Patent Application
20210284953
This disclosure relates to methods of culturing bacterial cells using synthetic quorum-regulated lysis, and more particularly to a co-lysis system. This invention also relates to uses of synthetic synchronized lysis circuits.
Microbial ecologists are increasingly turning to small, synthesized ecosystems1-5 as a reductionist tool to probe the complexity of native microbiomes6, 7. Concurrently, synthetic biologists have gone from single-cell gene circuits8-11 to controlling whole populations using intercellular signaling12-16.
Provided herein are methods of co-culturing by quorum sensing, bacterial strains useful in co-culture systems and methods, co-culture systems, and pharmaceutical compositions, drug delivery systems, and methods of treating disease related thereto. In some embodiments, the co-lysis systems are provided that operate in an orthogonal or essentially orthogonal manner.
In some embodiments, methods of maintaining a co-culture by quorum sensing are provided that include: co-culturing at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) bacterial strains at certain ratios (e.g., 1:1000, 1:900, 1:800, 1:750, 1:700, 1:650, 1:600, 1:550, 1:500, 1:450, 1:400, 1:350, 1:300, 1:250, 1:200, 1:150, 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1) during a period of time (e.g., at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours; or 12, 24, 48, 72, 96, or more hours; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days); wherein: at least one of the at least two bacterial strains has a growth advantage compared to at least one other bacterial strain; at least one of the at least two bacterial strains comprises a lysis plasmid and an activator plasmid.
In some embodiments, the at least two bacterial strains include a first bacterial strain and a second bacterial strain. In some embodiments, each of the first and second bacterial strains comprises a lysis plasmid having a lysis gene under the control of an activatable promoter; and an activator plasmid having an activator gene, the expression of which promotes the accumulation of a quorum sensing molecule, wherein both the activatable promoter of the lysis gene and the expression of the activator gene is activated by the quorum sensing molecule, wherein the quorum-sensing molecule of the first strain is different from the quorum-sensing molecule of the second strain, and wherein each quorum-sensing molecule of the first and second strains has no or substantially no effect on the activatable promoter of the lysis gene of the other strain.
As used herein, “substantially no effect” means no measurable effect on the activatable promoter, as measured by the expression of the activatable promotor of a fluorescent protein.
In some embodiments, the at least two bacterial strains (e.g., at least a first bacterial strain and a second bacterial strain) are metabolically competitive. In some embodiments, at least one of the at least two bacterial strains (e.g., at least one of a first bacterial strain and a second bacterial strain) are E. coli, S. typhimurium, or a bacterial variant thereof. In some embodiments, at least one of the at least two bacterial strains (e.g., at least one of a first bacterial strain and a second bacterial strain) are Gram-negative bacterial strains, e.g., a Salmonella strain, an Acetobacter strain, an Enterobacter strain, a Fusobacterium strain, a Helicobacter strain, a Klebsiella strain, or an E. coli strain. In some embodiments, the at least two bacterial strains (e.g., at least a first bacterial strain and a second bacterial strain) are Gram-positive bacterial strain, e.g., a Actinomyces strain, a Bacillus strain, a Clostridium strain, an Enterococcus strain, or a Lactobacillus strain. In some embodiments, the at least two bacterial strains are both Gram negative bacterial strains or both Gram positive strains. In some embodiments, at least one of the at least two bacterial strains (e.g., at least a first bacterial strain and a second bacterial strain) is a Gram negative bacterial strain. In some embodiments, at least one of the at least two bacterial strains (e.g., at least a first bacterial strain and a second bacterial strain) is a Gram positive bacterial strain. In some embodiments, at least one of the at least two bacterial strains (e.g., at least a first bacterial strain and a second bacterial strain) comprising the lysis plasmid and the activator plasmid does not have a growth advantage compared to at least one other bacterial strain.
