bacteriocin in the coding mixture in communication with fluid reservoir fluid; allow in vitro transcription / translation solution to be incubated with discrete coding substrates of antimicrobial peptides and / or bacteriocin in the coding specified mixture, thereby produce antimicrobial peptides and / or bacteriocin in the specified mixture; allow antimicrobial peptides and / or bacteriocin to flow into fluid reservoir through valve; and control the flow of the fluid in the fluid reservoir, wherein the flow includes the movement of antimicrobial peptides and / or bacteriocin in the fluid reservoir, thereby produce the specified mixture of antimicrobial peptides and / or bacteriocin in the fluid reservoir. In some embodiments, the microfluidic device is included in a cartridge, and the system includes a coupling for placing the cartridge in data communication with the processor. In some embodiments, the system also includes the reservoir of in vitro transcription / translation solution. In some embodiments, the system also includes a reservoir of chemical and / or bacteriophage antibiotics, which are configured to mix with a specified antimicrobial peptide and / or bacteriocin mixture. In some embodiments, the system also includes antibiotics, which include chemical antibiotics and / or bacteriophages, are essentially composed of or are composed of them. In some embodiments, the in vitro transcription / translation solution is lyophilized. In some embodiments, the system is used to produce a specified mixture of bacteriocins. Therefore, the processor can be configured to, based on a specified mixture of antimicrobial peptides and / or bacteriocins (as described herein), configure the valve to place the discrete coding substrate (rather than other discrete coding substrates) of the bacteriocin in the coding mixture in communication with the fluid reservoir fluid; allow the in vitro transcription / translation solution to be incubated with the discrete coding substrate of the antimicrobial peptide and / or bacteriocins in the coding specified mixture, thereby producing the bacteriocin in the specified mixture. In some embodiments, the system is used to produce a specified mixture of bacteriocins that do not include antimicrobial peptides. In some embodiments, the system is used to produce a specified mixture of antimicrobial peptides. In some embodiments, the system is used to produce a specified mixture of antimicrobial peptides. In some embodiments, the system is used to produce a specified mixture of antimicrobial peptides. In some embodiments, the system is used to produce a specified mixture of antimicrobial peptides that does not include a bacteriocin. In some embodiments, the system is used to produce a specified mixture of a bacteriocin and an antimicrobial peptide.

[0019] Some embodiments include the microfluidic device for producing the specified mixture of antimicrobial peptides and / or bacteriocin.Described device can include the discrete coding substrates of encoding antimicrobial peptides and / or bacteriocin separately, and be arranged on the valve on the fluid path connected to discrete coding substrate separately.Each valve can be configured to regulate the flow to or flow from discrete coding substrate.Described device can be configured to be placed in and be communicated with fluid reservoir and / or in vitro transcription / translation solution fluid.In some embodiments, described device also includes fluid reservoir or in vitro transcription / translation solution.Some embodiments include the microfluidic device for producing the specified mixture of bacteriocin.Described device can include the discrete coding substrates of encoding bacteriocin separately, and be arranged on the valve on the fluid path leading to or from discrete coding substrate separately, and it is configured to place discrete coding substrate in and be communicated with fluid reservoir and / or in vitro transcription / translation solution fluid. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of a conventional method for preparing a composition comprising bacteriocins. As shown, multiple strains are used to produce a mixture of bacteriocins. However, it is considered that there is a high probability that different bacteriocins will have different concentrations ([A] ≠ [B] ≠ [C] ≠ [D]). In addition, it is considered that the produced bacteriocins may be toxic to the host, thereby causing production problems.

[0022] Figure 2 Schematic diagram of an embodiment of a nucleic acid encoding a propolypeptide comprising a bacteriocin and a cleavage site according to some embodiments of the present invention. As shown, a single bacterium can produce a propolypeptide encoded by a synthetic gene. It is contemplated that the molecular ratio of a bacteriocin can be altered by placing multiple copies of the bacteriocin in the propolypeptide.

[0023] Figure 3 is a schematic diagram of an embodiment of a composition comprising a bacteriocin and a cleavage site according to some embodiments herein.

[0024] Figure 4A is a schematic diagram of an embodiment of a propolypeptide comprising a bacteriocin, a cleavage site, and a signal molecule according to some embodiments herein.

[0025] Figure 4B is a schematic diagram of an embodiment of a composition comprising a bacteriocin and a signal molecule after cleavage of a propolypeptide according to some embodiments herein.

[0026] Figure 5 is a schematic diagram depicting a microfluidic device according to some embodiments herein.

[0027] Figure 6 is a schematic diagram depicting a microfluidic device according to some embodiments herein.

[0028] Figure 7 is a flow chart illustrating a method for producing a specified mixture of antimicrobial peptides and / or bacteriocins according to some embodiments herein.

[0029] Figure 8 is a flow chart illustrating a method for preparing an antimicrobial peptide and / or bacteriocin according to some embodiments herein.

[0030] Details

[0031] According to some embodiments herein, methods and compositions for preparing bacteriocins in desired ratios are described. Some embodiments include compositions containing bacteriocins in precise ratios or stoichiometric amounts, which can be used to regulate populations of microbial organisms in many applications, such as industrial biotechnology manufacturing processes, pharmaceutical, biological and cosmetic manufacturing, and medical applications. In some embodiments, a single propolypeptide containing multiple bacteriocins separated by cleavage sites is encoded by a nucleic acid and produced by a genetically engineered microbial cell containing the nucleic acid (see, e.g., Figure 2 In some embodiments, the propolypeptide may also contain a signaling molecule that can regulate the growth of microbial cells and / or multicellular host cells. The bacteriocin in the propolypeptide may be in an inactive form, so that the microbial cells do not need immunity to these bacteriocins. The propolypeptide may be isolated, for example, by affinity purification. The cleavage site of the propolypeptide may be cleaved (see, e.g., Figure 3 ), thereby producing a mixture of active bacteriocins. Conceptually, propolypeptides can be envisioned as "spaghetti" chains containing multi-component bacteriocins, which can then be cleaved into individual bacteriocins. Note that in this application, propolypeptides may also be referred to as "spaghetti" for the sake of brevity. Some embodiments include microfluidic devices and methods for preparing specified mixtures of bacteriocins. The "specified mixture" of bacteriocins in the methods and microfluidic devices of some embodiments of the present invention can be specified according to parameters, including but not limited to the composition of the bacteriocins (so that specified bacteriocins are included and / or other bacteriocins are not included), the stoichiometry of the bacteriocins in the mixture, and optionally the presence of auxiliary proteins. The microfluidic device may include discrete encoding substrates (e.g., see Figure 5). The discrete coding substrates encoding the bacteriocin in the specified mixture can be placed in communication with the in vitro transcription / translation solution fluid, so that the discrete coding substrates encoding the bacteriocin in the specified mixture are incubated with the in vitro transcription / translation solution, thereby producing the bacteriocin in the specified mixture. The bacteriocin in the specified mixture can then be placed in communication with the fluid reservoir fluid of the microfluidic device, and the bacteriocin moves fluidically to the fluid reservoir, thereby producing the specified mixture of antimicrobial peptides and / or bacteriocin in the fluid reservoir. In some embodiments, each discrete coding substrate encodes different bacteriocins. In some embodiments, the discrete coding substrate encodes two or more different bacteriocins. In some embodiments, at least one or all discrete coding substrates encode "spaghetti" propolypeptides as described herein. Optionally, the discrete coding substrates can include or encode proteases as described herein, which can cut the cleavage site of the propolypeptide so that the discrete coding substrates can produce two or more bacteriocins of a specified stoichiometry. The microfluidic device can include an outlet, which is configured to place the specified mixture of antimicrobial peptides and / or bacteriocin in communication with tissue, wound, host microbial community or vessel fluid. A given mixture of bacteriocins can be used, for example, as a point-of-care antimicrobial composition, and / or tested for the effect of a given mixture on host tissue and / or host microbiota (eg, containing undesirable microbial organisms).

[0032] Advantageously, the relative amounts of the different bacteriocins in the propolypeptide will accurately determine the ratio of bacteriocins in the mixture after cleavage, and this ratio will be maintained regardless of the expression rate of the polypeptides within or between different microbial cells. In contrast, conventional methods in which bacteriocins are translated as separate peptides or produced by different cells (see Figure 1 ) may be subject to changes in transformation efficiency, genetic engineering efficiency and / or gene expression efficiency (e.g., if some bacteriocin encoding plasmids replicate and / or initiate transcription more efficiently than other plasmids), so that the final ratio of bacteriocins will not be as accurate as the embodiments herein. As another advantage, since each nucleic acid can encode a variety of bacteriocins, some embodiments herein provide genetic modification of host cells more effectively than conventional methods, which involve the introduction of a variety of constructs, each of which encodes a specific bacteriocin. As another advantage, since bacteriocins can be produced in their inactive form in propolypeptides, the "workhorse" microbial organisms that produce bacteriocins do not need to be configured with immunity to all, some or any of these bacteriocins, and can be easily used to produce a mixture of any of many different bacteriocins. Although in some embodiments, the microbial organisms produced can be naturally or artificially configured with immunity to one or more or all of the bacteriocins contained in the propolypeptides.

[0033] The population of microbial organisms targeted by the bacteriocin compositions of some embodiments herein can be in commercially useful environments, such as industrial cultures, fermenters, pharmaceutical, biological and cosmetic manufacturing; in microbial communities, such as human organs, animals and plants (e.g., roots or soil where plants grow); and in products, such as food (for humans and / or animals), medicines and cosmetics, in a certain amount. Without being limited by theory, it is considered that the populations of microbial organisms in any of these environments do not necessarily coexist in a stable state. For example, as reviewed in Jaramillo et al., Nature Microbiology 2: 17119 (2017), because the populations of microbial organisms interact with each other (directly or indirectly), they can affect each other in a positive or negative manner, but are not expected to exist in a stable state, which generally leads to challenges in maintaining co-cultivation. In some embodiments, when a population of certain microbial organisms reaches a number or density above a certain threshold, a mixture of bacteriocins produced by the methods of some embodiments herein and / or by using propolypeptides and / or nucleic acids and / or microbial cells can be added to the environment to target the microbial cells of the population. For example, according to some embodiments, if a population contains a large excess of microbial organism #1 and a small excess of microbial organism #2, a mixture comprising a relatively high ratio of bacteriocin #1 (targeting microbial organism #1) to bacteriocin #2 (targeting microbial organism #2) can be administered to the population to reduce the growth of microbial organisms #1 and #2, with a greater reduction targeting microbial organism #1.

[0034] Bacteriocins and antimicrobial peptides

[0035] As used herein, "bacteriocin" and variations of this root term have their usual and common meanings as understood by those skilled in the art in view of the present disclosure. It refers to a polypeptide that is secreted by a host cell and can neutralize at least one cell other than the single host cell that prepares the polypeptide, including cells associated with the host cell and other microbial cell clones. "Bacteriocin" also encompasses cell-free or chemically synthesized forms of such polypeptides. Cells expressing a specific "immunomodulator" (discussed in more detail herein) are immune to the neutralizing effect of a specific bacteriocin or bacteriocin group. Therefore, bacteriocins can neutralize cells and / or other microbial cells that produce bacteriocins, as long as these cells do not produce appropriate immunomodulators. Therefore, host cells can exert cytotoxic or growth-inhibiting effects on a variety of other microbial organisms by secreting bacteriocins. A detailed description of bacteriocins (including methods and compositions for controlling microbial cell growth using bacteriocins) can be found in, for example, U.S. Patent No. 9,333,227, which is incorporated herein by reference in its entirety. "Bacteriocin" is not limited by the source of the polypeptide, and for example, it is considered to encompass any bacteriocin, such as naturally occurring bacteriocins, synthetic bacteriocins, and variants and combinations thereof.

[0036] The bacteriocin of some embodiments is initially produced in a propolypeptide, which can then be cleaved as described herein to produce a separate bacteriocin. In some embodiments, the propolypeptide is produced by the translation machinery (e.g., ribosomes, etc.) of a microbial cell. In some embodiments, the propolypeptide is produced by an in vitro expression system. The propolypeptide can be cleaved (e.g., processed by a cleavage enzyme such as a naturally occurring or synthetic protease) to produce the polypeptide of the bacteriocin itself. Therefore, in some embodiments, the bacteriocin is produced by a precursor polypeptide. In some embodiments, the bacteriocin comprises a polypeptide that has been post-translationally modified (e.g., cleaved) or has one or more functional groups added. In some embodiments, a propolypeptide as described herein is chemically synthesized, which comprises a bacteriocin and a cleavage site, is essentially composed of it, or is composed of it.

[0037] "Antibiotic" and variations of this root term have their usual and ordinary meaning as understood by those skilled in the art in view of the present disclosure. It refers to a metabolite or intermediate of a metabolic pathway that can kill or prevent the growth of at least one microbial cell. Some antibiotics can be produced by microbial cells such as bacteria. Some antibiotics can be chemically synthesized. It should be understood that bacteriocins differ from antibiotics at least in that bacteriocins refer to gene products (which, in some embodiments, undergo additional post-translational processing) or synthetic analogs thereof, while antibiotics refer to intermediates or products of metabolic pathways or synthetic analogs thereof.

[0038] The neutralizing activity of bacteriocins can include, for example, preventing microbial reproduction or cytotoxicity. Some bacteriocins have cytotoxic activity (e.g., "bactericide" effect), and are therefore capable of killing microbial organisms, such as bacteria, yeasts, algae, synthetic microorganisms, etc. Some bacteriocins can inhibit the reproduction of microbial organisms, such as bacteria, yeasts, algae, synthetic microorganisms, etc., for example, by preventing the cell cycle (e.g., "bacteriostatic" effect). In some, the neutralizing effect includes, consists of, or consists essentially of: preventing microbial reproduction, inhibiting the reproduction of microbial organisms and / or cytotoxicity.

[0039] In some embodiments, based on the desired microbial regulation type and the specific strain or species of the targeted microbial organism, select a specific neutralization activity or activity range (such as cytotoxicity or microbial reproduction prevention). Therefore, in some embodiments, select a specific bacteriocin or a combination of bacteriocins. For example, in some embodiments, based on the regulated cell, the microbial cell is engineered to express a specific bacteriocin. In some embodiments, for example, if contaminated cells are to be killed, at least one cytotoxic bacteriocin is provided. In some embodiments, select a bacteriocin or a combination of bacteriocins that effectively resists the pollutants that are usually present in a specific culture or a specific geographical location or a specific type of culture grown in a specific geographical location. In some embodiments, for example, wherein the embodiment of the reversible regulation of the microbial cell ratio is desired, a bacteriocin that inhibits microbial reproduction is provided. Without being limited by any particular theory, many bacteriocins can have neutralization activity to microbial organisms, and the microbial organisms usually occupy the same niche as the species that produce bacteriocins. Therefore, in some embodiments, when the specific spectrum of bacteriocin activity is desired, the bacteriocin is selected from the host species that occupies the same (or similar) niche as one or more microbial organisms targeted by the bacteriocin. In some embodiments, a particular mixture and / or ratio is selected to target a single microbial organism (which may include targeting one or more microbial organisms of that type, such as clonally related microbial organisms). For example, a predetermined mixture and / or ratio of bacteriocins may more effectively target a particular type of microbial organism than a single bacteriocin.

[0040] In some embodiments, one or more bacteriocin activities are selected before the culture is grown, and a polypeptide progenitor of a bacteriocin containing a desired stoichiometry is prepared. Polynucleotides encoding propolypeptides can be prepared, for example, using nucleic acid synthesis and / or molecular cloning, and can be used to produce propolypeptides. Polynucleotides can be transcribed (if DNA) and translated using a variety of suitable systems, for example in microbial cells, or in an in vitro expression system. In some embodiments, bacteriocins (and their ratios) can be selected based on the ability of bacteriocins to neutralize one or more invading organisms, which may attempt to grow in a specific culture. In some embodiments, bacteriocins can be used to limit the ability of particularly useful microbial strains in the environment, for example in industrial raw materials, or in a fermenter, or in a food, drug or cosmetic manufacturing environment, or in a tissue environment such as the intestinal tract or the skin microbial community, or in the ability to maintain or regulate the microbial population in plants, plant roots and / or soil, or to maintain or maintain the quality of food, medicine or cosmetics. In some embodiments, one or more bacteriocin activities (and / or ratios) are selected based on one or more microbial strains or microbial strain populations in an existing environment. For example, in some embodiments, if a specific invader is identified in the environment, a set of neutralizing bacteriocins (and their ratios) can be selected to neutralize the identified invader. In some embodiments, bacteriocins are selected to neutralize all or substantially all microbial cells in the environment, for example to eliminate industrial cultures in a culture environment so that new industrial cultures can be introduced into the culture environment, or to prevent or inhibit contamination of a pharmaceutical or cosmetic manufacturing environment, or to prevent or minimize contamination or spoilage of food, pharmaceuticals, or cosmetics.

[0041] For example, in some embodiments, antifungal activity (such as anti-yeast activity) is desired. A number of bacteriocins with antifungal activity have been identified. For example, bacteriocins from Bacillus have been shown to have neutralizing activity against yeast strains (see Adetunji and Olaoye (2013) Malaysian Journal of Microbiology 9: 130-13, which is hereby incorporated by reference in its entirety), Enterococcus faecalis ( Enterococcus faecalis ) peptide (WLPPAGLLGRCGRWFRPWLLWLQ SGAQY KWLGNLFGLGPK, SEQ ID NO: 1) has been shown to have neutralizing activity against Candida species (see Shekh and Roy (2012) BMC Microbiology 12: 132, which is hereby incorporated by reference in its entirety), and bacteriocins from Pseudomonas have been shown to have neutralizing activity against fungi such as Curvularia lunula ( Curvularia lunata), Fusarium species, Helminthosporium species, and Biopolaris species have neutralizing activity (Shalani and Srivastava (2008) The Internet Journal of Microbiology. Volume 5, Number 2. DOI: 10.5580 / 27dd—accessed on the World Wide Web at archive.ispub.com / journal / the-internet-journal-of-microbiology / volume-5-number-2 / screening-for-antifungal-activity-of-pseudomonas-fluorescens-against-phytopathogenic-fungi.html#sthash.d0Ys03UO.1DKuT1US.dpuf, hereby incorporated by reference in its entirety). For example, staphylococcin AJ1316 from Bacillus subtilis (see Zuber, P et al. (1993) Peptide Antibiotics. In Bacillus subtilis and Other Gram-Positive Bacteria: Biochemistry, Physiology, and Molecular Genetics ed Sonenshein et al., pp. 897-916, American Society for Microbiology, incorporated herein by reference in its entirety) and Alirin B1 (see Shenin et al. (1995) Antibiot Khimioter 50: 3-7, incorporated herein by reference in its entirety) have been shown to have antifungal activity. Thus, in some embodiments, such as embodiments where neutralization of fungal microorganisms is desired, the bacteriocin comprises at least one of staphylococcin AJ1316 or Alirin B1.

[0042] For example, in some embodiments, the bacteriocin activity in the culture of a specific microorganism (or the set of different microorganisms) is desirable, and selects bacteriocin to neutralize the microorganism except the desired microorganism with a predetermined ratio. The bacteriocin produced by the desired microorganism can be selected usually, because the desired microorganism organism can produce the relevant immunomodulator for these bacteriocins, or can be easily engineered to produce immunomodulator. Therefore, the selected bacteriocin can target undesirable microbial cells, and hardly causes or does not cause the neutralization of the desired microorganism simultaneously. For example, in some embodiments, the bacteriocin of a specific ratio is selected to neutralize the invasion microorganism organism that is usually present in the cyanobacteria culture environment, while retaining cyanobacteria. The cluster of conservative bacteriocin polypeptides has been identified in multiple cyanobacteria species. For example, at least 145 putative bacteriocin gene clusters have been identified in at least 43 cyanobacterial species, such as Wang et al. (2011), Genome Mining Demonstrates the Widespread Occurrence of Gene Clusters Encoding Bacteriocins in Cyanobacteria. PLoS ONE 6(7): e22384, which is incorporated herein by reference in its entirety. Exemplary cyanobacterial bacteriocins are shown in Table 1.2, such as SEQ ID NO:420, SEQ ID NO:422, SEQ ID NO:424, SEQ ID NO:426, SEQ ID NO:428, SEQ ID NO:430, SEQ ID NO:432, SEQ ID NO:434, SEQ ID NO:436, SEQ ID NO:438, SEQ ID NO:440, SEQ ID NO:442, SEQ ID NO:444, SEQ ID NO:446, SEQ ID NO:448, and SEQ ID NO:450.

[0043] In some embodiments, microbial cells produce bacteriocins. In some embodiments, bacteriocins neutralize cells of different species or strains from microbial cells producing bacteriocins. In some embodiments, if these cells lack appropriate immunomodulators, bacteriocins neutralize cells of the same species or strains as host cells. Because bacteriocins can mediate the neutralization of host and non-host microbial organisms, it will be readily understood by those skilled in the art that bacteriocins are different from poison-antidote systems, which relate to host microorganisms that can only neutralize their own endogenous mechanisms. In other words, bacteriocins can neutralize cells other than the cells producing them (for example, bacteriocins can be selected and / or engineered to serve as niche protectors), and poison molecules only kill the individual cells producing them (for example, serving as suicide systems).

[0044] Many bacteriocins have been identified and characterized. Without being limited by theory, exemplary bacteriocins can be classified as "Class I" bacteriocins that are usually post-translationally modified and "Class II" bacteriocins that are usually unmodified. In addition, exemplary bacteriocins in each category can be classified into different subgroups, as summarized in Table 1.1, which is adapted from Cotter, PD et al., "Bacteriocins- a viable alternative to antibiotics" Nature Reviews Microbiology (2013) 11: 95-105, incorporated herein by reference in its entirety.

[0045] Without being limited by theory, bacteriocins can achieve neutralization of target microbial cells in a variety of ways. For example, bacteriocins can permeabilize the cell wall, thereby depolarizing the cell wall and interfering with respiration.

[0046] Table 1.1: Classification of exemplary bacteriocins

[0047] According to the embodiments of the present invention, a variety of bacteriocins can be used. Exemplary bacteriocins are shown in Table 1.2. In some embodiments, at least one bacteriocin is provided, which comprises the polypeptide sequence in Table 1.2, is essentially composed of it, or is composed of it. As shown in Table 1.2, some bacteriocins act as molecular pairs. Therefore, it should be understood that, unless otherwise explicitly stated, when discussing functional "bacteriocins" or "providing bacteriocins" and the like, functional bacteriocin pairs are included together with bacteriocins that act alone. With reference to Table 1.2, the "source organisms" listed in brackets represent alternative names and / or strain information of organisms known to produce the bacteriocins shown. However, in some embodiments of the present invention, bacteriocins are produced by desired microbial organisms, and are therefore not limited to examples of organisms known to produce the bacteriocins shown in Table 1.2.

[0048] Embodiments herein also include peptides and proteins identical to the bacteriocins described in Table 1.2. The term "identity" has its usual and common meaning as understood by those skilled in the art in view of the present disclosure. It is intended to include nucleic acid or protein sequence homology or three-dimensional homology. As used herein, "variants" of polypeptides, such as bacteriocins, signaling molecules, immunomodulators, tags (or any other component peptides of the original polypeptides described herein), have their usual and common meanings as understood by those skilled in the art in view of the present disclosure. It should be understood that the identities described herein include at least about 70%, such as at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the reference sequence, including ranges between any two of the listed values, such as 70%-100%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity. 5%-100%, 80%-100%, 85%-100%, 90%-100%, 95%-100%, 97%-100%, 99%-100%, 70%-99%, 75%-99%, 80%-99%, 85%-99%, 90%-99%, 95%-99%, 97%-99%, 70%-95%, 75%-95%, 80%-95%, 85%-95%, 90%-95%, 70%-90%, 75%-90%, 80%-90% and 85%-90%. There are several techniques for determining nucleic acid or polypeptide sequence homology and / or three-dimensional homology to a polypeptide. These methods are generally used to find the degree of identity that a sequence, domain or model has with a target sequence, domain or model. Percent identity can be determined using the BLAST software (Altschul, SF, et al., (1990) "Basic local alignment search tool." J. Mol. Biol. 215:403-410, accessible on the World Wide Web at blast.ncbi.nlm.nih.gov) with default parameters.

