Methods and compositions for making bacteriocins and antimicrobial peptides
AssigneeSyngulon SA
InventorPhilippe Gabant
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Abstract
Methods and compositions for making bacteriocins are described in some embodiments herein. In some embodiments, pro-polypeptide comprising the bacteriocins in the desired ratios in cis, and separated by cleavage sited can be produced by a microbial cell comprising a nucleic acid encoding the pro-polypeptide. In some embodiments microfluidic devices and methods for making specified mixtures of antimicrobial peptides and/or bacteriocins are described.
Claims34
A method of making bacteriocins, the method comprising expressing a nucleic acid comprising:
- a bacteriocin coding sequence and a second polypeptide coding sequence in a single reading frame, wherein the second polypeptide is a bacteriocin or signal molecule;
- and
- cleavage site coding sequences disposed between the bacteriocin coding sequence and the second polypeptide coding sequence in the single reading frame,
- thereby generating a pro-polypeptide comprising the bacteriocin, second polypeptide, and cleavage sites disposed therebetween.
An isolated nucleic acid comprising:
- a bacteriocin coding sequence and a second polypeptide coding sequence in a single reading frame, wherein the second polypeptide is a bacteriocin or a signal molecule;
- and
- cleavage site coding sequences disposed between the bacteriocin coding sequences and in the single reading frame.
An isolated pro-polypeptide comprising:
- two bacteriocins, and/or a bacteriocin and a signal molecule;
- cleavage sites disposed between the bacteriocins and/or the bacteriocin and the signal molecule;
- and
- an affinity tag.
A composition comprising two more bacteriocins in a ratio selected to target a microbial cell or populations of microbial cells,
- wherein each of the bacteriocins comprises, at its N-terminus, C-terminus, or N-terminus and C-terminus, a portion of a cleavage sequence that has been cleaved, wherein the portions of cleavage sequences at the N-, C-, or N- and C-termini of the bacteriocins are for cleavage sites of the same or different cleavage enzyme.
A method for producing a specified mixture of bacteriocins and/or antimicrobial peptides, the method comprising:
- selecting the mixture to comprise two or more different bacteriocins and/or antimicrobial peptides;
- in a microfluidic device comprising discrete coding substrates that each encode a bacteriocin or antimicrobial peptide:
- placing discrete coding substrates that encode the antimicrobial peptides or bacteriocins of the specified mixture in fluid communication with an in vitro transcription/translation solution;
- incubating the discrete coding substrates with the in vitro transcription/translation solution, thereby generating antimicrobial peptides and/or bacteriocins encoded by the discrete coding substrates;
- and
- mixing the antimicrobial peptides and/or bacteriocins in the microfluidic device, thereby producing the specified mixture of antimicrobial peptides and/or bacteriocins.
A microfluidic device for producing a specified mixture of antimicrobial peptides and/or bacteriocins, the device comprising:
- discrete coding substrates that each encode an antimicrobial peptide and/or bacteriocin;
- an in vitro transcription/translation solution;
- a fluidic reservoir;
- and
- valves each disposed on a fluidic path between a discrete coding substrate and the fluidic reservoir, each valve configured to regulate flow between the discrete coding substrate and the fluidic reservoir, wherein the device is configured to be placed in data communication with a processor configured to:
- based on the specified mixture of antimicrobial peptides and/or bacteriocins, configure the valves to place the discrete coding substrates that encode the antimicrobial peptides and/or bacteriocins of the mixture, but not other discrete coding substrates, in fluidic communication with the fluidic reservoir;
- permit incubation of the in vitro transcription/translation solution with the discrete coding substrates that encode the antimicrobial peptides and/or bacteriocins of the specified mixture, whereby the antimicrobial peptides and/or bacteriocins of the specified mixture are produced;
- permit flow of the antimicrobial peptides and/or bacteriocins through the valves into the fluidic reservoir;
- and
- control flow of fluid in the fluidic reservoir, wherein the flow comprises movement of the antimicrobial peptides and/or bacteriocins in the fluidic reservoir, thereby producing the specified mixture of antimicrobial peptides and/or bacteriocins in the fluidic reservoir.
A microfluidic device for producing a specified mixture of antimicrobial peptides and/or bacteriocins, the device comprising:
- discrete coding substrates that each encode an antimicrobial peptide and/or bacteriocin;
- valves each disposed on a fluidic path connected to a discrete coding substrate, each valve configured to regulate flow to or from the discrete coding substrate, and wherein the device is configured to be placed in fluid communication with a fluidic reservoir of an in vitro transcription/translation solution.
Dependencies are inferred from the claim text.