[0049] A wide range of functional bacteriocins can incorporate the features of the bacteriocins disclosed herein, thereby providing a high degree of identity with the bacteriocins in Table 1.2. In some embodiments, the bacteriocin has at least about 50% identity to any of the polypeptides in Table 1.2, such as at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145 8%, 99% or 100% identity, including ranges between any two of the listed values, for example, 70%-100%, 75%-100%, 80%-100%, 85%-100%, 90%-100%, 95%-100%, 97%-100%, 99%-100%, 70%-99%, 75%-99%, 80%-99%, 85%-99%, 90%-99%, 95%-99%, 97%-99%, 70%-95%, 75%-95%, 80%-95%, 85%-95%, 90%-95%, 70%-90%, 75%-90%, 80%-90%, and 85%-90%.

[0050] In some embodiments, a bacteriocin having the same identity as the bacteriocin in Table 1.2 is provided, wherein the bacteriocin has been modified to remove one or more cleavage sites for the propolypeptide described herein. Without being limited by theory, it is contemplated that modifying the bacteriocin to remove the cleavage site (e.g., by making conservative substitutions, such as a small non-polar amino acid to a small non-polar amino acid, such as Leu→Ile, or a small polar amino acid to a small polar amino acid, such as Ser→Thr) can prevent the bacteriocin itself from being cleaved when the propolypeptide is cleaved as described herein. Therefore, in some embodiments, the bacteriocin in the propolypeptide as described herein (e.g., a bacteriocin or a variant thereof in Table 1.2) does not contain any cleavage site for the bacteriocin (or other element) used to separate the propolypeptide.

[0051] Table 1.2: Exemplary bacteriocins

[0052] As used herein, "bacteriocin polynucleotide" has its usual and ordinary meaning as understood by those skilled in the art in view of the present disclosure. It refers to a polynucleotide encoding a bacteriocin. As used herein, "bacteriocin coding sequence" has its usual and ordinary meaning as understood by those skilled in the art in view of the present disclosure. It refers to a nucleic acid coding sequence (e.g., RNA or DNA) encoding the polypeptide sequence of a bacteriocin. In some embodiments, the host cell comprises at least one bacteriocin.

[0053] Antimicrobial peptides are peptides that give innate immune activity to kill or stop the growth of microorganisms. " Antimicrobial peptides " used herein (comprising the variant of this root term) has its common and common implication as those of ordinary skill in the art understand in view of the present disclosure. Classically, antimicrobial peptides have been described as peptides produced by the innate immune system of invertebrates and vertebrates. Therefore, although bacteriocin has been classically referred to as a class of microbial gene products of target microorganisms, antimicrobial peptides have been classically referred to as a class of invertebrates and vertebrate gene products of target microorganisms. The example of antimicrobial peptides that are applicable to the method, microfluidic device and system of some embodiments of this paper is known in the art, and can be found in the Antimicrobial Peptide Database (Antimicrobial Peptide Database) that for example is accessed with aps.unmc.edu / AP / on the World Wide Web, which is incorporated herein by reference in its entirety. Identified and classified more than 1000 antimicrobial peptides and variants thereof. The antimicrobial peptide database is described in Wang et al. (2016), Nucleic Acids Res. 44 (Database issue): D1087-D1093, which is incorporated herein by reference in its entirety. Examples of antimicrobial peptides include bacteriocins, antibacterial, antiviral, anti-HIV, antifungal, antiparasitic and anticancer peptides, such as Dermaseptin-B2, Abaecin, Ct-AMP1, Andropin, Aurein1.1, Lactoferricin B and Heliomicin. The methods, compositions, systems and microfluidic devices of some embodiments include naturally occurring antimicrobial peptides or nucleic acids encoding them. The methods, compositions, systems and microfluidic devices of some embodiments include non-naturally occurring antimicrobial peptides or nucleic acids encoding them. The methods, compositions, systems and microfluidic devices of some embodiments include antimicrobial peptides that contain mutations or variations in naturally occurring antimicrobial peptides, or nucleic acids encoding them. The methods, compositions, systems, and microfluidic devices of some embodiments comprise antimicrobial peptides or nucleic acids encoding them that comprise, consist essentially of, or consist of non-naturally occurring peptide sequences.

[0054] It is also contemplated that the method, system and microfluidic device of some embodiments herein can be combined with naturally occurring antimicrobial peptides, variants of naturally occurring antimicrobial peptides and / or synthetic antimicrobial peptides. Therefore, the antimicrobial peptides in the method, system and device of some embodiments can comprise the following, be basically composed of or be composed of: naturally occurring antimicrobial peptides, variants of naturally occurring antimicrobial peptides and / or synthetic antimicrobial peptides. In some embodiments, the variant antimicrobial peptides are combined with reference antimicrobial peptides (for example frog skin antimicrobial peptide-B2, Abaecin, Ct-AMP1, Andropin, Aurein 1.1, Lactoferrin peptide B or Heliomicin) has at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, including ranges between any two recited values, such as 70%-100%, 75%-100%, 76%-100%, 77%-100%, 78%-100%, 79%-100%, 80%-100%, 81%-100%, 82%-83%, 84%-85%, 86%-87%, 88%-89%, 90%-91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity. 100%, 80%-100%, 85%-100%, 90%-100%, 95%-100%, 97%-100%, 99%-100%, 70%-99%, 75%-99%, 80%-99%, 85%-99%, 90%-99%, 95%-99%, 97%-99%, 70%-95%, 75%-95%, 80%-95%, 85%-95%, 90%-95%, 70%-90%, 75%-90%, 80%-90% and 85%-90%. No matter where bacteriocin is mentioned in this paper, it is clearly considered that any antimicrobial peptide as described herein can replace one, two or more or all of the bacteriocins (although it should be understood that in some contexts, as bacteriocin immunomodulators, antimicrobial peptides will have characteristics different from bacteriocins and interact differently). Therefore, some embodiments of the method described herein and microfluidic device only include bacteriocin, some embodiments of the method described herein and microfluidic device only include antimicrobial peptides, and some embodiments of the method described herein and microfluidic device include the combination of bacteriocin and antimicrobial peptides. Therefore, in the method and composition of some embodiments, propolypeptide comprises antimicrobial peptides instead of bacteriocin. Therefore, in the method and composition of some embodiments, propolypeptide comprises one or more antimicrobial peptides and one or more bacteriocins.

[0055] Bacteriocin immunomodulators

[0056] Exemplary bacteriocin immunomodulators are shown in Table 2. Although the immunomodulators in Table 2 are naturally occurring, it will be appreciated by those skilled in the art that variants of the immunomodulators in Table 2, naturally occurring immunomodulators other than the immunomodulators in Table 2, or synthetic immunomodulators may be used according to some embodiments herein. In some embodiments, the microbial cell that produces the propolypeptide comprising the bacteriocin does not produce an immunomodulator against at least one bacteriocin. Without being limited by theory, it is considered that in some embodiments, the bacteriocin in the propolypeptide is inactive and therefore has little or no ability to neutralize the cells that produce them (or cells associated with the cell clones that produce them), so that the cell does not need immunity against these bacteriocins in the propolypeptide.

[0057] In some embodiments, a specific immunomodulator or a specific combination of immunomodulators confers immunity to a specific bacteriocin, a specific classification or class of bacteriocin, or a specific combination of bacteriocins. Exemplary bacteriocins are identified in Table 2, and immunomodulators can confer immunity to the exemplary bacteriocins. Although Table 2 identifies the "source organism" of exemplary immunomodulators, according to some embodiments herein, these immunomodulators can be easily expressed in other naturally occurring, genetically modified or synthetic microorganisms to provide the desired bacteriocin immunoactivity. Therefore, "immunomodulator" as used herein has its usual and common meaning as understood by those skilled in the art in view of the present disclosure, and not only relates to the structure clearly provided herein, but also relates to a structure having substantially the same effect as the "immunomodulator" structure described herein, including fully synthetic immunomodulators and immunomodulators that provide immunity to bacteriocins that are functionally equivalent to bacteriocins disclosed herein.

[0058] Exemplary polynucleotide sequences encoding the polypeptides in Table 2 are shown in Table 2. Those skilled in the art will readily appreciate that the genetic code is degenerate and that codon usage can vary based on the specific organism in which the gene product is expressed, and therefore, a specific polypeptide can be encoded by more than one polynucleotide. In some embodiments, polynucleotides encoding bacteriocin immunomodulators are selected based on the codon usage of the organism in which the bacteriocin immunomodulator is expressed. In some embodiments, polynucleotides encoding bacteriocin immunomodulators are codon optimized based on the specific organism in which the bacteriocin immunomodulator is expressed. A wide range of functional immunomodulators can incorporate the features of the immunomodulators disclosed herein, thereby providing a great degree of identity with the immunomodulators in Table 2. In some embodiments, the immunomodulator has at least about 50% identity to any of the polypeptides in Table 2, such as at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, or a range of identity defined by any two of the foregoing values.

[0059] Table 2: Exemplary bacteriocin immunomodulators

[0060] Promoter

[0061] Promoters are well known in the art. Promoters can be used to drive the transcription of one or more genes. In some embodiments, promoters drive the expression of polynucleotides encoding desired gene products as described herein. In some embodiments, promoters drive the expression of polynucleotides encoding propolypeptides comprising two or more bacteriocins as described herein. In some embodiments, promoters drive the expression of immunomodulator polynucleotides as described herein. In some embodiments, promoters drive the expression of polynucleotides encoding propolypeptides comprising two or more bacteriocins in microbial cells, but microbial cells do not express immunomodulators for one or more of these bacteriocins (for example, cells may lack promoters driving immunomodulator transcription, or may lack nucleic acids encoding immunomodulators). Some promoters can always drive transcription ("constitutive promoters"). Some promoters can only drive transcription ("conditional promoters") under selected environments, for example, depending on whether there are environmental conditions, compounds, gene products, cell cycle stages, etc.

[0062] Those skilled in the art will appreciate that, according to the desired expression activity, appropriate promoters can be selected and placed in cis with the nucleic acid sequence to be expressed. Exemplary promoters with exemplary activity and useful in some embodiments of this invention are provided in Tables 3.1-3.11 herein. Those skilled in the art will appreciate that some promoters are compatible with specific transcription mechanisms (e.g., RNA polymerase, general transcription factors, etc.). Therefore, although compatible "species" have been identified for some promoters described herein, it is considered that in some embodiments, these promoters can easily play a role in microorganisms (e.g., species with compatible endogenous transcription mechanisms, species containing genetic modifications of compatible transcription mechanisms, or fully synthetic microbial organisms containing compatible transcription mechanisms) outside the identified species.

[0063] The promoters in Tables 3.1-3.11 herein are publicly available from the Biobricks Foundation. Note that the Biobricks Foundation encourages the use of these promoters under the BioBrick™ Public Agreement (BPA).

[0064] It is understood that any "encoding" polynucleotide described herein (e.g., a bacteriocin polynucleotide, an immunity polynucleotide, or a nucleotide encoding a propolypeptide comprising two or more bacteriocins) is typically expressed under the control of a desired promoter. In some embodiments, a single "encoding" polynucleotide is under the control of a single promoter. In some embodiments, two or more "encoding" polynucleotides, such as two, three, four, five, six, seven, eight, nine, or ten polynucleotides are under the control of a single promoter.

[0065] Generally, the translation initiation of a specific transcript is regulated by a specific sequence of 5' of the 5' end or 5' end of the coding sequence of the transcript. For example, the coding sequence can start with a start codon configured to be paired with a starter tRNA. Although naturally occurring translation systems generally use Met (AUG) as a start codon, it is readily understood that the starter tRNA can be engineered to combine any desired triplet, and therefore, in certain embodiments, the triplet except AUG can also function as a start codon. In addition, sequences near the start codon, such as Kozak sequences ((gcc) gccRccAUGG, SEQ ID NO: 542, wherein R represents "A" or "G") in a typical eukaryotic translation system or Shine-Delgarno sequences (GGAGGU, SEQ ID NO: 543) in an internal ribosome entry site (IRES) or a typical prokaryotic translation system, can promote ribosome assembly. Thus, in some embodiments, a transcript comprising a "coding" polynucleotide sequence, such as a bacteriocin polynucleotide or an immunity polynucleotide, or a nucleotide encoding a polypeptide pronucleotide comprising two or more bacteriocins, comprises an appropriate start codon and a translation initiation sequence. In some embodiments, for example, if two or more "coding" polynucleotide sequences are located in cis on the transcript, each polynucleotide sequence comprises an appropriate start codon and a translation initiation sequence. In some embodiments, for example, if two or more "coding" polynucleotide sequences are located in cis on the transcript, the two sequences are controlled by a single translation initiation sequence, and either of the two sequences provides a single polypeptide capable of functioning with two cis-encoded polypeptides.

[0066] Table 3.1: Exemplary metal-sensitive promoters

[0067] Table 3.2: Exemplary Cell Signaling Response Promoters

[0068] Table 3.3: Exemplary constitutive E. coli σ 70 Promoter

[0069] Table 3.4: Exemplary constitutive E. coli σ s Promoter

[0070] Table 3.5: Exemplary constitutive E. coli σ 32 Promoter

[0071] Table 3.6: Exemplary constitutive Bacillus subtilis σ A Promoter

[0072] Table 3.7: Exemplary constitutive Bacillus subtilis σ B Promoter

[0073] Table 3.8: Exemplary constitutive promoters from various prokaryotes

[0074] Table 3.9: Exemplary constitutive promoters from bacteriophage T7

[0075] Table 3.10: Exemplary constitutive promoters from yeast

[0076] Table 3.11: Exemplary constitutive promoters from various eukaryotic organisms

[0077] The above-mentioned promoters are provided by way of non-limiting examples only. Those skilled in the art will readily recognize that many variants of the above-mentioned promoters and many other promoters (including promoters isolated from naturally occurring organisms, variants thereof, and fully synthetic promoters) may be readily used according to some embodiments herein.

[0078] Cleavage site

[0079] As used herein, "cleavage site" has its usual and common meaning as understood by those skilled in the art in view of the present disclosure. It refers to a polypeptide sequence that mediates polypeptide cleavage (e.g., by hydrolysis of a peptide bond) to separate a single polypeptide into two or more discrete polypeptides. In some embodiments, the cleavage site comprises, consists of, or consists essentially of a common polypeptide sequence for cleavage by a "cleavage enzyme" such as a peptidase. In some embodiments, the cleavage enzyme is a wild-type, variant, or synthetic cleavage enzyme, such as a wild-type, variant, or synthetic endopeptidase. Many exemplary cleavage enzymes and their corresponding cleavage sites are described herein. For reference, the cleavage site is described with reference to the following formula (I): Pn – P4 – P3 – P2 – P1-||cut||-P1' – P2' – P3' – P4' – Pm' (I) The amino acid residues in the substrate that undergo cleavage are designated as P1, P2, P3, P4, etc. in the N-terminal (or "upstream") direction from the cleaved bond. Likewise, the residues in the C-terminal (or "downstream") direction of the cleavage site are designated as P1', P2', P3', P4', etc.

[0080] Exemplary cleavage enzymes and their cleavage sites useful according to some embodiments herein are described in Table 4 below.

[0081] Note that many cutting enzymes cut at the consensus sequence, so a specific cutting enzyme can cut at two or more specific polypeptide sequences falling within the scope of a consensus sequence (or multiple consensus sequences) of the cutting enzyme. Therefore, for convenience herein, unless otherwise clearly stated, when two cutting sites are cut by the same cutting enzyme under the same conditions, even if they are two different sequences (they may fall within the scope of the cutting enzyme consensus sequence), they can also be referred to as "identical". For example, for the purpose of being cut by caspase 2, sequences DVADL (SEQ ID NO: 739) and DVADI (SEQ ID NO: 740) can be referred to as "identical" cutting sites, because they all fall within the scope of the consensus sequence of caspase 2, even if these cutting sites are not identical. On the other hand, also for convenience herein, unless otherwise clearly stated, when two cutting sites are each cut by different cutting enzymes, but not by the same cutting enzyme, they can be referred to as "different".

[0082] Table 4: Exemplary cleavage enzymes and cleavage sites

[0083] Additional information regarding cleavage sites can be found on the World Wide Web at web.expasy.org / peptide_cutter / peptidecutter_enzymes.html, which is incorporated herein by reference in its entirety.

[0084] In some embodiments, the cleavage site comprises, consists essentially of, or consists of a cleavage site in Table 4. Thus, in some embodiments, the cleavage site comprises, consists essentially of, or consists of a cleavage site for Arg-C protease, Asp-N endopeptidase, BNPS-skatole, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, chymotrypsin-high specific, clostripain (clostridial peptidase B), CNBr, enterokinase, factor Xa, formic acid, glutamyl endopeptidase, granzyme B, hydroxylamine, iodosylbenzoic acid, LysC, neutrophil elastase, NTCB (2-nitro-5-thiocyanatobenzoic acid), pepsin (pH 1.3), pepsin (pH > 2), proline-endopeptidase, proteinase K, staphylococcal peptidase I, thermolysin, thrombin, or trypsin. Thus, in the methods of some embodiments herein, one or more of the listed enzymes are used to cleave the cleavage site.

[0085] In some embodiments, the cleavage site comprises a chemically sensitive or pH-sensitive joint, consisting essentially of or consisting of it. This joint can be cut in the presence of suitable chemicals (in the case of chemically sensitive joints) or at a suitable pH (in the case of pH joints). In some embodiments, when pH is in a suitable range, pH-sensitive joints undergo enzyme-mediated cutting. For example, pH-sensitive joints such as valine-citrulline (vc) dipeptides are cut in an acidic pH (e.g., in the range of 4.8-6) by cathepsin B in a pH-sensitive manner (see, e.g., Jain et al. .Pharmaceutical Research, 32: 3526-3540, (2015), which are incorporated herein by reference in their entirety). In some embodiments, when pH is in a specific range, pH-sensitive joints undergo cleavage events. For example, an adjustable pH-sensitive joint based on a phosphoramidate backbone has also been described, which can undergo hydrolysis at a pH less than 7.4, which is described in PCT Publication No. WO2016028700, which is incorporated herein by reference in its entirety. In some embodiments, the chemically sensitive linker undergoes a cleavage event in the presence of a chemical substance. For example, a linker comprising, consisting essentially of, or consisting of a disulfide bond can be released upon introduction of glutathione (see, e.g., Jain et al. Pharmaceutical Research, 32: 3526-3540, (2015)).

[0086] signaling molecule

[0087] In some embodiments, the propolypeptide comprises one or more signal molecules. As used herein, "signal molecule" has its usual and common meaning as understood by those skilled in the art in view of the present disclosure. It refers to such secretory molecules that can regulate, induce or inhibit the activity or process in the cell or different cells that produce it (the target cell can be a microbial cell or a non-microbial cell, such as a multicellular organism such as an animal or plant cell). Exemplary signal molecules include, but are not limited to, signal peptides, quorum sensing molecules (e.g., quorum sensing peptides), signal transduction receptor ligands, growth factors, hormones and cytokines. In some embodiments, the signal molecule comprises the following, consists essentially of the following or consists of the following: signal peptides, quorum sensing molecules (e.g., quorum sensing peptides), signal transduction receptor ligands, growth factors, hormones or cytokines. In some embodiments, the signal molecule comprises the following, consists essentially of the following or consists of the following: a combination of two or more of a signal peptide, a quorum sensing molecule (e.g., a quorum sensing peptide), a signal transduction receptor ligand, a growth factor, a hormone and a cytokine, which may include a combination of two or more molecules of the same type (e.g., a combination of two signal peptides or a combination of two receptor ligands) and a combination of two different types of molecules (e.g., a combination of a cytokine and a hormone). In some embodiments, the signal molecule is used for microbe-host dialogue, and thus, the signal molecule is selected to target one or more cells of a host organism, such as a plant or animal. In some embodiments, the signal molecule stimulates, inhibits, increases or decreases the production of bacteriocins and / or the growth rate of a subpopulation of a bacterial population. In some embodiments, the signal molecule comprises, consists of or consists essentially of a quorum sensing peptide or a variant thereof as described herein.

[0088] Propolypeptides, methods and / or exemplary quorum sensing peptides encoded by nucleic acids suitable for use in some embodiments include, but are not limited to, quorum sensing peptides such as those shown in Table 5 below, including variants of these peptides, and combinations of two or more of any of these peptides. Without being limited by theory, it is contemplated that microbial cells, such as Gram-positive bacteria, use quorum sensing peptides to coordinate intercellular communication. A review of quorum sensing peptides can be found in Rajput et al., PLoS One DOI: 10.1371 / journal.pone.0120066, March 17, 2015, pp. 1-16, which is hereby incorporated by reference in its entirety. Quorum sensing peptides can induce activation of downstream response regulators and / or transcription factors in target microbial cells. It should be noted that in some embodiments, downstream response regulators or transcription factors can be configured to activate or inhibit transcription of target nucleic acids of interest. Therefore, in some embodiments, target microbial cells are genetically engineered to express or inhibit transcription (and subsequent expression) of target gene products when stimulated by signal peptides of propolypeptides.

[0089] In some embodiments, the signal molecule of the propolypeptide comprises, consists essentially of, or consists of a signal molecule (e.g., a quorum sensing molecule, such as a quorum sensing peptide) that stimulates the transcription of one or more bacteriocin polynucleotides in the target microbial cell. The target microbial cell can be genetically engineered to place one or more bacteriocin polynucleotides under the control of a transcription factor that responds to a quorum sensing molecule. In some embodiments, the transcription of one or more bacteriocin polynucleotides is configured to be induced by a transcription factor (e.g., a transcription activator) that responds to a quorum sensing molecule. Therefore, signaling by the signal molecule (e.g., a quorum sensing molecule) of the propolypeptide can stimulate the target microbial cell to produce one or more additional bacteriocins.

[0090] In some embodiments, the target microbial cell is genetically engineered to place one or more molecules of the poison-antidote system under the control of a transcription factor or a response regulator of a response signal molecule (e.g., a quorum sensing molecule such as a quorum sensing peptide), thereby inducing a suicide response of the target cell after the signal molecule performs signal transduction. A poison-antidote system different from bacteriocins can be used to achieve this suicide response, which in turn can be used for the inhibition and / or selective growth of microbial cells. Exemplary poison-antidote systems are described in U.S. Patents Nos. 5,910,438, 6,180,407, 7,176,029, and 7,183,097, each of which is incorporated herein by reference in its entirety. In some embodiments, the poison-antidote system comprises a cytotoxic (poison) polypeptide and a corresponding antitoxin (antidote) polypeptide in a single cell. As used herein, "poison polynucleotide" has its usual and common meaning as understood by those skilled in the art in view of the present disclosure, and may refer to a polynucleotide encoding a poison polypeptide. "Antidote polynucleotide" has its usual and common meaning as understood by those skilled in the art in view of the present disclosure, and may refer to a polynucleotide encoding an antidote polypeptide. Thus, in some embodiments, the target microbial cell is configured to induce transcription of a poison polynucleotide in response to a signal molecule such as a quorum sensing molecule, thereby inducing a suicide response in the target microbial cell. In some embodiments, the target microbial cell is configured to transcribe an inhibitory antibody polynucleotide in response to a signal molecule such as a quorum sensing molecule (while continuing to express the corresponding poison polypeptide), thereby inducing a suicide response in the target microbial cell.

[0091] In some embodiments, the quorum sensing peptide is naturally occurring. In some embodiments, the quorum sensing peptide comprises, consists essentially of, or consists of a variant of a naturally occurring quorum sensing peptide. In some embodiments, the quorum sensing peptide comprises, consists essentially of, or consists of a synthetic peptide. Information about quorum sensing peptides, including exemplary sequences, can be found in the quorumpeps database, which can be accessed on the World Wide Web at quorumpeps.ugent.be., which is hereby incorporated by reference in its entirety.

[0092] Table 5

[0093] Cytokines are a class of signaling molecules that are usually produced by cells such as cells of the immune system and can induce responses of other cells. Many different cytokines can be used in the propolypeptides, methods of some embodiments herein and / or can be encoded by the nucleic acids of some embodiments herein. It is contemplated that propolypeptides comprising bacteriocins and cytokines according to some embodiments can be used to induce antimicrobial activity (via bacteriocins) and host responses, such as immune cell suppression or immune cell stimulation (via cytokines). In some embodiments, cytokines include, are essentially composed of, or are composed of: naturally occurring cytokines, variants of naturally occurring cytokines, or synthetic cytokines. Many suitable cytokines can be used in the propolypeptides, methods, compositions, cells according to some embodiments herein, or encoded by the nucleic acids according to some embodiments herein, including but not limited to IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IFN-α, IFN-β, IFN-γ, TNF-α, TNF-β, TGF-β1, M-CSF, G-CSF and GM-CSF, variants of any of these, or any combination of two or more of these.

[0094] Hormones are a class of signaling molecules that are typically produced by cells of a multicellular organism and send signals to other cells that often circulate through different tissues and / or organs of a multicellular organism. Many different hormones can be used in the propolypeptides, methods, and / or by the nucleic acid encodings of some embodiments herein. It is contemplated that propolypeptides comprising bacteriocins and hormones according to some embodiments can be used to induce antimicrobial activity (via bacteriocins) and host responses, such as cell growth or proliferation (via hormones). In some embodiments, cytokines include, consist essentially of, or consist of naturally occurring hormones, variants of naturally occurring hormones, or synthetic hormones.Exemplary hormones suitable for use in some embodiments of the propolypeptides, methods and / or encoded by nucleic acids include, but are not limited to, protein and peptide hormones such as activin and inhibin, adiponectin, adipose-derived hormone, adrenocorticotropic hormone, agouti gene, agouti signaling peptide, allatostatin, amylin, amylin family, angiotensin, ANGPTL8, asprosin, atrial natriuretic peptide, big gastrin, bovine growth hormone, bradykinin, brain-derived neurotrophic factor, calcitonin, ciliary neurotrophic factor, corticotropin-releasing hormone, crustacean neurohormone Family, endothelin, enteroglucagon, erythroferrone, fellutamide, FGF15, FGF15 / 19, FGF19, FNDC5, follicle-stimulating hormone, gastrin, gastric inhibitory peptide, ghrelin, glucagon-like peptide-1, gonadotropin, gonadotropin-releasing inhibitor, gonadotropin-releasing hormone, granulocyte colony-stimulating factor, growth hormone, growth hormone-releasing hormone, hepcidin, human chorionic gonadotropin, human placental lactogen, incretin, insulin, insulin analogs, insulin aspart, insulin degludec (insulin degludec), insulin glargine, insulin lispro, insulin-like growth factor, insulin-like growth factor 1, insulin-like growth factor 2, leptin, limostatin, liraglutide, minigastrin I, luteinizing hormone, melanocortin, melanocyte stimulating hormone, α-melanocyte stimulating hormone, β-melanocyte stimulating hormone, γ-melanocyte stimulating hormone, minigastrin, N-terminal prohormone of brain natriuretic peptide, nerve growth factor, neuropeptide VF precursor, neurotrophin-3, neurotrophin-4, NPH insulin, obestatin , osteocalcin, parathyroid hormone, peptide hormone, peptide YY, plasma renin activity, pramlintide, preprohormone, prolactin, relaxin, relaxin family peptide hormones, renin, salmon calcitonin, sauvagine, secretin, secretin family, sincalide, stanniocalcin, teleost leptin, temporin, thyroid stimulating hormone, thyrotropin-releasing hormone, urocortin, urocortin II, urocortin III, vasoactive intestinal polypeptide, vitellogenin, variants of any of these, or any combination of two or more of these.

[0095] Affinity Tags

[0096] In the propolypeptide, method and nucleic acid encoding propolypeptide of some embodiments herein, the propolypeptide or its component peptide (such as bacteriocin or signal molecule) comprises affinity tag.Optionally, one or more cleavage sites can be located between the remainder of the affinity tag and the propolypeptide to promote affinity purification and remove the affinity tag. Affinity tag can be used for purification, for example, by contacting with molecules bound to affinity tags fixed on a solid phase such as beads. Exemplary affinity tags suitable for propolypeptide, method and nucleic acid encoding of some embodiments herein can include the following, consisting essentially of or consisting of: His-tag, glutathione-S-transferase (GST) tag, FLAG tag, strep tag, maltose binding protein (MBP), chitin binding protein (CBP), myc tag, HA tag, NE tag and V5 tag, any variant of these or any combination of two or more of these.

[0097] Microbial Organisms

[0098] In some embodiments, genetically engineered microorganisms are provided. As used herein, genetically engineered "microbial organisms," "microorganisms," and variations of these root terms (such as pluralization, etc.) have their usual and ordinary meanings as understood by those skilled in the art in view of the present disclosure. They encompass genetic modifications of any naturally occurring species or completely synthetic prokaryotic or eukaryotic single-cell organisms and archaeal species. Thus, this expression may refer to cells of bacterial species, fungal species, and algae.

[0099] The exemplary microorganisms that can be used according to the embodiments of this paper include but are not limited to bacteria, yeast and algae, such as photosynthetic microalgae. In addition, the fully synthetic microbial genome can be synthesized and transplanted into a single microbial cell to produce a synthetic microorganism that can continuously replicate itself (see Gibson et al., (2010), "Creation of aBacterial Cell Controlled by a Chemically Synthesized Genome," Science 329:52-56, incorporated herein by reference in its entirety). Therefore, in some embodiments, microorganism is fully synthetic. The desired combination of genetic elements (including elements (such as bacteriocins, immunomodulators, poisons, antidotes and industrially useful molecules) of regulating gene expression and encoding gene products) can be assembled into partially or fully synthesized microorganisms on the chassis (chassis) desired. The description of the genetically engineered microbial organism for industrial applications can also be found in Wright et al., (2013) "Building-in biosafety for synthetic biology" Microbiology 159: 1221-1235.

[0100] According to the embodiments herein, multiple bacterial species and bacterial strains can be used, and genetically modified variants or synthetic bacteria can be provided based on the " chassis " of known species. The exemplary bacteria with industrially applicable characteristics that can be used according to the embodiments herein include but are not limited to Bacillus species (such as Bacillus coagulans, Bacillus subtilis and Bacillus licheniformis), Paenibacillus species, Streptomyces species, Micrococcus species, Corynebacterium species, Acetobacter species, Cyanobacteria species, Salmonella species, Rhodococcus species. Pseudomonas species, Lactobacillus species, Enterococcus species, Alcaligenes species, Klebsiella species, Paenibacillus species, Arthrobacter species, Corynebacterium species, Brevibacterium species, Thermus aquaticus (Thermus aquaticus), Pseudomonas stutzeri (Pseudomonas stutzeri), Clostridium thermocellus (Clostridiumthermocellus) and Escherichia coli.

[0101] According to the embodiments herein, a variety of yeast species and strains can be used, and genetically modified variants or synthetic yeasts can be provided based on the "chassis" of known species. Exemplary yeasts with industrially applicable properties that can be used according to the embodiments herein include, but are not limited to, yeast species (e.g., Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces boulardii), Candida species (e.g., Candida utilis, Candida krusei), fission yeast species (e.g., Schizosaccharomyces pombe, Schizosaccharomyces japonicas), Pichia pastoris or Hansenula species (e.g., Pichia pastoris or Hansenula polymorpha) and Brettanomyces species (e.g., Brettanomyces claussenii).

[0102] According to the embodiments herein, multiple algae species and bacterial strains can be used, and genetically modified variants or synthetic algae can be produced based on the "chassis" of known species. In some embodiments, algae comprises photosynthetic microalgae. The exemplary algae species that can be used for biofuel and can be used according to the embodiments herein include Brown grape algae (Botryococcusbraunii), Chlorella species, Dunaliella tertiolecta (Dunaliella tertiolecta), Gracilaria species, Pleurochrysis carterae (Pleurochrysis carterae) and Sargassum species. In addition, many algae can be used for food, fertilizer, waste neutralization, environmental restoration and carbohydrate manufacturing (for example, biofuel).

[0103] Design of propolypeptides and nucleic acids encoding propolypeptides

[0104] A variety of methods can be used to design the polypeptide progeny of the selected bacteriocin comprising the desired ratio according to some embodiments herein. For example, one option is to design a nucleic acid encoding sequence, which is included in the bacteriocin, cleavage site and other features of the polypeptide progeny in a single open reading frame, such as affinity tags and / or signal molecules. Then the polynucleotide with the sequence can be sequenced. In addition, computing tools can be used to promote the design of the polypeptide progeny (and nucleic acids encoding them) according to some embodiments. For example, as described in Nielsen et al., "Genetic circuit design automation" Science (2016) 352: aac7341 (incorporated herein by reference in its entirety), a computer-based design environment Cello allows users to write the Verilog code automatically converted to DNA sequence. Therefore, in some embodiments, text-based methods can be easily used to generate nucleic acids encoding the desired elements of the polypeptide progeny, such as the bacteriocin of a specific combination and / or ratio and the cleavage site of the isolated bacteriocin, and in a manner that the bacteriocin is not cut by the cleavage enzyme cutting of the cleavage site. In some embodiments, the polypeptide progeny comprises the selected bacteriocin and antimicrobial peptide of the desired ratio. In some embodiments, the polypeptide progeny comprises the selected antimicrobial peptide of the desired ratio. In some embodiments, the propolypeptide comprises a desired ratio of a selected bacteriocin.

[0105] Genetic modification of microbial organisms for expression of nucleic acids

[0106] The technology of genetically modifying microorganisms is well known in the art. In the methods and compositions of some embodiments, the microorganism is genetically modified to include a nucleic acid sequence that regulates the expression of a propolypeptide of a bacteriocin, such as a bacteriocin described herein, and encoding it. In the methods and compositions of some embodiments herein, a polynucleotide encoding a propolypeptide can be delivered to the microorganism, and the polynucleotide can be stably integrated into the chromosome of these microorganisms, or can be absent from the genome, such as present in a plasmid, an extrachromosomal array, an episome, a minichromosome, etc.

[0107] Exemplary vectors for genetically modifying microbial cells include, but are not limited to, plasmids, viruses (including bacteriophages), and transposable elements. In addition, it will be appreciated that the entire microbial genome containing the desired sequence can be synthesized and assembled in the cell (see, e.g., Gibson et al., (2010), Science 329: 52-56). Therefore, in some embodiments, a microbial genome (or portion thereof) having a desired feature such as a bacteriocin polynucleotide is synthesized and introduced into a microbial cell.

[0108] In some embodiments, a box is provided for inserting one or more desired bacteriocins and / or immunomodulator polynucleotides into a polynucleotide sequence (e.g., inserting a box encoding a propolypeptide comprising a bacteriocin into an expression vector). Exemplary boxes include, but are not limited to, Cre / lox boxes or FLP / FRT boxes. In some embodiments, the box is located on a plasmid so that a plasmid with a desired polynucleotide encoding a desired propolypeptide can be easily introduced into a microbial cell. In some embodiments, the box is located at a desired position in the microbial cell genome.

[0109] In some embodiments, plasmid conjugation can be used to introduce a desired plasmid from a "donor" microbial cell into a recipient microbial cell. Goñi-Moreno et al., (2013) Multicellular Computing Using Conjugation for Wiring. PLoS ONE 8(6): e65986, incorporated herein by reference in its entirety. In some embodiments, plasmid conjugation can genetically modify a recipient microbial cell by introducing a conjugated plasmid from a donor microbial cell into a recipient microbial cell. Without being limited by any particular theory, conjugated plasmids containing identical or functionally identical replicating genomes generally cannot coexist in the same microbial cell. Therefore, in some embodiments, plasmid conjugation "reprograms" a recipient microbial cell by introducing a new conjugated plasmid to replace another conjugated plasmid present in the recipient cell. In some embodiments, plasmid conjugation is used to engineer (or reengineer) a microbial cell having a specific nucleic acid encoding a propolypeptide or a combination of different nucleic acids encoding different propolypeptides. According to some embodiments, a plurality of conjugated plasmids comprising different nucleic acids are provided, the nucleic acid comprising a plurality of different propolypeptides. The plasmid may comprise additional genetic elements as described herein, such as promoters, translation start sites, and the like. In some embodiments, a variety of conjugative plasmids are provided in a collection of donor cells so that donor cells containing the desired plasmid can be selected for plasmid conjugation. In some embodiments, a specific combination and / or ratio of bacteriocins are selected, and appropriate donor cells (encoding a specific propolypeptide) are conjugated with target microbial cells to introduce a conjugative plasmid containing the combination into a recipient cell.

[0110] Pro-peptide

[0111] As used herein, "propolypeptide" has its usual and ordinary meaning as understood by those skilled in the art in view of the present disclosure. It refers to a precursor polypeptide comprising, consisting essentially of, or consisting of: at least two component peptides (e.g., at least one bacteriocin and at least one signal molecule, or two or more bacteriocins and optionally one or more other peptides disclosed herein (e.g., signal peptides, quorum sensing molecules such as quorum sensing peptides, signal transduction receptor ligands, growth factors, hormones, cytokines, or any combination of two or more of the listed items)) in a desired ratio and at least one cleavage site for separating the component peptides of the propolypeptide. Figure 2 An exemplary propolypeptide according to some embodiments is schematically illustrated in FIG. The component peptides can be separated from each other by a cleavage site, so that upon cleavage, the component peptides are separated from each other and are present in a ratio determined by the number of copies of each component peptide in the propolypeptide (see Figure 3 ). In some embodiments, one or more component peptides of a propolypeptide are in an inactive form and become active after being cleaved from the surrounding polypeptide sequence. Without being limited by theory, it is contemplated that in some embodiments, at least some or in some embodiments all of the bacteriocins are inactive when they are part of a propolypeptide, so that advantageously, the propolypeptide can be produced and / or modified by a microbial cell that does not require immunity to those inactive bacteriocins. It is expressly contemplated that the propolypeptides described herein can be used in conjunction with any of the methods and kits described herein. In addition, it should be understood that for any propolypeptide described herein, one or more polynucleotides (to the extent that there are multiple possible codons) can be readily understood based on the understanding of the genetic code by those skilled in the art.

[0112] Figure 4A1 is a schematic diagram illustrating a propolypeptide 100 according to some embodiments of the present invention. The propolypeptide may include bacteriocins 110, 112, 114. The bacteriocins may be the same or different (e.g., all bacteriocins may be the same, or some bacteriocins may be the same while other bacteriocins are different, or all bacteriocins may be different from each other). The propolypeptide may also include one or more cleavage sites 120, 122, 124, and 126 disposed between the bacteriocins and / or other polypeptides in the propolypeptide. In some embodiments, the propolypeptide further includes a tag 130. In some embodiments, the propolypeptide further includes a signal molecule 140 as described herein, such as a quorum sensing molecule such as a quorum sensing peptide. In some embodiments, the cleavage sites 120, 122, 124, and 126 are disposed between the bacteriocin, the tag, and the signal molecule. In some embodiments, the tag can be used to purify the propolypeptide. In some embodiments, the cleavage sites (e.g., 120, 122, 124, and 126) include two or more common sites for enzymes (e.g., endopeptidases). It is contemplated that in some embodiments, the cleavage site contains two or more cleavage consensus sequences (which may be identical or different), at least one of which is immediately downstream of the C-terminus of the upstream peptide and / or at least one of which is immediately upstream of the N-terminus of the downstream polypeptide, so that any residual portion (vestige) of the cleavage site can be substantially eliminated or completely eliminated from the peptide. In some embodiments, the "cleavage site" comprises two or more consensus sequences, at least one of which is configured to be immediately upstream or downstream of the peptide (e.g., bacteriocin) so that any residual portion of the cleavage site is removed from the peptide when cleaving. In some embodiments, the "cleavage site" comprises two consensus sequences, one of which is configured to be immediately downstream of the upstream peptide (e.g., bacteriocin) and the other immediately upstream of the downstream peptide (e.g., bacteriocin) so that any residual portion of the cleavage site is removed from the C-terminal portion of the upstream peptide and the N-terminal portion of the downstream peptide when cleaving the two cleavage sites. Optionally, an additional cleavage site is located between the two cleavage sites.

[0113] Figure 4B1 is a schematic diagram illustrating a composition comprising a bacteriocin 101 prepared from a propolypeptide 100 according to some embodiments herein. The composition may comprise bacteriocins 110, 112, 114. The bacteriocins may be the same or different (e.g., all bacteriocins may be the same, or some bacteriocins may be the same while other bacteriocins are different, or all bacteriocins may be different from each other). Optionally, some bacteriocins 110, 112, 114 may also comprise residual portions of cleavage sites 121, 123, 125, 127. In some embodiments, the composition further comprises a tag 130 and / or a signal molecule 140 as described herein. Some bacteriocins 110, 112, 114 and / or tags 130 and / or signal molecules 140 may also comprise residual portions of cleavage sites 121, 123, 125, 127. In some embodiments, after cleavage, the bacteriocin comprises no more than 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids as the residual portion of the cleavage site, including the range between any two listed values. In some embodiments, the bacteriocin does not comprise any residual portion of the cleavage site. In some embodiments, the bacteriocin comprises a residual portion of the cleavage site at its N-terminus, at its C-terminus, or at its N- and C-termini. It is contemplated that in some embodiments, the signal molecule and / or tag also comprises a residual portion as described herein. In some embodiments, the signal molecule and / or tag does not comprise any residual portion described herein.

[0114] In some embodiments, the propolypeptide comprises, consists essentially of, or consists of at least one bacteriocin, at least one signal molecule, and one or more cleavage sites disposed between the bacteriocin and the signal molecule, such that each bacteriocin or signal molecule can be separated from the remainder of the propolypeptide by cleavage at the cleavage site. In some embodiments, the bacteriocin and the signal molecule are in a desired ratio.

[0115] In some embodiments, the propolypeptide comprises a first component peptide that comprises, consists essentially of, or consists of a bacteriocin, at least one additional component peptide (bacteriocin or signal molecule) and a cleavage site disposed between the component peptides. The propolypeptide may also comprise an affinity tag. In some embodiments, the affinity tag is separated from the bacteriocin (and / or other component peptides) in the propolypeptide by the cleavage site, so that after affinity purification of the propolypeptide, the affinity tag can be cleaved to remove it from the propolypeptide. In some embodiments, the propolypeptide comprises, consists essentially of, or consists of at least two bacteriocins and optionally at least one signal molecule. In some embodiments, the isolated propolypeptide comprises three or more bacteriocins, for example, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 8, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 bacteriocins, including ranges between any two of the listed values, such as 3-10, 3-20, 3-30, 3-50, 5-10, 5-20, 5-30, 5-50, 7-10, 7-20, 7-30, 7-50, 10-20, 10-30 or 10-50 bacteriocins. In some embodiments, the propolypeptide comprises at least 5 bacteriocins.

[0116] It is also contemplated that the propolypeptide may comprise a signal molecule that may facilitate communication with a microbial cell or a host organism. Therefore, in the propolypeptide, method, and nucleic acid of some embodiments, the propolypeptide also comprises a signal molecule. The signal molecule may be separated from the other component peptides of the propolypeptide by a cleavage site. In some embodiments, the signal molecule comprises, is essentially composed of, or is composed of a quorum sensing molecule (e.g., a quorum sensing peptide), a cytokine, or a hormone as described herein. In some embodiments, the target cell is configured to produce additional gene products, such as bacteriocins, in response to a quorum sensing molecule. The target cell may be genetically engineered so that one or more bacteriocin polynucleotides are under the control of a transcription factor that responds to a quorum sensing molecule, such that when a quorum sensing molecule sends a signal, the bacteriocin polynucleotide is transcribed, and the target cell produces additional bacteriocins.

[0117] In some embodiments, one or more cleavage sites are set in a frame between any two adjacent component peptides (e.g., bacteriocin, signal molecule) of the propolypeptide. In some embodiments, one or more cleavage sites are set in a frame between any two adjacent bacteriocins of the propolypeptide. Note that in the propolypeptides, methods and nucleic acids of some embodiments, the component peptides (e.g., bacteriocin and / or signal molecule) or affinity tags located at or near the N- or C-terminus of the propolypeptide can be located near the cleavage site to separate them from the rest of the propolypeptide, but they are not on the side of the cleavage site on both sides. For example, for a bacteriocin at the N-terminus of a bacteriocin, there may be a cleavage site in the direction from the C-terminus of the bacteriocin, but there is no cleavage site on the N-terminal side of the bacteriocin. In some embodiments, for example, if the component peptide or affinity tag is near the N- or C-terminus of the propolypeptide, but there is an additional N- or C-terminal sequence outside the component peptide or affinity tag, the component peptide can include cleavage sites on both sides to facilitate the removal of the additional N- or C-terminal sequence. In some embodiments, some additional N- or C-terminal sequences can be retained on the component polypeptide.

[0118] It should be noted that in some embodiments, the cleavage site can include the sequence targeted by the cleavage enzyme. In order to minimize or avoid the cleavage of bacteriocin or other component peptides (e.g., signal molecules) in the propolypeptide, it is considered that in some embodiments, the component peptide itself does not include the cleavage site of the cleavage enzyme that targets the cleavage site between the component peptides. Therefore, in some embodiments, the bacteriocin does not include the cleavage site of the cleavage enzyme for the cleavage site between the targeting bacteriocin. In some embodiments, two or more cleavage enzymes are used to target all cleavage sites between the component peptides of the propolypeptide, but the bacteriocin does not include the cleavage site targeted by any one of these cleavage enzymes. In some embodiments, at least one cleavage site is used for the first cleavage enzyme, another cleavage site is used for the second cleavage enzyme that is different from the first cleavage enzyme, and the bacteriocin does not include the cleavage site for any one of the first or second cleavage enzymes.

[0119] In some embodiments, the signal molecule does not contain a cleavage site for a cleavage enzyme that targets the cleavage site between the bacteriocin and the signal molecule. Note that, in some embodiments, cleavage is performed after affinity purification of the propolypeptide. Therefore, it is acceptable for the affinity tag itself to contain one or more cleavage sites, because the affinity tag may be dispensable when cleavage is performed.

[0120] In some embodiments, the cleavage sites of the component peptides separating the propolypeptide are all targeted by the same cleavage enzyme, so that a single enzyme can be used to cut the propolypeptide into a separate bacteriocin (and optional other peptides) of a desired ratio. In some embodiments, the cleavage sites of the component peptides separating the propolypeptide are jointly targeted by a combination of cleavage enzymes, so the number of cleavage enzymes is less than the number of cleavage sites in the propolypeptide. In some embodiments, one, two, three, four, five, six, seven, eight, nine or ten cleavage enzymes are jointly targeted to separate the cleavage sites of the component peptides of the propolypeptide. In some embodiments, the cleavage site comprises a chemically sensitive or pH-sensitive linker. Therefore, in some of these embodiments, the cleavage sites of the component peptides separating the propolypeptide are jointly cut by the same chemistry and / or the same pH conditions.

[0121] In some embodiments, the bacteriocin itself comprises a tag, such as an affinity tag, a signal sequence (for secretion, internalization, nuclear localization, etc.) or a stability tag. Optionally, the tag can be cut from the pro-polypeptide. Therefore, the bacteriocin can comprise a cleavage site between itself and the tag. After the component peptide is cut from the pro-polypeptide, it may be useful for the tag to remain attached to the bacteriocin. Therefore, in some embodiments, the cleavage site between the bacteriocin and the tag can be targeted by a different cleavage enzyme than the cleavage site that separates the component peptides from each other (and / or cut under different chemical or pH conditions).

[0122] In some embodiments, the propolypeptide further comprises a post-translational or co-translational modification, such as glycosylation, acetylation, methylation, pegylation, SUMOylation, ubiquitination, or two or more of any these.

[0123] In some embodiments, the polypeptide precursor comprises a certain amount of two or more different component peptides (e.g., bacteriocins and / or signal molecules) in a desired ratio or a portion of a desired ratio. For example, the ratio of the first bacteriocin to the second (different) bacteriocin or signal molecule can be about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 2:3, 2:5, 2:7, 2:9, 3:4, 3:5, 3:7, 3:8, 3:10, 4:5, 4:7, 4:9, 5:6, 5:7, 5:8, 5:9, 6:7, 7:8, 7:9, 7:10, 8:9 or 9:10. In some embodiments, the polypeptide precursor comprises a certain amount of two or more different bacteriocins in a desired ratio or a portion of a desired ratio. In some embodiments, the desired bacteriocin ratio is selected to target an undesirable microbial organism or an undesirable population of microbial organisms. In some embodiments, the desired bacteriocin ratio is selected to balance the microbial population in an animal, human organ, plant root, and / or soil. In some embodiments, the bacteriocin and / or signal molecule is present in a portion of the desired ratio, and the desired ratio can be achieved by adding additional bacteriocin and / or signal molecule in an appropriate ratio (e.g., if the products of two different pro-polypeptides are combined, they can produce a desired ratio of component polypeptides, such as bacteriocins and / or signal molecules).

[0124] In some embodiments, the propolypeptide is no more than 20,000 amino acids in length, such as no more than 20,000, 15,000, 10,000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 amino acids in length, including any two of the listed In some embodiments, the length of the propolypeptide is no more than 5000 amino acids. In some embodiments, the length of the propolypeptide is no more than 2000 amino acids.

[0125] Nucleic Acids

[0126] The methods, kits, microbial cells and / or nucleic acids of some embodiments of the present invention include nucleic acids encoding propolypeptides. In some embodiments, the nucleic acid encodes a bacteriocin and a signal molecule in a single reading frame, and encodes a cleavage site coding sequence disposed between the bacteriocin and signal molecule coding sequences. In some embodiments, the isolated nucleic acid comprises two bacteriocin coding sequences in a single reading frame. The isolated nucleic acid may also comprise a cleavage site coding sequence disposed between the bacteriocin coding sequences and in a single reading frame. In the methods, nucleic acids, microbial cells and / or kits of some embodiments of the present invention, the nucleic acid encodes any propolypeptide described herein. In some embodiments, the kit comprises nucleic acids constituting one or more propolypeptides as described herein. In some embodiments, the kit further comprises a microbial cell capable of expressing the propolypeptide.

[0127] As described herein, for the nucleic acid of some embodiments, it is advantageous for the nucleic acid-encoded bacteriocin (or other polypeptide) to remain intact after cutting. Therefore, in some embodiments, the nucleic acid encodes the cleavage site of the cleavage enzyme, and its bacteriocin and / or signal molecule coding sequence (respectively) encodes a bacteriocin and / or signal molecule that does not comprise the cleavage site of the cleavage enzyme. In some embodiments, the cleavage site coding sequence of the nucleic acid encodes the cleavage site of the cleavage enzyme, and the bacteriocin coding sequence encodes a bacteriocin that does not comprise the cleavage site of the cleavage enzyme.

[0128] In some embodiments, the nucleic acid comprises three or more bacteriocin encoding sequences in a single reading frame, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 In some embodiments, the nucleic acid comprises at least three bacteriocin sequences in a single reading frame. In some embodiments, the nucleic acid comprises at least 5 bacteriocin sequences in a single reading frame. In some embodiments, at least two bacteriocin coding sequences encode bacteriocins that are different from each other.

[0129] In some embodiments, one or more cleavage site coding sequences are set in the frame between any two adjacent bacteriocin coding sequences of the isolated nucleic acid. As described herein, it may be advantageous for a single cleavage enzyme to be able to separate multiple or all component peptides of a propolypeptide. Therefore, in some embodiments, each bacteriocin coding sequence (and, if present, a signal molecule coding sequence) is separated from each other by an in-frame cleavage site coding sequence of the same enzyme. Therefore, after translation of the nucleic acid, the resulting propolypeptide can be cleaved into its component peptides by a single enzyme.

[0130] In some embodiments, the cleavage site encoding sequence encodes a cleavage site for a first cleavage enzyme, and another cleavage site encoding sequence encodes another cleavage site for a different cleavage enzyme. In some embodiments, the bacteriocin encoded by the bacteriocin encoding sequence does not include a cleavage site for either the first or second cleavage enzyme. In some embodiments, the bacteriocin encoded by the isolated nucleic acid does not include a cleavage site for any cleavage enzyme that cleaves interspersed cleavage sites.

[0131] In some embodiments, all bacteriocin coding sequences (and, if present, signal molecule coding sequences) are separated from each other by in-frame cleavage site coding sequences that are co-cleaved by a combination of two, three, four, five, six, seven, eight, nine, or ten cleavage enzymes. Thus, upon translation of the nucleic acid, this combination of cleavage enzymes can be used to cleave the propolypeptide into its component peptides.

[0132] In some embodiments, the cleavage site encoding sequence encodes a cleavage site for a cleavage enzyme as described herein (e.g., in Table 4). In some embodiments, the cleavage site encoding sequence encodes a chemical and / or pH sensitive cleavage site as described herein.

[0133] In some embodiments, the component peptide (e.g., bacteriocin and / or signal molecule) coding sequences of the isolated nucleic acid are present in a desired ratio or a portion of a desired ratio. In some embodiments, three or more bacteriocin sequences are present in a desired ratio or a portion of a desired ratio. In some embodiments, the desired ratio is selected to target an undesirable microbial organism or a population of undesirable microbial organisms. In some embodiments, the desired bacteriocin ratio is selected to balance the microbial community in animals, human organs, or plant roots and / or soil.

[0134] In some embodiments, the isolated nucleic acid further comprises a coding sequence for a signal molecule in a single reading frame. The cleavage site coding sequence can be disposed between the coding sequence for the signal molecule and the adjacent bacteriocin coding sequence. In some embodiments, the signal molecule comprises a quorum sensing molecule, a signal transduction receptor ligand, a growth factor, a hormone, or a cytokine. In some embodiments, the signal molecule can be wild type, mutant, or synthetic.

[0135] Microbial cells for producing bacteriocin

[0136] The methods, kits and microbial cells of some embodiments include microbial cells containing isolated nucleic acids as described herein. Such microbial cells can be used to produce polypeptide progenitors containing bacteriocins of the desired ratio as described herein. Microbial cells can include promoters as described herein. Promoters can be operably connected to isolated nucleic acids. Therefore, microbial cells can be configured to transcribe isolated nucleic acids and translate them into polypeptide progenitors containing bacteriocins. As described herein, when the bacteriocin of the polypeptide progenitor is part of the polypeptide progenitor, they can be inactive. Therefore, in some embodiments, the isolated microbial cells do not produce functional immunomodulators of bacteriocins encoded by isolated nucleic acids. For example, the microbial cell can lack the coding sequence of an immunomodulator, or if it does contain the coding sequence of an immunomodulator, the coding sequence can be transcriptionally silent (e.g., due to lack of a promoter), and / or the coding sequence can be mutated so that any immunomodulator produced by the coding sequence is non-functional. In some embodiments, the promoter operably connected to the nucleic acid includes any promoter of Tables 3.1-3.11, is essentially composed of it or is composed of it.

[0137] Method for preparing bacteriocin

[0138] Some embodiments include methods for preparing bacteriocins. The methods can be used to prepare compositions comprising bacteriocins (and optionally other molecules such as signal molecules) in a desired ratio. In some embodiments, the method for preparing bacteriocins comprises expressing a nucleic acid. The nucleic acid can contain two bacteriocin coding sequences in a single reading frame, or a bacteriocin and a signal molecule coding sequence in a single reading frame (see, e.g., Figure 8 ). The nucleic acid may also include a cleavage site encoding sequence disposed between the bacteriocin encoding sequences (or between the bacteriocin and the signal molecule encoding sequences) and in a single reading frame. The nucleic acid may be expressed to produce a propolypeptide comprising a bacteriocin and a cleavage site disposed therebetween. In some embodiments, a nucleic acid encoding a bacteriocin (and / or a signal molecule) as described herein is expressed by a gene expression system to produce a propolypeptide as described herein.

[0139] In some embodiments, for example, when the bacteriocin is produced in a semi-controlled environment such as biopharmaceutical production, the propolypeptide comprises a bacteriocin coding sequence, a signal molecule coding sequence, and a cleavage site disposed between the bacteriocin and signal molecule coding sequences. It is contemplated that such a propolypeptide can be used to produce a composition comprising a desired ratio of bacteriocin and a signal molecule, which can be used to inhibit genetic drift of microbial organisms (via bacteriocins; for example, if the immunity of the target organism is related to their maintenance of a certain genetic state; this method is described in more detail in U.S. Patent No. 9,333,227. In addition, the signal molecule can promote the growth, proliferation or production of the desired product by producing cells in the environment (e.g., genetically engineered microbial cells or mammalian cell cultures such as CHO or BHK cells).

[0140] In some embodiments, the method further comprises cutting the cleavage site, thereby separating the bacteriocin (and / or signal molecule) from each other. When separating bacteriocin (and / or signal molecule), a composition comprising bacteriocin (or a combination of bacteriocin and signal molecule) is produced. In some embodiments, the cleavage site is cut by a combination of a cleavage enzyme or a cleavage enzyme as described herein (e.g., in Table 4). In some embodiments, the cleavage site is cut by exposing a peptide comprising a pH-sensitive or chemically sensitive linker to a pH or chemical condition that induces cleavage. In some embodiments, the original polypeptide is stored for a period of time before cutting, for example, at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months or 3 months, including the range between any two listed values.

[0141] In some embodiments, when bacteriocins are part of a propolypeptide, they are inactive. Thus, in some embodiments, the microbial cell does not produce a functional immunomodulator of at least one bacteriocin, for example, the microbial cell may lack a coding sequence for an immunomodulator, or if it does contain a coding sequence for an immunomodulator, the coding sequence may be transcriptionally silent (e.g., due to lack of a promoter), and / or the coding sequence may be mutated such that any immunomodulator produced by the coding sequence is inactive. In some embodiments, the microbial cell comprises Escherichia coli or Bacillus subtilis.

[0142] In some embodiments, at least one bacteriocin is inactive when it is part of a propolypeptide.

[0143] In some embodiments, nucleic acid is expressed in vitro. Cell-free expression systems can be used for in vitro transcription and / or translation of nucleic acid. In some embodiments, the in vitro expression system comprises, consists of, or is substantially composed of a cell extract. In some embodiments, the in vitro expression system comprises RNA polymerase, ribosomes, tRNA (and corresponding amino acids), energy sources, and enzyme cofactors. The in vitro expression system can also include enzymes for co-translation or post-translational modification, and / or cellular components that mediate protein folding, such as heat shock proteins.

[0144] In some embodiments, the method further comprises separating the propolypeptide before cutting. The propolypeptide can be separated from a microbial cell, or from an in vitro expression system as described herein. In some embodiments, separation comprises purifying the propolypeptide. In some embodiments, separation comprises affinity purification of the propolypeptide. Affinity purification can be performed using affinity tags as described herein. The nucleic acid can encode the affinity tag on the propolypeptide. The affinity tag can be bound by a suitable binding agent specific to the affinity tag (e.g., an anti-myc antibody for a myc tag, or a GSH-coated bead for GST, or nickel or cobalt for a His tag). The binding agent can be fixed on a solid phase so that when the affinity tag is bound, the propolypeptide will be fixed on the solid phase. The solid phase can be removed from the microbial cell and / or in vitro expression system. Optionally, the solid phase can be washed. The affinity-tagged propolypeptide can then be released from the solid phase. For example, the propolypeptide can be separated by immunoprecipitation, affinity chromatography, etc.

[0145] In some embodiments, the nucleic acid comprises at least three bacteriocin encoding sequences in a single reading frame, for example at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 bacteriocins, including ranges between any two of the recited values, such as about 3-10, 3-20, 3-30, 3-50, 5-10, 5-20, 5-30, 5-50, 7-10, 7-20, 7-30, 7-50, 10-20, 10-30 or 10-50 bacteriocins. Thus, the nucleic acid can produce a propolypeptide comprising a specified number of bacteriocins.

[0146] In some embodiments, at least two bacteriocins are different from each other. In some embodiments, two or more bacteriocins are the same. In some embodiments, at least two bacteriocins are different from each other, and at least two bacteriocins are the same.

[0147] When the propolypeptide is cut, a composition comprising a bacteriocin can be produced. In some embodiments, the composition comprises a desired ratio of bacteriocins. In some embodiments, at least a portion of the desired ratio is achieved by the ratio of bacteriocin coding sequences in a single reading frame of the nucleic acid. Without being limited by theory, it is considered that in some embodiments, the nucleic acid encodes two or more different bacteriocin coding sequences of the desired relative amount in cis, and the corresponding propolypeptide will also have these bacteriocin ratios. For example, if the nucleic acid contains two coding sequences of "bacteriocin A" and three coding sequences of "bacteriocin B", each sequence is separated by a cleavage site, the resulting propolypeptide will contain a ratio of bacteriocin A to bacteriocin B of 2:3. When cut, the solution will contain bacteriocins in these ratios. Note that in some embodiments, additional proteins or polypeptides (such as signal molecules) encoded by the nucleic acid are also produced in a desired ratio. Therefore, a solution containing bacteriocins and signal molecules in a predetermined ratio or a range of ratios can be produced. In some embodiments, a predetermined ratio is achieved by the ratio of bacteriocin coding sequences in a single reading frame of the nucleic acid.

[0148] In some embodiments, the nucleic acid encodes a portion of the desired ratio of bacteriocin (and optional additional peptides, such as signal molecules). Additional components can be added to achieve the final desired ratio. For example, a second different pro-polypeptide can contain a second ratio of bacteriocin and / or signal molecules. Two different polypeptides can provide the final desired ratio of the target bacteriocin (and optional other peptides, such as signal molecules) together. Therefore, in some embodiments, the desired ratio is further achieved by comprising a certain ratio of bacteriocin coding sequences and further comprising a second nucleic acid of a cleavage site between bacteriocin coding sequences.

[0149] The desired ratio can be selected for a variety of applications. In some embodiments, the desired bacteriocin ratio is selected to target an undesirable microbial organism or a population of undesirable microbial organisms. For example, if an environment such as a culture medium, a feedstock, a fermenter, a bioreactor, or a microbiome contains an undesirable population of microbial organisms or is at risk of containing it, a certain ratio of bacteriocins can be selected to target those undesirable microbial organisms. In some embodiments, the desired bacteriocin ratio is selected to balance the microbial community population of an animal (e.g., horse, cattle, sheep, pig, donkey, dog, cat, or non-human primate), a human organ (e.g., skin or intestinal tract), or a plant root and / or a soil microbiome, or to preserve products such as food (human or non-human animal), medicine, or cosmetics. In some embodiments, the desired ratio includes a ratio of about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 2:3, 2:5, 2:7, 2:9, 3:4, 3:5, 3:7, 3:8, 3:10, 4:5, 4:7, 4:9, 5:6, 5:7, 5:8, 5:9, 6:7, 7:8, 7:9, 7:10, 8:9 or 9:10 between two bacteriocins (e.g., a first bacteriocin and a second bacteriocin, a first bacteriocin and a third bacteriocin, a second bacteriocin and a third bacteriocin, or a third bacteriocin and a fourth bacteriocin, or a fourth bacteriocin and a fifth bacteriocin) in a propolypeptide, wherein the first (or second, or third, or fourth) bacteriocin is different from the second (or third, or fourth, or fifth) bacteriocin. Note that different bacteriocin pairs in a propolypeptide may have different ratios from each other. Therefore, it is contemplated that the ratios of three or more bacteriocins to each other can be determined by the individual (paired) ratios of the bacteriocins to each other. For example, the ratio of the first bacteriocin to the second bacteriocin can be 1:2, and the ratio of the second bacteriocin to the third bacteriocin can be 2:5, so that the ratios of the first bacteriocin to the second bacteriocin and the third bacteriocin are 1:2:5, respectively. In some embodiments, the ratios of the first bacteriocin to the second bacteriocin and the third bacteriocin are expected to include about 1:1:2, 1:2:2, 1:1:3, 1:2:3, 1:3:3, 2:2:3 or 2:3:3 of the first bacteriocin to the second bacteriocin and the third bacteriocin.

[0150] As described herein, it may be advantageous for the bacteriocin to remain intact after the propolypeptide is cleaved. Therefore, in some embodiments, the propolypeptide bacteriocin does not contain any cleavage sites that separate the bacteriocin (or other optional polypeptides such as signal molecules). When the cleavage site is cleaved, the bacteriocin can remain intact. In some embodiments, the cleavage site is for a single cleavage enzyme, and the cleavage enzyme does not cut within the bacteriocin. In some embodiments, at least one cleavage site is used for a first cleavage enzyme, another cleavage site is used for a second cleavage enzyme, and both the first cleavage enzyme and the second cleavage enzyme do not cut within the bacteriocin.

[0151] In some embodiments, the composition produced by the method comprises a signal molecule as described herein. The nucleic acid can encode a signal molecule in the same reading frame as other components of the propolypeptide (e.g., bacteriocin). The cleavage site sequence is disposed between the signal molecule and the bacteriocin encoding sequence. Therefore, when the cleavage site is cut, the signal molecule can be separated from the bacteriocin. In some embodiments, the signal molecule comprises, is essentially composed of, or is composed of a quorum sensing molecule, a signal transduction receptor ligand, a growth factor, a hormone or a cytokine, or a combination of two or more of these. In some embodiments, the signal molecule is wild type, mutant, or synthetic.

[0152] In some embodiments, the propolypeptide is no more than 20,000 amino acids in length, such as no more than 20,000, 15,000, 10,000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 amino acids in length, including any two of the listed In some embodiments, the length of the propolypeptide is no more than 5000 amino acids. In some embodiments, the length of the propolypeptide is no more than 2000 amino acids.

[0153] As described herein, the ratio of bacteriocins (and other polypeptides such as signal molecules) can be further adjusted by using two or more different propolypeptides to obtain a specific ratio and / or combination of molecules (e.g., bacteriocins, signal molecules). In some embodiments, the method includes expressing a second nucleic acid encoding a second propolypeptide, the second propolypeptide comprising two bacteriocins and a cleavage site disposed between the bacteriocins. The second propolypeptide can be different from the first propolypeptide, so that the cleavage of the second propolypeptide produces a different ratio of bacteriocins and / or signal molecules than the first propolypeptide after cleavage.

[0154] In some embodiments, the method also includes chemically modified bacteriocin. Exemplary chemical modifications include but are not limited to glycosylation, acetylation, methylation, pegylation, ubiquitination, ubiquitination or any of these two or more. In some embodiments, bacteriocin is co-translationally chemically modified. In some embodiments, bacteriocin is post-translationally chemically modified. For example, microbial cells may include or may be genetically engineered to include enzymes for co-translationally modified or post-translationally modified. For example, an in vitro expression system may include enzymes for co-translationally modified or post-translationally modified. For example, an in vitro expression system may include enzymes for post-translationally modified, and / or after separating propolypeptides from an expression system (in vitro or microbial cells), propolypeptides may be contacted with enzymes producing desired post-translationally modified. In some embodiments, post-chemically modified bacteriocin after propolypeptides are cut.

[0155] Some embodiments include compositions comprising bacteriocins, which can be prepared according to the methods of some embodiments herein, such as Figure 3 As shown. In some embodiments, the composition is produced by cleavage of a propolypeptide as described herein. It is contemplated that bacteriocins can be released by cleavage of a propolypeptide (e.g., via an endopeptidase). Depending on the predetermined and / or desired bacterial killing result, the result can be a mixture of the correct molecular blend. In some embodiments, a host cell can produce a mixture of multiple bacteriocins in a single fermentation. It is contemplated that there will be no toxic effects during the production process (therefore, the host cell does not need to be immunized). Optionally, purification of the propolypeptide (and / or "cleaved" bacteriocin) can be performed via a tag. Optionally, molecular regulation between bacteriocins can be performed.

[0156] Compositions comprising bacteriocins

[0157] In some embodiments, compositions comprising bacteriocins in a desired ratio are provided. Optionally, the composition may also comprise additional polypeptides, such as signaling molecules, in a desired ratio with the bacteriocin and / or with each other.

[0158] In some embodiments, the composition comprises two or more bacteriocins, and its ratio is selected as a target microbial cell or a microbial cell colony. Each bacteriocin can include a part of a cleavage sequence that has been cut at its N-terminus, C-terminus, or N-terminus and C-terminus. Consider that when the polypeptide precursor comprising multiple bacteriocins as described herein is cut at its cleavage site, at least some bacteriocins will include the residual part of the cleavage site at its N- and / or C-terminus, depending on the cleavage site. Therefore, it is considered that the bacteriocin in the composition not only exists in the expected ratio with very high accuracy, but also that the bacteriocin is structurally different because many or all bacteriocins will include the N- and / or C-terminal residual part of the cleavage site (e.g., partial cleavage site). In some embodiments, the cleavage sequence portion at the N-, C- or N- and C-terminal of the bacteriocin is used for the cleavage site of the same cleavage enzyme. In some embodiments, the portion of the cleavage sequence at the N-, C- or N- and C-terminal of the bacteriocin is used for the cleavage site of two or more different cleavage enzymes.

[0159] In some embodiments, some or all of the bacteriocins in the composition further comprise a tag. In some embodiments, the tag is selected from an affinity tag, a signal sequence, or a stability tag. In some embodiments, the cleavage site is disposed between the tag and the bacteriocin.

[0160] In some embodiments, the composition further comprises a signal molecule as described herein. The signal molecule may also be in a desired ratio. The signal molecule may also comprise a residual portion of a cleavage site at its N- and / or C-terminus.

[0161] In some embodiments, the ratio of the bacteriocin in the composition is selected to target an undesirable microbial organism or a population of undesirable microbial organisms in an environment, such as an industrial manufacturing environment, a fermentation tank, a food, drug or cosmetic manufacturing environment, or a product to be preserved (e.g., a food, drug or cosmetic). In some embodiments, the ratio of the bacteriocin is selected to balance the microbiome population in an animal, human organ or plant root and / or soil, or to preserve a product (e.g., a food, drug or cosmetic). In some embodiments, the composition is formulated for topical or oral administration to a human subject.

[0162] Coding base

[0163] According to the method and microfluidic device and system of some embodiments as described herein, coding substrate is provided.Coding substrate can comprise the nucleic acid of protein or peptide as described herein such as antimicrobial peptide and / or bacteriocin (and optional auxiliary protein).Coding substrate in the method and microfluidic device of some embodiments encodes antimicrobial peptide and / or bacteriocin, and can be used for producing the specified mixture of antimicrobial peptide and / or bacteriocin.Coding substrate can be discrete, and can be placed in fluid isolation from each other (it can be referred to as "discrete" coding substrate at this).For example, in the method or microfluidic device of some embodiments, several discrete coding substrates can be used for producing the specified mixture of antimicrobial peptide and / or bacteriocin by in vitro transcription / translation, wherein (i) only encodes the discrete coding substrate of antimicrobial peptide and / or bacteriocin in the specified mixture to contact in vitro transcription / translation solution, and incubate with it, (ii) only the coding substrate of antimicrobial peptide and / or bacteriocin in the specified mixture is placed in the fluid reservoir fluid that forms the specified mixture to be communicated with, or (i) and (ii). For example, valve as described herein can control in vitro transcription / translation solution to flow to antimicrobial peptides and / or bacteriocin from discrete coding substrates (or chambers that hold discrete coding substrates). In the method and microfluidic device of some embodiments, different coding substrates encoding antimicrobial peptides and / or bacteriocins that are different from each other can be contained in a separate chamber (microfluidic device as described herein), each of which can be communicated with in vitro transcription / translation solution fluid by valve. Therefore, in the method and microfluidic device of some embodiments, the discrete coding substrate of microfluidic device is included in a separate chamber. In some embodiments, the discrete coding substrate comprises beads, is basically composed of or is composed of. For example, the beads of antimicrobial peptides and / or bacteriocin in the coding specified mixture (rather than the beads encoding other antimicrobial peptides and / or bacteriocin) can be placed in communication with in vitro transcription / translation solution fluid by opening and / or closing valves to direct fluid to appropriate beads. In some embodiments, different discrete coding substrates encode different antimicrobial peptides and / or bacteriocins from each other. Some embodiments include a system that comprises a processor. Described system can be configured to be placed in and be communicated with microfluidic device fluid as herein described and / or data communication.Optionally, described system comprises pump and / or reagent (for example, in vitro transcription / translation solution) reservoir, can be placed in and be communicated with microfluidic device fluid.In some embodiments, microfluidic device is designed to be inserted into the cylinder in the system.In some embodiments, coding substrate encodes bacteriocin, but does not encode antimicrobial peptide.In some embodiments, coding substrate encodes antimicrobial peptide, but does not encode bacteriocin.In some embodiments, coding substrate encodes the combination of antimicrobial peptide and bacteriocin.

[0164] According to the method and microfluidic device and system of some embodiments described herein, the coding substrate is a discrete coding substrate. In some embodiments, the discrete coding substrate is included in the chamber. Therefore, in some embodiments, a plurality of discrete coding substrates are included in a separate chamber separately. In some embodiments, two or more discrete coding substrates are included in the same chamber so that a single chamber includes two or more discrete coding substrates, therefore mixture and / or the stoichiometry of antimicrobial peptides and / or bacteriocin can be produced. In some embodiments, the chamber comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 100, 1000, 5000, 10,000, 500,000, 1,000,000, 10,000,000, or 100,000,000 discrete encoded substrates, including ranges between any two of the recited values, such as 1-3, 1-5, 1-10, 1-50, 2-3, 2-5, 2-10, 2-20, 2-50, 2-100, 10-50, 50-100, 50- 500, 50–1000, 100–500, 100–1000, 500–1000, 1000–5000, 5000–10,000, 10,000–50,000, 50,000–100,000, 100,000–500,000, 500,000–1,000,000, 1,000,000–10,000,000, 10,000,000–100,000,000, or 100,000,000–1,000,000,000 discrete coding substrates.

[0165] According to the methods and microfluidic devices and systems of some embodiments described herein, the chambers in the microfluidic devices including the discrete encoded substrates are microscale chambers. For example, the chambers can each have a volume of no more than 1, 5, 10, 20, 50, 100, 250, or 500 microliters, including ranges between any two of the listed values, such as 1-5 microliters, 1-10 microliters, 1-20 microliters, 1-50 microliters, 1-100 microliters, 1-500 microliters, 5-10 microliters, 5-20 microliters, 5-50 microliters, 5-100 microliters, 5-500 microliters, 10-20 microliters, 10-50 microliters, 10-100 microliters, 10-500 microliters, 50-100 microliters, or 50-500 microliters. In some embodiments, chamber comprises the following material or product selected from, is basically composed of or is composed of: hole, nanopore, film, matrix, plastics, metal, glass, polymer, polysaccharide and paramagnetic compound or two or more combination in these.According to the method and microfluidic device of some embodiments described herein, coding substrate comprises the following material or product selected from, is basically composed of or is composed of: chip, bead, nanoparticle, hole, nanopore, film, matrix, plastics, metal, glass, polymer, polysaccharide and paramagnetic compound or two or more combination in these.Some embodiments comprise the microfluidic device comprising independent microchamber, comprise one or more beads separately, and described microchamber comprises coding substrate separately, is basically composed of or is composed of.Bead can comprise the nucleic acid of coding antimicrobial peptide and / or bacteriocin separately. In some embodiments, the device comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-50, 50-100, 100-500, 500-1000, or 1000-5000 chambers, or any number therebetween.

[0166] According to the methods and microfluidic devices and systems of some embodiments described herein, the coding substrate includes nucleic acids fixed thereon. For example, the nucleic acid can be covalently bound to the coding substrate, hybridized with the coding substrate, and / or associated with the coding substrate via one or more forces, such as electrostatic forces. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises RNA.

[0167] In some embodiments, the coding substrate also encodes or comprises an auxiliary protein, such as a protein for stabilizing antimicrobial peptides and / or bacteriocins, a protein with antiprotease activity (e.g., serpentin), a protein for stabilizing antimicrobial peptides and / or bacteriocins, a protein for disrupting microbial biofilms (e.g., a biofilm disruptor, such as Dispersin B), a pheromone protein, a protein that attracts non-pathogenic microbial organisms, or a protein that enhances the growth or reproduction of non-pathogenic microbial organisms in a microbiome in a subject. Without being limited by theory, it is contemplated that such auxiliary proteins may, for example, enhance the expression and / or activity of antimicrobial peptides and / or bacteriocins by stabilizing antimicrobial peptides and / or bacteriocins in a particular environment, by inhibiting proteases from a particular environment (which would degrade the antimicrobial peptides and / or bacteriocins), and / or by degrading fibrils of microbial biofilms to enhance bacterial contact of the antimicrobial peptides and / or bacteriocins. In some embodiments, the discrete coding substrate encodes an antimicrobial peptide and / or bacteriocin in addition to an auxiliary protein. In some embodiments, the first coding substrate encodes an auxiliary protein, and the second coding substrate encodes an antimicrobial peptide and / or a bacteriocin. In some embodiments, the auxiliary protein is selected with an antimicrobial peptide and / or a bacteriocin. For example, if the mixture of antimicrobial peptides and / or bacteriocin is used in an environment containing a specific protease (e.g., trypsin), then the coding substrate encoding the protease inhibitor and the coding substrate encoding the mixture of antimicrobial peptides and / or bacteriocin can be selected. In some embodiments, the coding substrate encodes an auxiliary protein with antiprotease activity. In some embodiments, the auxiliary protein with antiprotease is a protein for stabilizing antimicrobial peptides and / or bacteriocin. In some embodiments, one or more discrete coding substrates encode auxiliary proteins, which attract non-pathogenic microbial organisms, or enhance the growth or reproduction of non-pathogenic microbial organisms in the microbial community in an object (e.g., skin, intestines, gastrointestinal tract, mammary glands, placenta, tissue, biological fluids, semen, uterus, vagina, follicles, lungs, saliva, oral cavity, mucosa, conjunctiva or bile duct). In some embodiments, the accessory protein is a pheromone peptide that induces an antimicrobial peptide and / or bacteriocin of a desired microbial organism, such as a non-pathogenic microbial organism (although in some embodiments, a pathogenic microbial organism is a desired microbial organism). Thus, for example, in some embodiments, the discrete coding substrate encodes an accessory protein that induces or causes bacteria to secrete an antimicrobial peptide and / or bacteriocin that inhibits the growth or reproduction of another bacteria. In some embodiments, one or more discrete coding substrates encode proteins that attract non-pathogenic microbial organisms, or enhance the growth or reproduction of non-pathogenic microbial organisms in the microbial community in the object. In some embodiments, bacteriocins are used instead of antimicrobial peptides.

[0168] Inhibition of growth or reproduction has its usual and common meaning as understood by those skilled in the art in view of the present disclosure. It refers to the reduction or cessation of microbial organism proliferation (or a reduction in the rate of microbial biological proliferation), for example, the cessation of the cell cycle and / or the killing of microbial organisms. Similarly, enhancement of growth or reproduction refers to the increase of the proliferation or proliferation rate of microbial organisms. Any method known in the art can be used to detect inhibition or enhancement of growth or reproduction.

[0169] In vitro transcription / translation solution

[0170] According to the method, system and microfluidic device of some embodiments described herein, transcription / translation solution can be used for producing antimicrobial peptides and / or bacteriocin encoded by coding substrate.Therefore, transcription / translation solution can be used for producing peptides or antimicrobial peptides and / or bacteriocin in the method or microfluidic device of the specified mixture for producing antimicrobial peptides and / or bacteriocin.The term "in vitro transcription / translation solution" encompasses in vitro transcription solution and / or in vitro translation solution that are enough to produce antimicrobial peptides and / or bacteriocin from coding substrate.For example, in the embodiment that coding substrate comprises RNA transcripts encoding antimicrobial peptides and / or bacteriocin, it is considered that comprising translation solution, being basically composed of it or being composed of it "in vitro transcription / translation solution" is enough (because will understand that RNA is transcript).For example, in the embodiment that coding substrate comprises DNA encoding antimicrobial peptides and / or bacteriocin, it is considered that "in vitro transcription / translation solution" comprises transcription solution (for DNA is transcribed into RNA) and translation solution (for RNA is translated into polypeptide).In some embodiments, transcription and translation solution are together in single solution. In some embodiments, the transcription and translation solutions are in separate solutions, such as in vesicles suspended in a single solution, and / or in separate solutions that are administered sequentially. In some embodiments, the components of the in vitro transcription / translation solution are lyophilized and configured to reconstitute into the in vitro transcription / translation solution upon the addition of water. In some embodiments, the in vitro transcription / translation solution is reconstituted by adding water to the lyophilized components.

[0171] Translation solution can be used for translating nucleic acid according to method, system and / or microfluidic device as described herein.Suitable translation solution can comprise the reagent (for convenience, it can be referred to as " translation reagent " in this article) for in vitro translation, basically be composed of it or be composed of, and therefore can be configured for the in vitro translation of transcript such as RNA.Some embodiments include the transcription solution comprising the reagent (for convenience, it can be referred to as " transcription reagent " in this article) for transcription, therefore be configured for in vitro transcription and translation, for example transcribe and translate the nucleic acid of coding antimicrobial peptide and / or bacteriocin or other peptide as described herein.According to the method and microfluidic device of some embodiments, consider that in vitro transcription and translation in single solution (as also comprising the transcription solution of translation solution as described herein) can promote the effective in vitro generation of antimicrobial peptide and / or bacteriocin or other peptide.Therefore, according to the method and microfluidic device of some embodiments as described herein, in vitro transcription / translation solution comprises in vitro transcription reagent and / or in vitro translation reagent.

[0172] According to the methods and microfluidic devices of some embodiments described herein, the translation solution comprises, consists essentially of, or consists of one or more translation reagents or in vitro translation reagents. Examples of translation reagents include, but are not limited to, ribosomes, buffers, amino acids, tRNA (which may be conjugated to amino acids), lysates or extracts such as E. coli lysates or E. coli extracts, and cofactors or metal ions such as Mg. 2+ , or a combination of two or more of any of the listed items. According to the methods, systems and kits of some embodiments described herein, the translation solution also includes a transcription solution, and is therefore configured for in vitro transcription and translation. As described herein, the transcription solution also including the translation solution covers a single solution suitable for in vitro transcription and translation. Therefore, the transcription solution also including the translation solution covers a single transcription / translation solution. It should be understood that some components of the transcription and / or translation solution, such as ribosomes, may not be liquids and may potentially be separated from the transcription and / or translation solution, such as by filtering and / or centrifugation. The translation solution in the methods and microfluidic devices of some embodiments described herein (and which may be included in the translation solution as described herein) may include one or more transcription reagents, be substantially composed of it or be composed of it. Examples of transcription reagents include RNA polymerase, buffer, nucleic acid mixture (e.g., NTPs including ATP, GTP, CTP and UTP), cofactors or metal ions such as Mg 2+, transcription inducers (such as transcription factors, IPTG or lactose), polyadenylation enzymes, capping enzymes, lysates or extracts such as bacterial lysates or extracts such as E. coli lysates or E. coli extracts, SP6 polymerase, T3 polymerase, T7 RNA polymerase, or a mixture of two or more of any of the listed items. The transcription solution can be used to transcribe a template, such as a candidate nucleic acid as described herein. The translation solution in the methods and microfluidic devices of some embodiments includes a combination of one or more transcription reagents and one or more translation reagents. In some embodiments, the microfluidic device and / or system includes a reservoir containing an in vitro transcription / translation solution as described herein. In some embodiments, the reservoir contains an in vitro transcription solution and an in vitro translation solution, which can be combined to produce the in vitro transcription / translation solution.

[0173] In some embodiments, the translation solution comprises a post-translational modification enzyme. Examples of post-translational modification enzymes include, but are not limited to, cleavage enzymes, kinases, phosphatases, glycosyltransferases, or a mixture of any two of the listed items.

[0174] In accordance with some embodiments of the systems, methods, and microfluidic devices described herein, the translation solution is provided to the encoding substrate in a microliter scale. For example, the volume of the translation solution can be 1 µl–1000 µl, 1 µl–50 µl, 1 µl–500 µl, 1 µl–900 µl, 50 µl–100 µl, 50 µl–500 µl, 50 µl–1000 µl, 100 µl–200 µl, 100 µl–500 µl, 100 µl–1000 µl, 200 µl–500 µl, 200 µl–1000 µl, 500 µl–900 µl, or 500 µl–1000 µl.

[0175] According to the system, method and microfluidic device of some embodiments described herein, in vitro transcription / translation solution is lyophilized.In some embodiments, in vitro transcription / translation solution is configured to reconstruct in a solution such as water.Therefore, the microfluidic device of some embodiments can stably store a period of time at ambient temperature, and the effect of in vitro transcription / translation solution will not be significantly affected.Therefore, it is considered that the microfluidic device of some embodiments is suitable for instant processing, and can be used to prepare the specified mixture of antimicrobial peptides and / or bacteriocin as needed (for example, in response to specific damage, infection and / or surgical procedure).

[0176] Microfluidic Devices and Systems

[0177] The fluid system comprising a microfluidic device can be used to produce a specified mixture of antimicrobial peptides and / or bacteriocin according to the method and microfluidic device and system of some embodiments described herein. In some embodiments, the system is configured to receive a microfluidic device comprising a discrete coding substrate as described herein, so that the system can guide the microfluidic device to produce a specified mixture of antimicrobial peptides and / or bacteriocin via in vitro transcription / translation. In some embodiments, the system is configured to be connected to and / or data communicated with the device fluid. For example, a disposable cartridge can include a microfluidic device used in combination with the system described herein, consisting essentially of it, or consisting of it. Due to the consideration that the specified mixture of antimicrobial peptides and / or bacteriocin can be customized for a specific application, such as interacting with a specific microbial community of a specific object or targeting a specific infection, it is considered that a single-use disposable microfluidic device can minimize the stoichiometry and composition of the specified mixture of antimicrobial peptides and / or bacteriocin, and can be used to maintain the sterility of the medical application of the object. Therefore, in some embodiments, the microfluidic device is sterile. In the method and microfluidic device of some embodiments, the fluid device includes a microfluidic device. Because it is understood that a "microfluidic device" is a "fluidic device", wherever a "fluidic device" is mentioned herein, a microfluidic device is expressly included. In addition, unless otherwise expressly stated, any disclosure of a fluidic device (such as a microfluidic device) herein should be understood to be applicable to the microfluidic devices of some embodiments, and methods comprising the microfluidic devices as described herein.

[0178] In some embodiments, a microfluidic device for producing a specified mixture of antimicrobial peptides and / or bacteriocin is provided. In some embodiments, the device comprises a discrete coding substrate as described herein, which encodes antimicrobial peptides and / or bacteriocin separately. The device may also comprise in vitro transcription / translation solution as described herein. The device may also comprise a fluid reservoir, one or more valves, and / or may be configured to be placed in data communication with a processor as described herein. The valve may regulate the fluid communication between transcription / translation solution, discrete coding substrates and / or the fluid reservoir. The processor may be configured to control the valve so that the coding substrate of the antimicrobial peptides and / or bacteriocin in the specified mixture is placed in in vitro transcription / translation solution fluid communication, so as to produce the antimicrobial peptides and / or bacteriocin in the specified mixture. The processor may be configured to control the valve (and in some embodiments, fluid flow) in the microfluidic device so that the antimicrobial peptides and / or bacteriocin in the mixture are placed in fluid communication with the fluid reservoir, so that the antimicrobial peptides and / or bacteriocin in the specified mixture flow to the fluid reservoir, thereby providing the specified mixture of antimicrobial peptides and / or bacteriocin in the fluid reservoir. Optionally, the antimicrobial peptide and / or bacteriocin in the specified mixture are actively mixed in the fluid reservoir. In some embodiments, the antimicrobial peptide and / or bacteriocin in the specified mixture are passively mixed (rather than actively mixed) in the fluid reservoir. In some embodiments, the microfluidic device comprises a processor. In some embodiments, the microfluidic device itself does not comprise a processor, but is configured to be placed in and carries out data communication with the processor. In some embodiments, the microfluidic device is used to produce the specified mixture of bacteriocin (rather than the antimicrobial peptide). In some embodiments, the microfluidic device is used to produce the specified mixture of antimicrobial peptide (rather than bacteriocin). In some embodiments, the microfluidic device is used to produce the specified mixture of bacteriocin and antimicrobial peptide.

[0179] Some embodiments of the methods and microfluidic devices described herein include a microfluidic device for producing a specified mixture of antimicrobial peptides and / or bacteriocins. The device may include: a discrete coding substrate, each of which encodes a bacteriocin and / or an antimicrobial peptide; an in vitro transcription / translation solution; a fluid reservoir; a valve, each of which is arranged on a fluid path between the discrete coding substrate and the fluid reservoir, and each valve is configured to regulate the flow between the discrete coding substrate and the fluid reservoir. The device can be configured to be placed in data communication with a processor, which is configured to: based on a specified mixture of antimicrobial peptides and / or bacteriocins, configure the valve to place the discrete coding substrates (rather than other discrete coding substrates) encoding the antimicrobial peptides and / or bacteriocins in the mixture in communication with the fluid reservoir; allow in vitro transcription / translation solution to be incubated with the discrete coding substrates encoding the antimicrobial peptides and / or bacteriocins in the mixture, so that the antimicrobial peptides and / or bacteriocins in the specified mixture are produced; allow the antimicrobial peptides and / or bacteriocins to flow into the fluid reservoir through the valve; and control the flow of the fluid in the fluid reservoir, wherein the flow includes the movement of the fluid containing the specified antimicrobial peptides and / or bacteriocins in the fluid reservoir, thereby producing the specified mixture of antimicrobial peptides and / or bacteriocins. In some embodiments, the microfluidic device includes a processor. In some embodiments, the microfluidic device is used to produce a specified mixture of bacteriocins (rather than antimicrobial peptides). In some embodiments, the microfluidic device is used to produce a specified mixture of antimicrobial peptides (rather than bacteriocins). In some embodiments, the microfluidic device is used to produce a specified mixture of bacteriocins and antimicrobial peptides.

[0180] According to the method and microfluidic device of some embodiments described herein, the microfluidic device includes discrete coding substrate, but does not include fluid reservoir and / or in vitro transcription / translation solution.Therefore, the microfluidic device can be placed in and communicated with system fluid as described herein and data communication, and the system can provide fluid reservoir and / or in vitro transcription / translation solution, so that the specified mixture of antimicrobial peptides and / or bacteriocin can be produced.Therefore, in some embodiments, the system includes fluid reservoir and / or in vitro transcription / translation solution as described herein.In some embodiments, the microfluidic device does not include fluid reservoir, in vitro transcription / translation solution and / or any other components in the system, and is separated from these components of the system until it is placed in data communication and / or fluid communication with the system.In some embodiments, the microfluidic device is configured to be attached to the fluid reservoir, in vitro transcription / translation solution and / or another component in the system.For example, the device can include a barrel, which is configured to be inserted into the system, so that the discrete coding substrate of the barrel is placed in and communicated with the (barrel) external fluid of the fluid reservoir, in vitro transcription / translation solution and / or other components in the system.In some embodiments, the barrel is consumable. For example, barrel can comprise discrete coding substrate, be configured to be inserted into system as herein described to produce the antimicrobial peptide of appointment and / or the mixture of bacteriocin, be discarded then, can comprise another barrel of other discrete coding substrate and be inserted into system to produce the different mixtures of antimicrobial peptide of appointment and / or bacteriocin afterwards.In some embodiments, barrel is configured to receive the in vitro transcription / translation solution from system, makes the discrete coding substrate of described in vitro transcription / translation solution contact barrel, and described discrete coding substrate encodes the bacteriocin and / or the antimicrobial peptide in the specified mixture.In some embodiments, microfluidic device is used to produce the specified mixture of bacteriocin (rather than antimicrobial peptide).In some embodiments, microfluidic device is used to produce the specified mixture of antimicrobial peptide (rather than bacteriocin).In some embodiments, microfluidic device is used to produce the specified mixture of bacteriocin and antimicrobial peptide.

[0181] Also contemplated are embodiments in which the system and / or microfluidic device is dry.In some embodiments, the system or microfluidic device includes a lyophilized reagent configured to dissolve in a fluid (eg, water).

[0182] Figure 5It is the schematic diagram of the microfluidic device 500 of some embodiments of this paper.Microfluidic device 500 can include discrete coding substrate 510a, 510b, 510c (although three discrete coding substrates are shown by example, the microfluidic device of the embodiment of this paper can include the discrete coding substrate of other quantities, as described herein).Described device can include fluid reservoir 540, and it can for example receive antimicrobial peptide and / or bacteriocin produced from discrete coding substrate 510a, 510b, 510c, therefore can include the specified mixture of antimicrobial peptide and / or bacteriocin as described herein.Valve 520a, 520b, 520c and / or 570a, 570b, 570c can regulate flow direction and the fluid flow from discrete coding substrate 510a, 510b, 510c.In some embodiments, microfluidic device also includes outlet 590, and it can allow to flow out microfluidic device from fluid reservoir 540. For example, a specified mixture of antimicrobial peptides and / or bacteriocins can flow to a tissue, wound, microbiome, and / or vessel as described herein of a subject through outlet 590. In some embodiments, a processor 595 as described herein is in data communication with the device and controls flow through valves 520a, 520b, 520c and / or 570a, 570b, 570c and / or 550 and / or 580. In some embodiments, the device itself does not include a processor 595 and is configured to be placed in data communication with a processor 595 as described herein to control flow through valves 520a, 520b, 520c and / or 570a, 570b, 570c and / or 550 and / or 580. Consider according to the microfluidic device and method of some embodiments of this paper, the generation of antimicrobial peptide and / or bacteriocin in the specified mixture can be by only making the subset of discrete coding substrate 510a, 510b, 510c first contact with in vitro transcription / translation solution (making only some discrete coding substrates be used to produce antimicrobial peptide and / or bacteriocin), and / or by only allowing the subset of antimicrobial peptide and / or bacteriocin produced from discrete coding substrate 510a, 510b, 510c to enter fluid reservoir 540 to realize. Note that only making the subset of discrete coding substrate contact with in vitro transcription / translation solution can improve efficiency by avoiding using in vitro transcription / translation solution on the coding substrate of antimicrobial peptide and / or bacteriocin in the specified mixture that is not encoded. In some embodiments, microfluidic device is a part of the barrel 591 that does not include the reservoir of in vitro transcription / translation solution. Barrel 591 can be configured to place microfluidic device in and carry out data communication with the system that comprises processor 595. Optionally, barrel can also be placed in and communicated with system fluid. In some embodiments, the microfluidic device is part of a cartridge 592 that includes a reservoir 530 of an in vitro transcription / translation solution. The cartridge 592 can be configured to place the microfluidic device in data communication with a system that includes a processor 595.In some embodiments, a microfluidic device comprises. Figure 6 The microfluidic device 600 shown consists essentially of or consists of a discrete coded substrate 610a, 610b, 610c, a channel 660, and valves 620a, 620b, 620c, 670a, 670b, 670c, and / or 680, similar to Figure 5 In the microfluidic device 500. The microfluidic device optionally does not include. In some embodiments, the microfluidic device 600 can be placed in the system fluid communication with the reservoir 530 or the fluid reservoir 540 comprising the in vitro transcription / translation solution, or with the heating element 555 data communication. For example, the disposable cartridge can include the microfluidic device 600, which is basically composed of or composed of it, and can be configured to be placed in the system fluid communication with the system fluid comprising the in vitro transcription / translation solution 530 and the fluid reservoir 540. The microfluidic device 600 can also be placed in the processor 595 and / or the heating element 555 with the system and carry out data communication. In some embodiments, the microfluidic device is used to produce the specified mixture of bacteriocin (rather than antimicrobial peptide). In some embodiments, the microfluidic device is used to produce the specified mixture of antimicrobial peptide (rather than bacteriocin). In some embodiments, the microfluidic device is used to produce the specified mixture of bacteriocin and antimicrobial peptide.

[0183] In some embodiments, valves 520a, 520b, and 520c regulate the flow between discrete coding substrates 510a, 510b, 510c and fluid reservoir 540. Valves 570a, 570b, and 570c are optional, and / or valves 550 and 580 are optional, and / or channel 560 is optional. In some embodiments, reservoir 530 of in vitro transcription / translation solution can be in fluid communication with fluid reservoir 540. Optionally, valve 550 is disposed between reservoir 530 of in vitro transcription / translation solution and fluid reservoir 540 and regulates the flow of in vitro transcription / translation solution to fluid reservoir 540. In some embodiments, Figure 6 The microfluidic device 600 is part of a cartridge.

[0184] In some embodiments, for example, if only the antimicrobial peptides and / or bacteriocins in a specified mixture are allowed to enter the fluid reservoir 540 from the coding substrates 510a, 510b, 510c, then valves 570a, 570b and 570c regulate the flow between the reservoir 530 of the in vitro transcription / translation solution and the discrete coding substrates 510a, 510b, 510c, and valves 520a, 520b, 520c are optional, and / or valve 550 is optional, and / or valve 580 is optional. The channel 560 can connect the reservoir of the in vitro transcription / translation solution 530 to the discrete coding substrates 510a, 510b, 510c, wherein there is an optional isolation valve 580 between the reservoir 530 and the channel 560, and / or there are optional isolation valves 570a, 570b, 570c between the channel 560 and the discrete coding substrates 510a, 510b, 510c. In some embodiments, valves 570a, 570b and 570c regulate the flow between the reservoir of the in vitro transcription / translation solution 530 and the discrete coding substrates 510a, 510b, 510c, and valves 520a, 520b, 520c regulate the flow between the discrete coding substrates 510a, 510b, 510c and the fluid reservoir 540. Optionally, channel 560 may connect reservoir 530 to discrete coded substrates 510a, 510b, 510c. Valve 580 is optional and / or valve 550 is optional.

[0185] The microfluidic device can also include an outlet 590. The outlet 590 can be placed in fluid reservoir (and any mixture of antimicrobial peptides and / or bacteriocin therein) and communicated with the tissue of the object, the wound of the object as described herein and / or the microbial community fluid. In some embodiments, the outlet 590 includes a valve. In some embodiments, the outlet 590 can be placed in a vessel (e.g., a test tube, a bag or a hole) fluid communication with a specified mixture for storing antimicrobial peptides and / or bacteriocin.

[0186] In some embodiments, the reservoir 530 of in vitro transcription / translation solution is in fluid communication with channel 560. Optional valve 580 controls from reservoir 530 to channel 560. In some embodiments, valves 570a, 570b and 570c regulate the flow of transcription / translation solution from channel 560 to discrete coding substrates 510a, 510b, 510c. For example, in order to produce an antimicrobial peptide and / or a specified mixture of bacteriocin comprising antimicrobial peptides and / or bacteriocin encoded by discrete coding substrates 510a and 510b (but not 510c), valves 570a and 570b can be opened, while valve 570c is closed, to allow transcription / translation solution 530 to flow into discrete coding substrates 510a and 510b, but not into 510c. In some embodiments, discrete encoded substrates 510a, 510b, and / or 510c are incubated with an in vitro transcription / translation solution to produce a specified mixture of antimicrobial peptides and / or bacteriocins, which is released into fluid reservoir 540 via valves 520a, 520b, and / or 520c.

[0187] In some embodiments, in vitro transcription / translation solution flows to discrete coding substrate 510a, 510b and / or 510c by fluid reservoir 540. The reservoir 530 of in vitro transcription / translation solution can be communicated with fluid reservoir 540 fluids, optionally by isolating valve 550 regulating flow. Optionally, valve 520a, 520b, 520c regulates the flow from fluid reservoir 540 to discrete coding substrate 510a, 510b and 510c. Therefore, in vitro transcription / translation solution can be incubated with suitable discrete coding substrate to produce antimicrobial peptide and / or bacteriocin, and then antimicrobial peptide and / or bacteriocin can flow to fluid reservoir 540 by valve 520a, 520b and 520c. Valve 570a, 570b, 570c, passage 560 and valve 580 can be optional. In some embodiments, discrete coded substrates 510a, 510b, and / or 510c are incubated with an in vitro transcription / translation solution to produce a specified mixture of antimicrobial peptides and / or bacteriocins, which is released into fluid reservoir 540 via valves 520a, 520b, and / or 520c. In some embodiments, the specified mixture of antimicrobial peptides and / or bacteriocins flows through outlet 590 to a wound, tissue, microbiome, and / or vessel as described herein of a subject.

[0188] In some embodiments, valve 550 regulates the flow from reservoir 530 of in vitro transcription / translation solution to fluid reservoir 540, and valves 520a, 520b and / or 520c regulate the flow from discrete coding substrates 510a, 510b and / or 510c to fluid reservoir 540. Valves 570a, 570b, 570c and 580 and channel 560 are optional. Therefore, in vitro transcription / translation solution can flow from fluid reservoir 540 to discrete coding substrates 510a, 510b and / or 510c, can be incubated with discrete coding substrates 510a, 510b and / or 510c, to produce antimicrobial peptides and / or bacteriocins, and antimicrobial peptides and / or bacteriocins can flow to fluid reservoir 540. In some embodiments, the processor 595 regulates the flow of the in vitro transcription / translation solution to discrete coding substrates 510a, 510b and / or 510c through valves 520a, 520b and 520c, so that only the discrete coding substrates encoding the antimicrobial peptides and / or bacteriocin in the specified mixture accept the in vitro transcription translation / solution. In some embodiments, the processor 595 allows the flow of the in vitro transcription / translation solution from the fluid reservoir 540 through valves 520a, 520b and 520c to the discrete coding substrates 510a, 510b and / or 510c encoding the antimicrobial peptides and / or bacteriocin in the specified mixture, and after the antimicrobial peptides and / or bacteriocin have been produced by in vitro transcription translation, the processor 595 regulates valves 520a, 520b, 520c, so that only the antimicrobial peptides and / or bacteriocin in the specified mixture enter the fluid reservoir 540.

[0189] Understandably, Figure 5 In some embodiments of the depicted fluid device 500, valves 550, 570a, 570b, 570c, 580 and channel 560 are optional. For example, some embodiments of the fluid device include valves 580, 570a, 570b and 570c, and flow device 560, but do not include valve 550. In some other embodiments, the fluid device includes valve 550, but does not include valves 570a, 570b, 570c or 580 or flow device 560. In some embodiments, the fluid device includes valves 550, 570a, 570b, 570c and 580 and flow device 560. In some embodiments, valve 580 is included, and valves 570a, 570b and 570c are not included. In some embodiments, valve 580 is not included, and valves 570a, 570b and 570c are included. It can also be understood that in some embodiments of the fluid device 500, the outlet 590 does not include a valve.

[0190] In some embodiments, outlet 590 allows a cleaning fluid to enter fluid reservoir 540, and any other valves may be opened to allow the cleaning fluid to flow into any other fluid connection of the device. In some embodiments, the washing fluid comprises a buffer or a detergent. It is understood that additional valves may be present in the microfluidic device to allow fluid to flow into or out of any fluid portion of the device.

[0191] In some embodiments, channel 560 comprises, consists essentially of, or consists of microchannels, tubes, pipes, and hoses, and thus, can contain fluids and / or direct fluid flow. In some embodiments, channel 560 comprises materials such as rubber, plastic, metal.

[0192] According to the methods and microfluidic devices of some embodiments described herein, the microfluidic devices are portable. For example, some embodiments of the device can be removed from the laboratory and brought into a natural environment where scientists test various mixtures of specified antimicrobial peptides and / or bacteriocins.

[0193] According to the methods and microfluidic devices of some embodiments described herein, the microfluidic device includes an outlet 590 through which a specified mixture of antimicrobial peptides and / or bacteriocins is delivered to the wound. For example, the outlet 590 can be connected to the wound via a membrane or a tube. Thus, in some embodiments, the device includes a wound dressing or a wound cleaning device.

[0194] In some embodiments, fluid is passively transferred from one component of the device to another component.The microfluidic device of some embodiments includes a pump 565 that actively pumps fluid from one component of the device to another component.

[0195] According to the methods and microfluidic devices of some embodiments described herein, the microfluidic device includes a heating element 555, such as a heating block. In some embodiments, the heating element is configured to heat components of the microfluidic device, such as the fluid reservoir 540 and / or the discrete coded substrates 510a, 510b, and / or 510c, to perform an incubation step. In some embodiments, the incubation step is at least about 0°C, 4°C, 25°C, 30°C, 37°C, 38°C, 40°C, including ranges between any two of the listed values, such as 0-40°C or 36-38°C or 4-40°C. In some embodiments, the in vitro transcription / translation solution is incubated for at least 1 second, 10 seconds, 30 seconds, 60 seconds, or at least 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes, including ranges between any two of the recited values, such as 1-30 seconds, 30-60 seconds, 1-2 minutes, 2-5 minutes, 5-10 minutes, 10-15 minutes, 15-30 minutes, or 30-60 minutes, 1-2 hours, 2-5 hours, 5-10 hours, 10-15 hours, 15-24 hours, 24-48 hours, or 48-72 hours. In some embodiments, the incubation with the in vitro transcription / translation solution includes more than one temperature at different times.

[0196] According to the method and microfluidic device of some embodiments described herein, the microfluidic device includes a reservoir configured to mix with a specified antimicrobial peptide and / or a mixture of bacteriocin and / or a phage antibiotic. In some embodiments, the microfluidic device comprises an antibiotic containing a chemical antibiotic and / or a phage. In some embodiments, the system includes a reservoir configured to mix with a specified antimicrobial peptide and / or a mixture of bacteriocin and / or a phage antibiotic. According to the method and microfluidic device of some embodiments described herein, the microfluidic device includes an antibiotic containing a chemical antibiotic and / or a phage.

[0197] Fluid Reservoir

[0198] In the method and microfluidic device of some embodiments herein, the microfluidic device includes a fluid reservoir 540. In some embodiments, the fluid reservoir is in communication with or configured to at least one discrete coding substrate fluid (optionally, separated by valve as described herein). In some embodiments, the fluid reservoir is in communication with discrete coding substrates as described herein or the chamber fluid accommodating discrete coding substrates. In some embodiments, the fluid reservoir is in communication with discrete coding substrates (or chambers) fluids by passage, pipeline, microfluidic pipeline, film, net, opening, passage or two or more of these. For example, the passage, pipeline, microfluidic pipeline, film, net, opening and / or passage of some embodiments may include materials such as rubber, glass, plastic, metal, organic compound or two or more of these. In some embodiments, the fluid reservoir is configured to receive antimicrobial peptides and / or bacteriocin in the specified mixture of bacteriocin. The specified antimicrobial peptides and / or bacteriocin can be mixed in the fluid reservoir, for example, by passive mixing and / or active mixing, to form a mixture. In some embodiments, the microfluidic device includes more than one fluid reservoir. In some embodiments, the volume of the fluid reservoir is at least 1 μl, such as at least 1 μl, 5 μl, 10 μl, 100 μl, 500 μl, or 1000 μl, including ranges between any two of the recited values, e.g.

[0199] 1 µl–1000 µl, 1 µl–50 µl, 1 µl–500 µl, 1 µl–900 µl, 50 µl–100 µl, 50 µl–500 µl, 50 µl–1000 µl, 100 µl–200 µl, 100 µl–500 µl, 100 µl–1000 µl, 200 µl–500 µl, 200 µl–1000 µl, 500 µl–900 µl, 500 µl–1000 µl, 1 ml–1000 ml, 1 ml–50 ml, 1 ml–500 ml, 1 ml–900 ml, 50 ml–100 ml, 50 ml–500 ml, 50 ml–1000 ml, 100 ml–200 ml, 100 ml–500 ml, 100 ml–1000 ml, 200 ml–500ml, 200ml–1000ml, 500ml–900ml, 500ml–1000ml.

[0200] valve

[0201] In the method and microfluidic device of some embodiments, the microfluidic device includes one or more valves. As used herein, "valve" covers mechanical valves and electromagnetic fields, conditional diffusion membranes, and other devices that allow and restrict liquid flow as understood in the art. In some embodiments, the valve separates the fluid reservoir from the discrete coding substrate and controls the flow of fluid between the discrete coding substrate and the fluid reservoir. Each valve can be arranged on the fluid path between the discrete coding substrate and the fluid reservoir. Each valve can be configured to regulate the flow between the discrete coding substrate and the fluid reservoir. In some embodiments, each discrete coding substrate is separated from the fluid reservoir by a valve, and the valve is configured to control the flow of fluid from the discrete coding substrate to the fluid reservoir. In some embodiments, the valve as described herein includes a hydraulic valve, a pneumatic valve, a manual valve, a solenoid valve, a motor valve or a socket ball valve (socket ball valve), or a combination of two or more of these. In some embodiments, the valve as described herein includes a two-way valve, a three-way valve, or a four-way valve. In some embodiments, valves as described herein include microfluidic valves or microvalves, such as solenoid microvalves, screw microvalves, pneumatic microvalves, or a combination of two or more of these. In some embodiments, the valve is binary, such that the flow through the valve is "on" or "off." In some embodiments, the valve regulates the flow rate through the valve.

[0202] In some embodiments, valve 520a, 520b, 520c are arranged between fluid reservoir 540 and coding substrate 510a, 510b, 510c.For example, in some embodiments, valve is arranged between fluid reservoir and coding substrate, and between coding substrate and fluid reservoir.Can open single valve between fluid reservoir and coding substrate to flush some or all coding substrates with transcription / translation fluid, then only open valve between target coding substrate and reservoir after incubation, make only target antimicrobial peptide and / or bacteriocin be included in the specified mixture of antimicrobial peptide and / or bacteriocin, allow it to flow into fluid reservoir.

[0203] In some embodiments, a separate valve is disposed between the transcription / translation solution and the discrete coding substrate so that the separate valve can be opened and closed to place only the target discrete coding substrate in contact with the transcription / translation solution, thereby producing a specified mixture of antimicrobial peptides and / or bacteriocins when the target discrete coding substrate is incubated with the transcription / translation solution. In view of the present disclosure, those skilled in the art will appreciate that valves have a variety of ways to regulate the inflow and outflow of discrete coding substrates so that only antimicrobial peptides and / or bacteriocins in a specified mixture of antimicrobial peptides and / or bacteriocins are obtained in the mixture in the fluid reservoir 540.

[0204] Smart bandage can include processor, sensor (such as pH and / or temperature sensor) and therapeutic agent source, wherein processor is configured to control the use of therapeutic agent.For example, sensor can detect inflammation, and processor can apply a certain amount of anti-inflammatory agent in response to detected inflammation.The example of suitable smart bandage can be found on the World Wide Web with www.cnet.com / news / this-smart-bandage-can-deliver-drugs-monitor-chronic-wounds, which is incorporated by reference in its entirety.In some embodiments, microfluidic device as described herein includes smart bandage.In some embodiments, microfluidic device or system are configured to be placed in communication with smart bandage fluid.For example, the port of microfluidic device can be placed in communication with the reservoir fluid of smart bandage, so that smart bandage can control the delivery of antimicrobial peptide and / or bacteriocin to object.In some embodiments, disposable tube includes microfluidic device, and this tube also includes smart bandage, and is communicated with fluid reservoir fluid, so that the specified mixture of antimicrobial peptide and / or bacteriocin can be delivered to object via smart bandage. Optionally, the disposable cartridge comprising the smart bandage and the microfluidic device comprises an adhesive for direct application to the tissue of an object (e.g., skin). Therefore, the microfluidic device of some embodiments is used for medical purposes. In some embodiments, the smart bandage monitors a wound or inflammation site, and a specified mixture of antimicrobial peptides and / or bacteriocins as described herein is delivered to the skin or wound site.

[0205] processor

[0206] Processor 595 can regulate the valve in the microfluidic device and method of some embodiments.Processor can be configured to, configure valve based on the specified mixture of antimicrobial peptide and / or bacteriocin, so that the discrete coding substrate (rather than other discrete coding substrate) of the antimicrobial peptide and / or bacteriocin in the coding mixture is placed in communication with the fluid reservoir fluid.In some embodiments, processor is configured to allow the discrete coding substrate of antimicrobial peptide and / or bacteriocin in vitro transcription / translation solution and the coding mixture to be incubated, so as to produce the specified antimicrobial peptide and / or bacteriocin in the mixture.In some embodiments, processor is configured to allow the specified antimicrobial peptide and / or bacteriocin to flow into the fluid reservoir by valve.In some embodiments, processor is configured to control the flow of the fluid in the fluid reservoir.In some embodiments, described flow comprises making the fluid that comprises the specified antimicrobial peptide and / or bacteriocin move into the fluid reservoir and / or move in the fluid reservoir, thereby produces the specified mixture of antimicrobial peptide and / or bacteriocin. In some embodiments, the microfluidic device comprises a processor. In some embodiments, the microfluidic device does not comprise a processor, but is configured to be placed in and carries out data communication with the processor.

[0207] Consider according to the method and microfluidic device of some embodiments of this paper, the specified mixture of antimicrobial peptide and / or bacteriocin can be formed by two or more submixtures.For example, the first submixture of bacteriocin A and B in ratio of 1:1 can be mixed with the second submixture of bacteriocin B and C in ratio of 1:1 with the same amount, thereby produce the mixture of bacteriocin A, B and C in ratio of 1:2:1.According to the method and microfluidic device of some embodiments described herein, the specified mixture of antimicrobial peptide and / or bacteriocin comprises two or more submixtures, it comprises the subset of antimicrobial peptide and / or bacteriocin separately, and processor is configured to allow every kind of submixture to flow into fluid reservoir, thereby produce the specified mixture of antimicrobial peptide and / or bacteriocin.In some embodiments, the specified mixture of antimicrobial peptide and / or bacteriocin comprises the summation of the submixture of the bacteriocin of specifying stoichiometry, and the combination of submixture produces the mixture of specifying stoichiometry.

[0208] Method for producing a specified mixture of antimicrobial peptides and / or bacteriocins

[0209] In some embodiments, a method for producing a specified mixture of antimicrobial peptides and / or bacteriocins is provided. The method may include producing a specified mixture of bacteriocins by in vitro transcription and translation of bacteriocins encoded by discrete coding substrates, but do not produce other bacteriocins, then mixing the selected bacteriocins from in vitro transcription and translation to produce a specified mixture of antimicrobial peptides and / or bacteriocins. In some embodiments, the method may include producing a bacteriocin by in vitro transcription and translation of bacteriocins encoded by discrete coding substrates, then mixing the bacteriocins in the specified mixture, but do not mix other bacteriocins (if any) to produce a specified mixture of antimicrobial peptides and / or bacteriocins. In some embodiments, the method for producing a specified mixture of antimicrobial peptides and / or bacteriocins is carried out on a microfluidic device as described herein. In some embodiments, the method is used to produce a specified mixture of bacteriocins (rather than antimicrobial peptides). In some embodiments, the method is used to produce a specified mixture of antimicrobial peptides (rather than bacteriocins). In some embodiments, the method is used to produce a specified mixture of bacteriocins and antimicrobial peptides.

[0210] Figure 7 is a flow chart illustrating some embodiments of a method for producing a specified mixture of antimicrobial peptides and / or bacteriocins 700. The method may include selecting a specified mixture comprising two or more different specified bacteriocins (710); placing a discrete encoding substrate encoding an antimicrobial peptide and / or bacteriocin in the mixture (but not other discrete encoding substrates) in fluid communication with an in vitro transcription / translation solution in a microfluidic device comprising discrete encoding substrates each encoding an antimicrobial peptide and / or bacteriocin (720); incubating the discrete encoding substrates with the in vitro transcription / translation solution to produce an antimicrobial peptide and / or bacteriocin encoded by the discrete encoding substrates (730); and mixing the bacteriocins in the microfluidic device to produce a specified mixture of antimicrobial peptides and / or bacteriocins (740).

[0211] In some embodiments, the method of producing a designated mixture of antimicrobial peptides and / or bacteriocins further comprises producing two or more submixtures, each of which contains a subset of the designated mixture of antimicrobial peptides and / or bacteriocins; and combining the submixtures to produce the designated mixture of antimicrobial peptides and / or bacteriocins. For example, the method may include performing (a) to (d) as follows to produce a first specified submixture of antimicrobial peptides and / or bacteriocins: (a) selecting a submixture comprising two or more different specified antimicrobial peptides and / or bacteriocins; (b) placing a discrete encoding substrate encoding the antimicrobial peptides and / or bacteriocins in the submixture (but not other discrete encoding substrates) in fluid communication with an in vitro transcription / translation solution in a microfluidic device comprising discrete encoding substrates each encoding an antimicrobial peptide and / or bacteriocin; (c) incubating the discrete encoding substrate with the in vitro transcription / translation solution to produce antimicrobial peptides and / or bacteriocins encoded by the discrete encoding substrate; (d) mixing the antimicrobial peptides and / or bacteriocins in the microfluidic device to produce a specified submixture of antimicrobial peptides and / or bacteriocins; repeating (a) to (d) to produce a second specified submixture of antimicrobial peptides and / or bacteriocins; and combining the first specified submixture of antimicrobial peptides and / or bacteriocins with the second specified submixture of antimicrobial peptides and / or bacteriocins to produce a specified mixture of antimicrobial peptides and / or bacteriocins.

[0212] In some embodiments of the method for producing the specified mixture of antimicrobial peptides and / or bacteriocin, selection also comprises the stoichiometry of selecting two or more different specified antimicrobial peptides and / or bacteriocins.In some embodiments, the specified mixture of antimicrobial peptides and / or bacteriocins comprises the specified stoichiometry, and the sub-mixture of combination causes the specified stoichiometry.For example, the first sub-mixture of bacteriocin " A " and the second sub-mixture of bacteriocin " A " and " B " can be combined in the ratio of 1: 1, to produce the specified mixture of antimicrobial peptides and / or bacteriocin " A " and " B " of 2: 1 in a ratio.

[0213] In some embodiments of the method for producing a specified mixture of antimicrobial peptides and / or bacteriocins, incubating a discrete coding substrate with an in vitro transcription / translation solution includes making the in vitro transcription / translation solution flow to each discrete coding substrate of the bacteriocin in the coding mixture. In some embodiments, the method includes placing a discrete coding substrate (rather than other discrete coding substrates) encoding the antimicrobial peptides and / or bacteriocin in the mixture in fluid communication with the in vitro transcription / translation solution, which includes (i) opening a valve so that the source of the in vitro transcription / translation solution is placed in fluid communication with the discrete coding substrate; (ii) closing a valve so as to inhibit the fluid communication between the source of the in vitro transcription / translation solution and other discrete coding substrates, or a combination of (i) and (ii). In some embodiments, mixing the antimicrobial peptides and / or bacteriocins in a microfluidic device includes opening a valve so that the discrete coding substrates are placed in fluid communication with a fluid reservoir, wherein the antimicrobial peptides and / or bacteriocins are mixed in the fluid reservoir.

[0214] In some embodiments, the method for producing a specified mixture of antimicrobial peptides and / or bacteriocins also includes in situ screening of antimicrobial peptides and / or bacteriocins for desired effects. In some embodiments, the screening is for suppressing the growth or reproduction of pathogenic microorganisms in the microbial community in an object (such as skin, intestines, gastrointestinal tract, mammary gland, placenta, tissue, biological fluid, semen, uterus, vagina, follicle, lung, saliva, oral cavity, mucosa, conjunctiva or bile duct). For example, pathogenic bacteria can be rapidly evolving bacteria or bacteria that show antibiotic resistance, such as MRSA. In some embodiments, the screening is for the mixture of antimicrobial peptides and / or bacteriocins to not have harmful effects on the microbial community in an object (such as skin, intestines, gastrointestinal tract, mammary gland, placenta, tissue, biological fluid, semen, uterus, vagina, follicle, lung, saliva, oral cavity, mucosa, conjunctiva or bile duct). In some embodiments, the screening is performed in real time. For example, can screen in 120 minutes, 60 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes or 1 minute of the specified mixture of the antimicrobial peptide and / or bacteriocin produced.In some embodiments, screening is for the stability of antimicrobial peptide and / or bacteriocin or for the destruction of microbial biofilm.In some embodiments, screening is for strengthening the growth or breeding of non-pathogenic microorganisms in the microbial community in object (as skin, intestine, gastrointestinal tract, mammary gland, placenta, tissue, biological fluid, semen, uterus, vagina, follicle, lung, saliva, oral cavity, mucosa, conjunctiva or biliary tract).

[0215] Some microbial organisms can evolve rapidly. Advantageously, the methods, devices, and systems described herein can be used to combat rapidly evolving microbial organisms by rapidly producing a mixture of specified bacteriocins that is tailored to the infection of a subject by the microbial organism. In some embodiments, the subject is infected with Staphylococcus aureus such as MRSA, influenza virus, West Nile virus, or Zika virus. In some embodiments, the subject is a diabetic and / or has an infection in an extremity such as a hand or foot.

[0216] Also envisioned that antimicrobial peptides and / or bacteriocins are used in combination with conventional chemical antibiotics and / or phage antibiotics. In some embodiments, method as described herein comprises treating microbial infection with a mixture of specified antimicrobial peptides and / or bacteriocins and a combination of chemical antibiotics and / or phages. Some embodiments comprise that a mixture of specified antimicrobial peptides and / or bacteriocins and a combination of chemical antibiotics and / or phage antibiotics are delivered to an object. In addition, in some embodiments, microfluidic devices as described herein and / or systems also comprise conventional antibiotics, for example phage antibiotics or small molecule antibiotics such as metabolites. In some embodiments, microfluidic devices and / or systems also comprise antibiotic reservoirs. The antibiotic may be selected from amoxicillin, amoxicillin / clavulanic acid (amoxicillin+clavulanic acid), ampicillin, benzathine penicillin, benzylpenicillin, benzylpenicillin, cephalexin, cefazolin, cefixime, cefotaxime, ceftriaxone, cloxacillin, penicillin, phenoxymethylpenicillin (penicillin V), piperacillin / tazobactam, procaine penicillin, ceftazidime α, meropenem α, aztreonam α, imipenem / cilastatin, amikacin, azithromycin, chloramphenicol, ciprofloxacin, clarithromycin, clindamycin, doxycycline, erythromycin, gentamicin, metronidazole, nitrofurantoin, spectinomycin, trimethoprim / sulfamethoxazole, trimethoprim, vastatin. Archaemycin, clofazimine, dapsone, rifampicin, ethambutol / isoniazid, ethambutol / isoniazid / pyrazinamide / rifampicin, ethambutol / isoniazid / rifampicin, isoniazid, isoniazid / pyrazinamide / rifampicin, isoniazid / rifampicin, pyrazinamide, rifabutin, rifampicin, rifapentine, amikacin, bedaquiline, capreomycin, clofazimine, cycloserine, delamanid, ethionamide, kanamycin, levofloxacin, linezolid, moxifloxacin, para-aminosalicylic acid, rifabutin, rifapentine, rifampicin, rifaximin, streptomycin, bacteriophage, or a combination of two or more of these antibiotics.

[0217] Some embodiments of the method for producing the specified mixture of antimicrobial peptides and / or bacteriocin also comprise that the specified mixture of antimicrobial peptides and / or bacteriocin is delivered to the wound via pipe or film, thereby clean or repair the wound.For example, the specified mixture of antimicrobial peptides and / or bacteriocin can be placed on incision or sore, then incision or sore can be closed and / or bandaged.

[0218] Some embodiments of the method for producing a specified mixture of antimicrobial peptides and / or bacteriocin include the washing of microfluidic devices. Washing can be carried out with washing fluid. For example, some embodiments include incubation steps with a transcription solution, followed by a washing step, followed by a separate incubation with a translation solution.

[0219] In some embodiments, the method also includes applying a specified mixture of antimicrobial peptides and / or bacteriocins to an object in need of treatment. For example, an object may have an infection, such as an infected wound, a surgical incision, and an infection of the limbs associated with diabetes, and / or a biofilm. The specified mixture of antimicrobial peptides and / or bacteriocins may be selected to target the microorganisms infected (or selected as a candidate to target the microorganisms infected), and the specified mixture may be produced in a microfluidic device as described herein. The specified mixture of antimicrobial peptides and / or bacteriocins may be applied to an object as described herein, such as at or near the site of infection. In some embodiments, the microfluidic device itself is directly applied to infection (for example, as a smart bandage as described herein). In some embodiments, the method also includes selecting an object in need of treatment of infection. The method may also include selecting a specified mixture of antimicrobial peptides and / or bacteriocins to target infection, and producing a specified mixture in a microfluidic device as described herein. It is also contemplated that a microfluidic device is used for treatment, suppression, prevention and / or reduction of the medical use of infection risk. In some embodiments, the method (or medical use) comprises administering to a subject a specified mixture of an antimicrobial peptide and / or bacteriocin and an antibiotic (eg, a small molecule antibiotic and / or a bacteriophage) as described herein.

[0220] In some embodiments, the microfluidic device is used for veterinary purposes, for example to produce a specified mixture of antimicrobial peptides and / or bacteriocins to treat infections in livestock or farm animals, and / or to promote animal growth. Advantageously, the use of a specified mixture of antimicrobial peptides and / or bacteriocins in farm animals can avoid the use of antibiotics in food production. As the World Health Organization's recommendations point out, doing so can reduce the development and proliferation of antibiotic-resistant microbial organisms.

[0221] In some embodiments, the microfluidic device is used in a small fermentor (e.g., a fermentor volume less than or equal to 100 liters, 50 liters, 40 liters, 30 liters, 20 liters, 10 liters, 5 liters, 2 liters, or 1 liter, including ranges between any two of the recited values), for example to produce a specified mixture of bacteriocins to target contaminating organisms in the small fermentor.

[0222] In some embodiments, microfluidic device is used for disinfecting medical device.For example, can select the specified mixture of antimicrobial peptide and / or bacteriocin with target pollutant, as MRSA on medical device, and can be applied to medical device on the spot, for example in hospital.In some embodiments, microfluidic device is used for pre-treatment implant with the specified mixture of antimicrobial peptide and / or bacteriocin, so that suppress or prevent infection and / or biofilm formation.

[0223] In some embodiments, the microfluidic device is used to defend against pathogens, such as in epidemics or bioterrorism defense. A specified mixture of bacteriocins against pathogens (e.g., bioterrorism agents) can be manually or automatically dispensed into a system as described herein, and a specified mixture of bacteriocins targeting pathogens (e.g., bioterrorism agents) can be produced in a microfluidic device at or near the location of the pathogen (e.g., bioterrorism agent).

[0224] In some embodiments, described method also comprises the specified mixture of antimicrobial peptide and / or bacteriocin and another kind of reagent or antimicrobial compound are mixed to produce final mixture.For example, the specified mixture of antimicrobial peptide and / or bacteriocin can be combined with chemistry or natural fluid.In some embodiments, the specified mixture of antimicrobial peptide and / or bacteriocin and antibiotic medicine or phage are combined to produce final mixture.In some embodiments, final mixture is to be used for treatment, as the part of the preparation of for example anti-pain treatment.

[0225] Embodiment 1: A nucleic acid is provided, which encodes a coding sequence of one copy of the bacteriocin subtilisin, two copies of the coding sequence of the bacteriocin bavariancin-MN, and one copy of the coding sequence of the quorum sensing factor BsEDF, all of which are in a single reading frame. The nucleic acid also encodes a cleavage site for caspase 2 in the same reading frame, which is located on the side of each bacteriocin coding sequence and BsEDF coding sequence. Therefore, the nucleic acid encodes a propolypeptide, which comprises a copy of subtilisin, two copies of bavariancin-MN, and one copy of BsEDF, each flanked by a caspase 2 cleavage site. The nucleic acid is expressed in Escherichia coli cells, which do not contain the coding sequence of the immunomodulator of subtilisin-A or bavariancin-MN, and therefore do not produce functional immunomodulators of these bacteriocins. The propolypeptide is produced by Escherichia coli and contains subtilisin and bavariancin-MN in inactive form. The propolypeptide is purified by its His tag using a nickel-containing substrate. The purified propolypeptide is then cleaved using caspase 2 to produce a composition comprising subtilisin, bavarianin-MN, and BsEDF in a ratio of 1:2:1. This composition is added to industrial feedstocks to prevent the proliferation of undesirable microbial organisms (via bacteriocins) and to control the growth of genetically modified Bacillus subtilis (via BsEDF).

[0226] Embodiment 2:

[0227] It was determined that the ratio of bacteriocin Mundticin, selamectin-P, thuringin-17 and plant lactobacillus J of 1:2:3:4 can be used to target undesirable microbial cell populations in animal food during storage. Nucleic acids encoding one copy of the coding sequence of Mundticin, two copies of the coding sequence of selamectin-P, three copies of the coding sequence of thuringin-17 and four copies of the coding sequence of plant lactobacillus J were synthesized. All bacteriocin coding sequences are in a single reading frame, and each bacteriocin coding sequence is separated from other bacteriocin coding sequences by a sequence encoding a granzyme B cleavage site. The nucleic acid also encodes GST at the 3' end of the open reading frame and immediately downstream of the granzyme B cleavage site coding sequence. Therefore, the nucleic acid encodes a propolypeptide that contains Mundticin, selamectin-P, thuringin-17, plant lactobacillus J in a ratio of 4:3:2:1, respectively, and a C-terminal GST tag. The nucleic acid is expressed in an in vitro translation system to produce a propolypeptide. The propolypeptide is purified using GSH-coated beads. The propolypeptide is then cleaved by granzyme B to produce a solution containing active Mundticin, selamectin-P, thuringin-17, and plant lactobacillus J in a ratio of 1:2:3:4, respectively. The cleavage site immediately adjacent to the GST tag is also cleaved so that none of the bacteriocins contain GST. The composition containing the bacteriocins in a ratio of 1:2:3:4 is added to animal food to target undesirable microbial cell populations in animal food.

[0228] Embodiment 3:

[0229] A microfluidic device comprising 244 chambers is provided. Each chamber comprises a discrete coding substrate. Each discrete coding substrate is a chip, each chip comprising 10,000-20,000 DNA molecules, each encoding a bacteriocin in Table 1.2. The DNA molecules in each chamber encode a separate bacteriocin compared to the DNA molecules in each other chamber. Each chamber is connected to a fluid reservoir via a valve. The fluid reservoir is also connected to a transcription / translation solution contained in a transcription / translation solution chamber via a valve. The device comprises a processor configured to open a valve connecting the fluid reservoir to the transcription / translation solution to allow the transcription / translation solution to flow into the fluid reservoir, close a valve connecting the fluid reservoir to the transcription / translation solution to prevent the fluid from flowing back into the transcription / translation solution chamber, open a valve connecting the fluid reservoir to a chamber containing a selected coding substrate to allow the transcription / translation solution to flow from the fluid reservoir into the chamber containing the selected coding substrate, and at 37 o C., thereby producing the bacteriocin from the selected encoding substrate, and opening one or more valves, such as a valve connecting the encoding substrate to a fluid reservoir, to release the bacteriocin into the fluid reservoir and / or container.

[0230] The device is electrically or wirelessly connected to a user input device, such as a phone, touch screen, keyboard, button, mouse or computer. The processor selects a bacteriocin based on user input entered into the user input device or according to a pre-programmed instruction set.

[0231] Embodiment 4:

[0232] The device as described herein is used to prepare a first specified mixture of bacteriocins. The bacteriocins are applied to a transdermal patch, which is applied to a subject with a burn. If the subject's burn does not heal, the device is used to prepare a second specified mixture of bacteriocins that is different from the first specified mixture of bacteriocins. The second specified mixture of bacteriocins is then applied to a new transdermal patch, which is applied to the burn. Multiple variations of the specified mixture of bacteriocins can be applied to burns of various subjects, depending on whether a particular formulation of the bacteriocin is expected to be beneficial to the healing of the burn, or whether it is desired to test a particular formulation.

[0233] Options

[0234] In addition to the above items, the following items are also described: 1. A method for preparing a bacteriocin, the method comprising expressing a nucleic acid comprising: A bacteriocin encoding sequence and a second polypeptide encoding sequence in a single reading frame, wherein the second polypeptide is a bacteriocin or a signal molecule; and a cleavage site encoding sequence disposed between the bacteriocin encoding sequence and the second polypeptide encoding sequence in said single reading frame, Thereby a propolypeptide comprising the bacteriocin, the second polypeptide and the cleavage site disposed therebetween is produced.

[0235] 2. The method of option 1, further comprising cleaving the cleavage site, thereby separating the bacteriocin and the second polypeptide from each other, thereby producing a composition comprising the bacteriocin and the second polypeptide.

[0236] 3. The method of any one of options 1 or 2, wherein the second polypeptide is the bacteriocin.

[0237] 4. A method as described in any of options 1 or 2, wherein the second polypeptide is the signaling molecule.

[0238] 5. The method of any one of options 1-4, wherein the expressing is performed by a microbial cell that does not produce a functional immunomodulator of at least one bacteriocin.

[0239] 6. The method of option 5, wherein the microbial cells do not produce any functional immunomodulatory agent of bacteriocin.

[0240] 7. The method of any one of options 1-4, wherein the expressing is performed in vitro.

[0241] 8. The method of any one of options 1-7, wherein at least one bacteriocin is inactive when it is part of the propolypeptide.

[0242] 9. The method of any one of options 2-8, further comprising isolating the propolypeptide prior to said cleavage.

[0243] 10. The method of option 9, wherein the isolating comprises affinity purifying the propolypeptide, wherein the affinity purification comprises binding an affinity tag encoded by the nucleic acid.

[0244] 11. The method of any one of options 1-8, wherein the nucleic acid comprises three bacteriocin encoding sequences in the single reading frame.

[0245] 12. The method of any one of options 1-3 or 5-11, wherein at least two of the bacteriocins are different from each other.

[0246] 13. The method of any one of options 2-12, wherein the composition comprises a desired ratio of bacteriocin, or a desired ratio of a signal molecule and a bacteriocin.

[0247] 14. The method of option 13, wherein at least a portion of the desired ratio is achieved by the ratio of bacteriocin encoding sequence or bacteriocin encoding sequence and signal molecule encoding sequence in a single reading frame of the nucleic acid.

[0248] 15. The method of any one of options 12-14, wherein the desired ratio is further achieved by a second nucleic acid comprising a certain ratio of bacteriocin encoding sequences and further comprising a cleavage site between the bacteriocin encoding sequences.

[0249] 16. The method of option 15, wherein the desired ratio is achieved by the ratio of bacteriocin encoding sequences in a single reading frame of the nucleic acid.

[0250] 17. The method of any one of options 12-16, wherein a desired bacteriocin ratio is selected to target an undesirable microbial organism or a population of undesirable microbial organisms, and / or

[0251] The desired bacteriocin ratio is selected to balance the microbiome population in an animal, human organ, plant root or soil.

[0252] 18. The method of any one of options 12-16, wherein the desired ratio of bacteriocin and signal molecule is selected to control genetic drift of target microbial cells and stimulate growth or production of producer cells.

[0253] 19. The method of any one of options 10-18, wherein the desired ratio comprises a ratio of the first bacteriocin to the second bacteriocin of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 2:3, 2:5, 2:7, 2:9, 3:4, 3:5, 3:7, 3:8, 3:10, 4:5, 4:7, 4:9, 5:6, 5:7, 5:8, 5:9, 6:7, 7:8, 7:9, 7:10, 8:9 or 9:10, wherein the first bacteriocin is different from the second bacteriocin.

[0254] 20. The method of any one of options 1-18, wherein the cleavage site is for a wild-type cleavage enzyme, a variant, or a synthetic cleavage enzyme, such as an endopeptidase.

[0255] 21. A method as described in any of options 1-20, wherein the cleavage site is for a cleavage enzyme selected from the following: Arg-C protease, Asp-N endopeptidase, BNPS-skatole, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, chymotrypsin-high specificity, clostripain (clostripain peptidase B), CNBr, enterokinase, factor Xa, formic acid, glutamyl endopeptidase, granzyme B, hydroxylamine, iodosyl benzoic acid, LysC, neutrophil elastase, NTCB (2-nitro-5-thiocyanatobenzoic acid), pepsin (pH 1.3), pepsin (pH > 2), proline endopeptidase, proteinase K, staphylococcal peptidase I, thermolysin, thrombin or trypsin.

[0256] 22. The method of any one of options 1-21, wherein the cleavage site is for a single cleavage enzyme, and wherein the cleavage enzyme does not cleave within the bacteriocin.

[0257] 23. The method of any one of options 1-22, wherein at least one cleavage site is for a first cleavage enzyme, another cleavage site is for a second cleavage enzyme, and neither the first cleavage enzyme nor the second cleavage enzyme cleaves within the bacteriocin.

[0258] 24. The method of any one of options 1-23, wherein the composition further comprises a signaling molecule, and wherein the nucleotide further comprises: a coding sequence for a signal molecule in said single reading frame; and A cleavage site sequence is disposed between the signal molecule and the bacteriocin encoding sequence.

[0259] 25. The method of any one of options 1-2 or 4-24, wherein the signal molecule is selected from: a quorum sensing molecule, a signal transduction receptor ligand, a growth factor, a hormone, and a cytokine, and wherein the signal molecule may be wild-type, mutant, or synthetic.

[0260] 26. The method of any one of options 1-25, wherein the propolypeptide is no more than about 2000 amino acids in length.

[0261] 27. The method of any one of options 1-26, further comprising expressing a second nucleic acid encoding a second propolypeptide, the second propolypeptide comprising two bacteriocins and a cleavage site disposed therebetween, wherein the second propolypeptide is different from the first propolypeptide.

[0262] 28. The method of any one of options 2-19 or 24-27, wherein cleavage of the propolypeptide comprises physical treatment of a peptide linker comprised by the cleavage site, wherein the peptide linker is chemically sensitive or pH sensitive.

[0263] 29. The method of any one of options 1-28, further comprising chemically modifying the bacteriocin.

[0264] 30. The method of option 29, wherein the bacteriocin is chemically co-translationally modified.

[0265] 31. The method of any one of options 2-30, further comprising chemically modifying the bacteriocin after the cleavage.

[0266] 32. An isolated nucleic acid comprising: a bacteriocin encoding sequence and a second polypeptide encoding sequence in a single reading frame, wherein the second polypeptide is a bacteriocin or a signal molecule; and A cleavage site encoding sequence is disposed between the bacteriocin encoding sequences and in the single reading frame.

[0267] 33. The isolated nucleic acid of option 32, wherein the second polypeptide is the bacteriocin.

[0268] 34. The isolated nucleic acid of claim 32, wherein the second polypeptide is the signaling molecule.

[0269] 35. The isolated nucleic acid of any one of options 32-34, wherein the cleavage site encoding sequence encodes a cleavage site for a cleavage enzyme, and wherein the bacteriocin encoding sequence does not comprise a cleavage site for the cleavage enzyme.

[0270] 35. The isolated nucleic acid of any one of options 32-35, wherein the nucleic acid comprises three bacteriocin encoding sequences in the single reading frame.

[0271] 36. The isolated nucleic acid of any one of options 32-35, wherein the nucleic acid comprises at least 5, 10, 15 or 20 bacteriocin sequences in the single reading frame.

[0272] 37. The isolated nucleic acid of any one of options 32-36, wherein the cleavage site encoding sequence is set in frame between any two adjacent bacteriocin and / or signal molecule encoding sequences.

[0273] 38. The isolated nucleic acid of any one of options 32-36, wherein at least two bacteriocin sequences encode bacteriocins that are different from each other.

[0274] 39. The isolated nucleic acid of option 35, wherein the three bacteriocin sequences are present in a desired ratio or a portion of a desired ratio.

[0275] 40. The isolated nucleic acid of option 39, wherein the desired ratio is selected to target an undesirable microbial organism or a population of undesirable microbial organisms.

[0276] 41. The isolated nucleic acid of option 39, wherein the desired bacteriocin ratio is selected to balance a microbiome population in an animal, human organ, plant, plant root, or soil.

[0277] 42. An isolated nucleic acid as described in any one of options 32-41, wherein the cleavage site is for a wild-type cleavage enzyme, a variant or a synthetic cleavage enzyme, such as an endopeptidase.

[0278] 43. An isolated nucleic acid as described in any of options 32-42, wherein the cleavage site is for a cleavage enzyme selected from the group consisting of: Arg-C protease, Asp-N endopeptidase, BNPS-skatole, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, chymotrypsin-high specificity, clostripain (clostripain peptidase B), CNBr, enterokinase, factor Xa, formic acid, glutamyl endopeptidase, granzyme B, hydroxylamine, iodosyl benzoic acid, LysC, neutrophil elastase, NTCB (2-nitro-5-thiocyanatobenzoic acid), pepsin (pH 1.3), pepsin (pH > 2), proline endopeptidase, proteinase K, staphylococcal peptidase I, thermolysin, thrombin or trypsin.

[0279] 44. An isolated nucleic acid as described in any of options 42-43, wherein the first cleavage site encoding sequence encodes a first cleavage site for a first cleavage enzyme, and wherein the second cleavage site encoding sequence encodes a cleavage site for a second cleavage enzyme different from the first cleavage enzyme, and wherein the bacteriocin does not comprise a cleavage site for either the first cleavage enzyme or the second cleavage enzyme.

[0280] 45. An isolated nucleic acid as described in any of options 32-41, wherein the cleavage site comprises a pH-sensitive or chemical-sensitive linker.

[0281] 46. ​​An isolated nucleic acid as described in any one of options 32, 33 or 35-44, wherein the isolated nucleic acid further comprises a coding sequence of a signal molecule in the single reading frame, wherein the cleavage site coding sequence is arranged between the coding sequence of the signal molecule and the adjacent bacteriocin coding sequence.

[0282] 47. An isolated nucleic acid as described in any one of options 32 or 24-46, wherein the signal molecule is selected from: a quorum sensing molecule, a signal transduction receptor ligand, a growth factor, a hormone and a cytokine, and wherein the signal molecule can be wild-type, mutant or synthetic.

[0283] 48. A microbial cell comprising a promoter operably linked to the isolated nucleic acid of any one of options 32-47, wherein the isolated microbial cell does not produce a functional immunomodulator of a bacteriocin encoded by the isolated nucleic acid.

[0284] 49. The microbial cell of option 48, wherein the cell does not produce a functional immunomodulator of any bacteriocin encoded by the isolated nucleic acid.

[0285] 50. An isolated propolypeptide comprising: two bacteriocins, and / or a bacteriocin and a signaling molecule; A cleavage site disposed between the bacteriocin and / or the bacteriocin and the signal molecule; and Affinity tags.

[0286] 51. The isolated propolypeptide of option 50, wherein the propolypeptide comprises the two bacteriocins.

[0287] 52. The isolated propolypeptide of option 50, wherein the propolypeptide comprises the bacteriocin and the signal molecule.

[0288] 53. The isolated propolypeptide of any one of options 50-52, wherein the propolypeptide comprises three bacteriocins.

[0289] 54. The isolated propolypeptide of any one of options 50-52, wherein the propolypeptide comprises at least 5, 10, 15 or 20 bacteriocins.

[0290] 55. An isolated propolypeptide as described in any of options 50, 51, 53 or 54, wherein the propolypeptide comprises a signal molecule.

[0291] 56. The isolated propolypeptide of any one of options 50-55, wherein the cleavage site is for a cleavage enzyme, and wherein the bacteriocin encoding sequence does not comprise a cleavage site for the cleavage enzyme.

[0292] 57. The isolated propolypeptide of any one of options 50-55, wherein the cleavage site is for a first cleavage enzyme, and wherein another cleavage site is for a second cleavage enzyme different from the first cleavage enzyme, and wherein the bacteriocin does not comprise a cleavage site for either the first cleavage enzyme or the second cleavage enzyme.

[0293] 58. The isolated propolypeptide of any one of options 50-57, further comprising a co-translational or post-translational modification.

[0294] 59. A composition comprising two or more bacteriocins in a ratio selected to target a microbial cell or a population of microbial cells, Each of the bacteriocins comprises a portion of a cleavage sequence at its N-terminus, C-terminus, or both N-terminus and C-terminus that has been cleaved, wherein the portion of the cleavage sequence at the N-, C-, or both N- and C-termini of the bacteriocin is a cleavage site for the same or different cleavage enzymes.

[0295] 60. The composition of option 59, wherein at least some of the bacteriocins further comprise a tag.

[0296] 61. The composition of option 60, wherein the tag is selected from an affinity tag, a signal sequence, or a stability tag.

[0297] 62. A composition as described in any of options 59-61, further comprising a signal molecule in a desired ratio with the bacteriocin, wherein the signal molecule comprises a portion of a cleavage sequence that has been cleaved at its N-terminus, C-terminus, or N-terminus and C-terminus, wherein the portion of the cleavage sequence at the N-, C-, or N- and C-termini of the signal molecule is used for a cleavage site for the same or different cleavage enzymes.

[0298] 63. The composition of any one of options 59-62, wherein the ratio of bacteriocins is selected to target an undesirable microbial organism or a population of undesirable microbial organisms.

[0299] 64. The composition of any one of options 59-63, wherein the ratio of bacteriocins is selected to balance the microbiome population in an animal, human organ, plant, plant root, or soil.

[0300] 65. The composition of option 64, wherein the composition is formulated for topical or oral administration to a human subject.

[0301] 66. The composition of option 64 or 65, wherein the composition is configured for balancing the microbiome population in an animal, a human organ, a plant root, or soil.

[0302] 67. A method for producing a specified mixture of bacteriocins and / or antimicrobial peptides, the method comprising: selecting a mixture comprising two or more different bacteriocins and / or antimicrobial peptides; In a microfluidic device comprising discrete encoding substrates each encoding a bacteriocin or an antimicrobial peptide: placing the discrete encoding substrates encoding the antimicrobial peptide or bacteriocin in the specified mixture in fluid communication with an in vitro transcription / translation solution; incubating the discrete encoding substrate with the in vitro transcription / translation solution, thereby producing the antimicrobial peptide and / or bacteriocin encoded by the discrete encoding substrate; and The antimicrobial peptides and / or bacteriocins in the microfluidic device are mixed to produce a specified mixture of the antimicrobial peptides and / or bacteriocins.

[0303] 68. The method of option 67, further comprising: producing two or more submixtures each comprising a subset of the specified mixture of antimicrobial peptides and / or bacteriocins, and The submixtures are combined to produce the specified mixture of antimicrobial peptides and / or bacteriocins.

[0304] 69. The method of option 67 or 68, wherein selecting further comprises selecting the stoichiometry of two or more different antimicrobial peptides and / or bacteriocins in the specified mixture.

[0305] 70. The method of any one of options 68-69, wherein the specified mixture of antimicrobial peptides and / or bacteriocins comprises a specified stoichiometry, and wherein combining the submixtures produces the specified stoichiometry.

[0306] 71. A method as described in any of options 67-70, wherein the discrete encoded substrates are included in separate chambers.

[0307] 72. A method as described in any of options 67-71, wherein the discrete encoding substrate includes a nucleic acid fixed thereon.

[0308] 73. A method as described in any one of options 67-72, wherein a discrete coding substrate encoding the antimicrobial peptides and / or bacteriocins in the specified mixture, but not other discrete coding substrates, is placed in fluid communication with the in vitro transcription / translation solution.

[0309] 74. The method of any one of options 67-73, wherein incubating the discrete encoding substrates with the in vitro transcription / translation solution comprises flowing the in vitro transcription / translation solution into each chamber.

[0310] 75. The method of any one of options 67-74, wherein the in vitro transcription / translation solution comprises an in vitro transcription reagent and / or an in vitro translation reagent.

[0311] 76. A method as described in any one of options 67-75, wherein placing the discrete coding substrate encoding the antimicrobial peptides and / or bacteriocin in the specified mixture in fluid communication with an in vitro transcription / translation solution comprises (i) opening a valve to place a source of the in vitro transcription / translation solution in fluid communication with the discrete coding substrate; (ii) closing a valve to inhibit fluid communication between the source of the in vitro transcription / translation solution and the other discrete coding substrates, or a combination of (i) and (ii).

[0312] 77. A method as described in any one of options 67-76, wherein mixing the antimicrobial peptides and / or bacteriocins in the microfluidic device comprises opening a valve to place the discrete encoded substrate in fluid communication with a fluid reservoir, wherein the antimicrobial peptides and / or bacteriocins are mixed in the fluid reservoir.

[0313] 78. The method of any one of options 67-77, further comprising in situ screening of the mixture of antimicrobial peptides and / or bacteriocins for a desired effect.

[0314] 79. The method of option 78, wherein the screening is for inhibiting the growth or reproduction of pathogenic microbial organisms in the microbiome of a subject, such as the skin, intestine, gastrointestinal tract, breast, placenta, tissue, biological fluid, semen, uterus, vagina, ovarian follicles, lungs, saliva, oral cavity, mucosa, conjunctiva, or biliary tract.

[0315] 80. The method of option 78, wherein the screening is for the absence of a deleterious effect of the mixture of antimicrobial peptides and / or bacteriocins on a microbial community in a subject, such as the skin, intestine, gastrointestinal tract, breast, placenta, tissue, biological fluid, semen, uterus, vagina, follicles, lung, saliva, oral cavity, mucosa, conjunctiva, or biliary tract.

[0316] 81. The method of option 78, wherein the screening is performed in real time.

[0317] 82. The method of option 78, wherein the screening is for stability of antimicrobial peptides and / or bacteriocins or for disruption of microbial biofilms.

[0318] 83. A method as described in option 82, wherein one or more of the discrete coding substrates encode an auxiliary protein with anti-protease activity.

[0319] 84. The method of option 78, wherein the screening is used to enhance the growth or proliferation of non-pathogenic microbial organisms in the microbiome of a subject, such as the skin, intestine, gastrointestinal tract, breast, placenta, tissue, biological fluid, semen, uterus, vagina, ovarian follicles, lung, saliva, oral cavity, mucosa, conjunctiva, or biliary tract.

[0320] 85. The method of option 84, wherein one or more of the discrete encoded substrates encodes an accessory protein that attracts the non-pathogenic microbial organisms or enhances the growth or reproduction of non-pathogenic microbial organisms in the microbiome of the subject.

[0321] 86. The method of any one of options 67-85, further comprising delivering the specified mixture of antimicrobial peptides and / or bacteriocins to the wound via a tube or a membrane, thereby cleaning or dressing the wound.

[0322] 87. A microfluidic device for producing a specified mixture of antimicrobial peptides and / or bacteriocins, the device comprising: Discrete coding substrates, each encoding an antimicrobial peptide and / or a bacteriocin; In vitro transcription / translation solution; a fluid reservoir; and valves, each disposed in a fluid path between the discrete coded substrate and the fluid reservoir, each valve configured to regulate flow between the discrete coded substrate and the fluid reservoir, wherein the device is configured to be placed in data communication with a processor, the processor being configured to: Based on the specified mixture of antimicrobial peptides and / or bacteriocins, configuring the valve to place the discrete encoded substrate encoding the antimicrobial peptides and / or bacteriocins in the mixture, but not other discrete encoded substrates, in fluid communication with the fluid reservoir; allowing the in vitro transcription / translation solution to be incubated with the discrete encoding substrate encoding the antimicrobial peptides and / or bacteriocins in the specified mixture, thereby producing the antimicrobial peptides and / or bacteriocins in the specified mixture; allowing the antimicrobial peptide and / or bacteriocin to flow through the valve into the fluid reservoir; and The flow of the fluid in the fluid reservoir is controlled, wherein the flow includes the movement of the antimicrobial peptides and / or bacteriocins in the fluid reservoir, thereby generating a specified mixture of the antimicrobial peptides and / or bacteriocins in the fluid reservoir.

[0323] 88. The microfluidic device of option 87, wherein: The specified mixture of antimicrobial peptides and / or bacteriocins comprises two or more sub-mixtures, each of which comprises a subset of antimicrobial peptides and / or bacteriocins; and The processor is configured to allow each submixture to flow into the fluid reservoir, thereby producing a specified mixture of the antimicrobial peptides and / or bacteriocins.

[0324] 89. A microfluidic device as described in option 88, wherein the specified mixture of antimicrobial peptides and / or bacteriocins comprises the sum of subsets of antimicrobial peptides and / or bacteriocins of specified stoichiometry, and wherein the combination of the sub-mixtures produces the specified stoichiometry.

[0325] 90. A microfluidic device as described in any of options 87-89, wherein the discrete encoded substrates are included in separate chambers.

[0326] 91. A microfluidic device as described in any of options 87-90, wherein the discrete encoding substrate includes nucleic acids immobilized thereon.

[0327] 92. A microfluidic device as described in option 91, wherein the discrete encoding substrate comprises a material or product selected from the group consisting of: a chip, a bead, a nanoparticle, a hole, a membrane, a matrix, a plastic, a metal, glass, a polymer, a polysaccharide, and a paramagnetic compound.

[0328] 93. A microfluidic device as described in any of options 87-92, wherein the in vitro transcription / translation solution comprises an in vitro transcription reagent and / or an in vitro translation reagent.

[0329] 94. A microfluidic device as described in any of Options 87-93, wherein the device is portable.

[0330] 95. A microfluidic device as described in any of options 87-94, wherein one or more of the discrete encoding substrates encode auxiliary proteins, the auxiliary proteins comprising proteins for stabilizing antimicrobial peptides and / or bacteriocins, proteins with anti-protease activity, or proteins for destroying microbial biofilms.

[0331] 96. A microfluidic device as described in any of options 87-95, wherein one or more of the discrete encoded substrates encode an auxiliary protein that attracts non-pathogenic microbial organisms or enhances the growth or reproduction of non-pathogenic microbial organisms in the microbiome of a subject, such as the skin, intestine, gastrointestinal tract, breast, placenta, tissue, biological fluid, semen, uterus, vagina, follicles, lungs, saliva, oral cavity, mucosa, conjunctiva or biliary tract.

[0332] 97. A microfluidic device as described in any of options 87-96, wherein the fluid reservoir is configured to be placed in communication with the tissue fluid of the subject.

[0333] 98. A microfluidic device as described in any of option 97, wherein the tissue contains a microbiome, and the tissue is such as skin, intestine, gastrointestinal tract, breast, placenta, tissue, biological fluid, semen, uterus, vagina, follicle, lung, saliva, oral cavity, mucosa, conjunctiva or biliary tract.

[0334] 99. A microfluidic device as described in any of options 87-98, wherein the fluid reservoir is configured to be placed in fluid communication with a wound of a subject.

[0335] 100. A microfluidic device as described in any of Options 97-99, which also includes a fluid channel such as a tube or a membrane, through which the fluid reservoir is connected to the microbial community or wound fluid, and through which the specified mixture of antimicrobial peptides and / or bacteriocins can be delivered to the microbial community or wound.

[0336] 101. A microfluidic device as described in any of options 87-100, wherein each discrete encoding substrate encodes a different antimicrobial peptide and / or bacteriocin.

[0337] 102. A microfluidic device as described in any of options 87-102, wherein the discrete encoded substrate comprises an isolated nucleic acid as described in any of options 32-49.

[0338] 103. The microfluidic device of any one of options 87-102, wherein the discrete encoding substrate encodes the isolated propolypeptide of any one of options 50-58.

[0339] 104. A microfluidic device as described in any of options 87-103, which also includes the processor.

[0340] 105. A system comprising: The microfluidic device of any of options 87-104, and A processor configured to: Based on the specified mixture of antimicrobial peptides and / or bacteriocins, configuring the valve to place the discrete encoded substrate encoding the antimicrobial peptides and / or bacteriocins in the mixture, but not other discrete encoded substrates, in fluid communication with the fluid reservoir; allowing the in vitro transcription / translation solution to be incubated with the discrete encoding substrate encoding the antimicrobial peptides and / or bacteriocins in the specified mixture, thereby producing the antimicrobial peptides and / or bacteriocins in the specified mixture; allowing the antimicrobial peptide and / or bacteriocin to flow through the valve into the fluid reservoir; and The flow of the fluid in the fluid reservoir is controlled, wherein the flow includes the movement of the antimicrobial peptides and / or bacteriocins in the fluid reservoir, thereby generating a specified mixture of the antimicrobial peptides and / or bacteriocins in the fluid reservoir.

[0341] 106. The system of option 105, wherein the microfluidic device is included in (or is a cartridge), the system comprising a connector for placing the cartridge in data communication with the processor.

[0342] 107. The system of any of options 105-106, further comprising a reservoir for an in vitro transcription / translation solution.

[0343] 108. A microfluidic device for producing a specified mixture of said antimicrobial peptides and / or bacteriocins, said device comprising: Discrete coding substrates each encoding an antimicrobial peptide and / or a bacteriocin; Valves are each disposed on a fluid path connected to a discrete encoded substrate, each valve being configured to regulate flow to or from the discrete encoded substrate, and wherein the device is configured to be placed in fluid communication with a fluid reservoir or an in vitro transcription / translation solution.

[0344] 109. The microfluidic device of option 108, further comprising a fluid reservoir or an in vitro transcription / translation solution.

[0345] 110. The method of option 78, wherein the desired effect comprises antimicrobial activity.

[0346] 111. The method of option 110 further comprising screening 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 different mixtures for microbial infection.

[0347] 112. The method of any one of options 67-86, further comprising delivering the mixture of the designated antimicrobial peptides and / or bacteriocins in combination with a chemical antibiotic and / or a phage antibiotic to the subject.

[0348] 113. The method of any one of options 67-86, wherein the in vitro transcription / translation solution is lyophilized and further comprising adding water to the in vitro transcription / translation solution.

[0349] 114. A microfluidic device as described in any of claims 87-104, further comprising a reservoir of a chemical or phage antibiotic configured to mix with the specified mixture of antimicrobial peptides and / or bacteriocins.

[0350] 115. A microfluidic device as described in any of options 87-104, further comprising an antibiotic, wherein the antibiotic comprises a chemical antibiotic and / or a bacteriophage.

[0351] 116. A microfluidic device as described in any of options 87-104, wherein the in vitro transcription / translation solution is lyophilized.

[0352] 117. The system of any of options 105-107, further comprising a reservoir of a chemical or bacteriophage antibiotic configured to be mixed with the mixture of designated antimicrobial peptides and / or bacteriocins.

[0353] 118. The system of any one of options 105-107, further comprising an antibiotic comprising a chemical antibiotic and / or a bacteriophage.

[0354] 119. The system of option 107, wherein the in vitro transcription / translation solution is lyophilized.

[0355] 120. The method of any one of options 67-86 and 110-113, wherein the method is used to prepare a specified mixture of antimicrobial peptides that does not contain bacteriocin.

[0356] 121. The method of any one of options 67-86 and 110-113, wherein the method is used to prepare a specified mixture of bacteriocins that does not contain an antimicrobial peptide.

[0357] 122. The method of any one of options 67-86 and 110-113, wherein the method is used to prepare a specified mixture of bacteriocin and antimicrobial peptides.

[0358] 123. A microfluidic device as described in any of options 87-104, 108, 109 and 114-116, wherein the device is used to produce a specified mixture of bacteriocins that does not contain antimicrobial peptides.

[0359] 124. The microfluidic device of any one of options 87-104, 108, 109 and 114-116, wherein the device is used to produce a specified mixture of antimicrobial peptides that does not contain bacteriocins.

[0360] 125. A microfluidic device as described in any of options 87-104, 108, 109 and 114-116, wherein the device is used to produce a specified mixture of antimicrobial peptides and bacteriocins.

[0361] 126. The system of any one of options 105-107 and 117-119, wherein the system is used to produce a specified mixture of bacteriocins that does not include an antimicrobial peptide.

[0362] 127. The system of any one of options 105-107 and 117-119, wherein the system is used to produce a specified mixture of antimicrobial peptides that does not comprise a bacteriocin.

[0363] 128. The system of any one of options 105-107 and 117-119, wherein the system is used to produce a specified mixture of antimicrobial peptides and bacteriocins.

[0364] In at least some embodiments described herein, one or more elements used in the embodiments can be used interchangeably in another embodiment, unless such replacement is technically infeasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described herein without departing from the scope of the claimed subject matter. All of these modifications and variations are intended to fall within the subject matter scope defined by the appended claims.

[0365] With respect to substantially any plural and / or singular terms used herein, those skilled in the art may translate from the plural to the singular and / or from the singular to the plural as appropriate to the context and / or application. For clarity, the different singular / plural permutations may be clearly listed herein.

[0366] Those skilled in the art will understand that, in general, the terms used herein, especially in the appended claims (e.g., the bodies of the appended claims), are generally intended to be "open" terms (e.g., the term "including" should be understood as "including but not limited to", the term "having" should be understood as "having at least", the term "includes" should be understood as "including but not limited to", etc.). Those skilled in the art will also understand that if a specific number of introduced claim recitations is intended, such intent is explicitly recited in the claim, and in the absence of such a recitation, such intent is not present. For example, to aid understanding, the following appended claims may include the use of the introductory phrases "at least one" and "one or more / one or more" to introduce claim recitations. However, the use of such phrases should not be interpreted as implying that the introduction of a claim recitation by the indefinite article "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted as meaning at least one" or "one or more"); the same is true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of introduced claim recitations is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted as meaning at least the recited number (e.g., without The base statement of "two statements" with other modifiers means at least two statements or two or more statements). In addition, in those cases where a convention similar to "at least one of A, B, and C, etc." is used, generally, such construction is intended to mean that a person skilled in the art will understand the convention (e.g., "a system having at least one of A, B, and C" would include but is not limited to systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those cases where a convention similar to "at least one of A, B, or C, etc." is used, generally, such construction is intended to mean that a person skilled in the art will understand the convention (e.g., "a system having at least one of A, B, or C" would include but is not limited to systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.).Those skilled in the art will also understand that almost any disjunctive word and / or phrase presenting two or more alternative terms, whether in the specification, claims or drawings, should be understood to contemplate the possibility of including one of these terms, one of these terms, or both of these terms. For example, the phrase "A or B" will be understood to include the possibility of "A" or "B" or "A and B".

[0367] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also described in terms of any individual member or subgroup of members of the Markush group.

[0368] As will be understood by those skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily considered to fully describe and enable the same range to be divided into at least two equal parts, three equal parts, four equal parts, five equal parts, ten equal parts, etc. As a non-limiting example, each range discussed herein can be easily divided into a lower third, a middle third, and an upper third, etc. As will be understood by those skilled in the art, all languages ​​such as "at most", "at least", "greater than", "less than", etc. include the narrated numbers, and refer to the ranges that can be subsequently divided into sub-ranges as discussed herein. Finally, as will be understood by those skilled in the art, the range includes each individual member. Therefore, for example, a group with 1-3 products refers to a group with 1, 2 or 3 products. Similarly, a group with 1-5 products refers to a group with 1, 2, 3, 4 or 5 products, etc.

[0369] Although various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the appended claims.

Claims

1. A method for preparing a bacteriocin, the method comprising expressing a nucleic acid comprising: A bacteriocin encoding sequence and a second polypeptide encoding sequence in a single reading frame, wherein the second polypeptide is a bacteriocin or a signal molecule; and a cleavage site encoding sequence disposed between the bacteriocin encoding sequence and the second polypeptide encoding sequence in said single reading frame, Thereby a propolypeptide comprising the bacteriocin, the second polypeptide and the cleavage site disposed therebetween is produced.

2. The method of claim 1, further comprising cleaving the cleavage site, thereby separating the bacteriocin and the second polypeptide from each other, and thereby producing a composition comprising the bacteriocin and the second polypeptide.

3. The method of claim 1, wherein the expressing is performed by a microbial cell that does not produce a functional immunomodulator of at least one of the bacteriocins.

4. The method of claim 1, wherein at least one of the bacteriocins is inactive when it is part of the propolypeptide.

5. The method of any one of claims 1 to 4, wherein the composition comprises a desired ratio of bacteriocin, or a desired ratio of a signal molecule and a bacteriocin.

6. The method of claim 5, wherein at least a portion of the desired ratio is achieved by the ratio of bacteriocin encoding sequences or the ratio of bacteriocin and signal molecule encoding sequences in a single reading frame of the nucleic acid.

7. The method of claim 5, wherein a desired ratio of bacteriocins is selected to target an undesirable microbial organism or a population of undesirable microbial organisms, and / or The desired ratio of bacteriocins is selected to balance the microbial flora in animals, human organs, plant roots or soil.

8. An isolated nucleic acid comprising: A bacteriocin encoding sequence and a second polypeptide encoding sequence in a single reading frame, wherein the second polypeptide is a bacteriocin or a signal molecule; and A cleavage site encoding sequence is disposed between the bacteriocin encoding sequences and in the single reading frame.

9. The isolated nucleic acid of claim 8, wherein the cleavage site encoding sequence encodes a cleavage site for a cleavage enzyme, and wherein the bacteriocin encoding sequence does not comprise a cleavage site for the cleavage enzyme.

10. A microbial cell comprising a promoter operably linked to the isolated nucleic acid of any one of claims 8-9, wherein the microbial cell does not produce a functional immunomodulator of a bacteriocin encoded by the isolated nucleic acid.

11. An isolated propolypeptide comprising: two bacteriocins, and / or a bacteriocin and a signaling molecule; A cleavage site disposed between the bacteriocin and / or the bacteriocin and the signal molecule; and Affinity tags.

12. The isolated propolypeptide of claim 11, wherein the cleavage site is for a cleavage enzyme, and wherein the bacteriocin encoding sequence does not comprise a cleavage site for the cleavage enzyme.

13. A composition comprising two or more bacteriocins in a ratio selected to target a microbial cell or a population of microbial cells, Each of the bacteriocins comprises a portion of a cleavage sequence that has been cleaved at its N-terminus, C-terminus, or both, wherein the portion of the cleavage sequence at the N-terminus, C-terminus, or both of the N-terminus and C-terminus of the bacteriocin is a cleavage site for the same or different cleavage enzymes.

14. The composition of claim 13, wherein at least some of the bacteriocins further comprise a tag.

15. The composition of claim 14, wherein the tag is selected from an affinity tag, a signal sequence, or a stability tag.

16. A method for producing a specified mixture of bacteriocins and / or antimicrobial peptides, the method comprising: selecting a mixture comprising two or more different bacteriocins and / or antimicrobial peptides; In a microfluidic device comprising discrete encoding substrates each encoding a bacteriocin or an antimicrobial peptide: placing the discrete encoding substrates encoding the antimicrobial peptide or bacteriocin in the specified mixture in fluid communication with an in vitro transcription / translation solution; incubating the discrete encoding substrate with the in vitro transcription / translation solution, thereby producing the antimicrobial peptide and / or bacteriocin encoded by the discrete encoding substrate; as well as The antimicrobial peptides and / or bacteriocins in the microfluidic device are mixed to produce a specified mixture of the antimicrobial peptides and / or bacteriocins.

17. The method of claim 16, further comprising: producing two or more submixtures each comprising a subset of the specified mixture of antimicrobial peptides and / or bacteriocins, and The submixtures are combined to produce the specified mixture of antimicrobial peptides and / or bacteriocins.

18. The method of claim 16, wherein selecting further comprises selecting the stoichiometry of two or more different antimicrobial peptides and / or bacteriocins in the specified mixture.

19. The method of claim 16, wherein the specified mixture of antimicrobial peptides and / or bacteriocins comprises a specified stoichiometry, and wherein combining the submixtures produces the specified stoichiometry.

20. The method of any one of claims 16-19, wherein the discrete encoded substrates are contained in separate chambers.

21. as described in any one of the method for claim 16-19, wherein the discrete coding substrates encoding the antimicrobial peptides and / or bacteriocin in the specified mixture, but not other discrete coding substrates, are placed in communication with the in vitro transcription / translation solution fluid.

22. The method of any one of claims 16-19, further comprising in situ screening of the mixture of antimicrobial peptides and / or bacteriocins for a desired effect.

23. The method of claim 22, wherein the screening is for enhancing the growth or proliferation of non-pathogenic microbial organisms in the microbiome of a subject, such as the skin, intestine, gastrointestinal tract, breast, placenta, tissue, biological fluid, semen, uterus, vagina, ovarian follicles, lung, saliva, oral cavity, mucosa, conjunctiva, or biliary tract.

24. A microfluidic device for producing a specified mixture of antimicrobial peptides and / or bacteriocins, the device comprising: Discrete coding substrates, each encoding an antimicrobial peptide and / or a bacteriocin; In vitro transcription / translation solution; Fluid reservoirs; as well as valves, each disposed in a fluid path between the discrete coded substrate and the fluid reservoir, each valve being configured to regulate flow between the discrete coded substrate and the fluid reservoir, wherein the device is configured to be placed in data communication with a processor, the processor being configured to: Based on the specified mixture of antimicrobial peptides and / or bacteriocins, configuring the valve to place the discrete encoded substrate encoding the antimicrobial peptides and / or bacteriocins in the mixture, but not other discrete encoded substrates, in fluid communication with the fluid reservoir; allowing the in vitro transcription / translation solution to be incubated with the discrete encoding substrate encoding the antimicrobial peptides and / or bacteriocins in the specified mixture, thereby producing the antimicrobial peptides and / or bacteriocins in the specified mixture; allowing the antimicrobial peptide and / or bacteriocin to flow through the valve into the fluid reservoir; as well as The flow of the fluid in the fluid reservoir is controlled, wherein the flow includes the movement of the antimicrobial peptides and / or bacteriocins in the fluid reservoir, thereby generating a specified mixture of the antimicrobial peptides and / or bacteriocins in the fluid reservoir.

25. The microfluidic device of claim 24, wherein: The specified mixture of antimicrobial peptides and / or bacteriocins comprises two or more sub-mixtures, each of which comprises a subset of antimicrobial peptides and / or bacteriocins; and The processor is configured to allow each submixture to flow into the fluid reservoir, thereby producing the designated mixture of the antimicrobial peptides and / or bacteriocins.

26. microfluidic device as claimed in claim 24, wherein the specified mixture of antimicrobial peptides and / or bacteriocin comprises the sum of subsets of antimicrobial peptides and / or bacteriocins of a specified stoichiometry, and wherein the combination of said submixtures produces said specified stoichiometry.

27. The microfluidic device of any one of claims 24-26, wherein the discrete encoded substrates are contained within separate chambers.

28. The microfluidic device of any one of claims 24-26, wherein the fluid reservoir is configured to be placed in fluid communication with a tissue of a subject.

29. The microfluidic device of any one of claims 24-26, further comprising the processor.

30. A system comprising: The microfluidic device of any one of claims 24 to 26, and A processor configured to: Based on the specified mixture of antimicrobial peptides and / or bacteriocins, configuring the valve to place the discrete encoded substrate encoding the antimicrobial peptides and / or bacteriocins in the mixture, but not other discrete encoded substrates, in fluid communication with the fluid reservoir; allowing the in vitro transcription / translation solution to be incubated with the discrete encoding substrate encoding the antimicrobial peptides and / or bacteriocins in the specified mixture, thereby producing the antimicrobial peptides and / or bacteriocins in the specified mixture; allowing the antimicrobial peptide and / or bacteriocin to flow through the valve into the fluid reservoir; as well as The flow of the fluid in the fluid reservoir is controlled, wherein the flow includes the movement of the antimicrobial peptides and / or bacteriocins in the fluid reservoir, thereby generating a specified mixture of the antimicrobial peptides and / or bacteriocins in the fluid reservoir.

31. The system of claim 30, wherein the microfluidic device is included in a cartridge, the system comprising a coupling for placing the cartridge in data communication with the processor.

32. The system of claim 30, wherein the in vitro transcription / translation solution is lyophilized.

33. A microfluidic device for producing a specified mixture of said antimicrobial peptides and / or bacteriocins, said device comprising: Discrete coding substrates, each encoding an antimicrobial peptide and / or a bacteriocin; Valves are each disposed in a fluid path connected to a discrete encoded substrate, each valve being configured to regulate flow to or from the discrete encoded substrate, and wherein the device is configured to be placed in fluid communication with a fluid reservoir of an in vitro transcription / translation solution.

34. The microfluidic device of claim 33, further comprising a reservoir of a chemical or bacteriophage antibiotic configured to mix with the mixture of designated antimicrobial peptides and / or bacteriocins.

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