In some embodiments, the lysis plasmid comprises a lysis gene, an activatable promoter, and optionally a reporter gene; and the activator plasmid comprises an activator gene, a degradation tag, and optionally a reporter gene. In some embodiments, the lysis gene is E from a bacteriophage ΦX174. In some embodiments, the activatable promoter is a LuxR- N-acyl homoserine lactone (AHL) activatable luxI promoter and the activator gene is a LuxI. In some embodiments, the activatable promoter is a RpaR- N-acyl homoserine lactone (AHL) activatable RpaI promoter and the activator gene is a RpaI. In some embodiments, the reporter gene is green fluorescent protein (GFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP) or a variant thereof. In some embodiments, the degradation tag is an ssrA-LAA degradation tag. In some embodiments, each of the at least two bacterial stains comprises the lysis plasmid and the activator plasmid. In some embodiments, each of the at least two bacterial strains (e.g., each of at least a first bacterial strain and a second bacterial strain) comprises a different reporter gene.
In some embodiments, the co-culture is inoculated at a ratio of 1:1000 (e.g.,1:900, 1:800, 1:750, 1:700, 1:650, 1:600, 1:550, 1:500, 1:450, 1:400, 1:350, 1:300, 1:250, 1:200, 1:150, 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1) of the bacterial strain having the growth advantage compared to the other bacterial strain.
In some embodiments, the plasmid is integrated into a genome of at least one of the at least two bacterial strains (e.g., at least a first bacterial strain and a second bacterial strain). In some embodiments, the plasmid further comprises a plasmid-stabilizing element. In some embodiments, the plasmid-stabilizing element is a toxin/antitoxin system or an actin-like protein partitioning system.
In some embodiments, the co-culturing occurs in a microfluidic device. In some embodiments, the co-culturing occurs in a cell culture vessel (e.g., a cell culture plate, a bioreactor).
In some embodiments, the period of time is 0 to 72 hours (e.g., o to 72; 0 to 60 hours; 0 to 48 hours; 0 to 36 hours; 0 to 24 hours; 0 to 16 hours; 0 to 14 hours; 0 to 12 hours; 0 to 10 hours; 0 to 8 hours; 0 to 6 hours; 0 to 4 hours; 0 to 2 hours; 2 to 72 hours; 2 to 60 hours; 2 to 48 hours; 2 to 36 hours; 2 to 24 hours; 2 to 16 hours; 2 to 14 hours; 2 to 12 hours; 2 to 10 hours; 2 to 8 hours; 2 to 6 hours; 2 to 4 hours; 4 to 72 hours; 4 to 60 hours; 4 to 48 hours; 4 to 36 hours; 4 to 24 hours; 4 to 16 hours; 4 to 14 hours; 4 to 12 hours; 4 to 10 hours; 4 to 8 hours; 4 to 6 hours; 6 to 8 hours; 6 to 10 hours; 6 to 12 hours; 6 to 14 hours; 6 to 16 hours; 6 to 18 hours; 6 to 20 hours; 6 to 22 hours 6 to 24 hours; 8 to 10 hours; 8 to 12 hours; 8 to 16 hours; 8 to 24 hours; 8 to 36 hours; 8 to 48 hours; 8 to 60 hours; 8 to 72 hours; 1 to 2 hours; 1 to 3 hours; 1 to 4 hours; 1 to 6 hours; 1 to 8 hours; 1 to 10 hours; 1 to 12 hours; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 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 or 72 hours).
In some embodiments, the co-culturing of the at least two bacterial strains (e.g., at least a first bacterial strain and a second bacterial strain) is in a constant lysis state; wherein the constant lysis state is characterized by a steady-state balance of growth and lysis of the at least two bacterial strains (e.g., at least a first bacterial strain and a second bacterial strain). In some embodiments, the co-culturing of the at least two bacterial strains (e.g., at least a first bacterial strain and a second bacterial strain) is oscillatory; wherein the oscillatory co-culturing indicates a high level of activator degradation in at least one of the two bacterial strains (e.g., at least a first bacterial strain and/or a second bacterial strain).
Provided herein are bacterial strains including a lysis plasmid and an activator plasmid; wherein the lysis plasmid comprises a lysis gene, an activatable promoter, and optionally a reporter gene; and the activator plasmid comprises an activator gene, a degradation tag, and optionally a reporter gene.
In some embodiments, the lysis gene is E from a bacteriophage ΦX174.
In some embodiments, the activatable promoter is a LuxR- N-acyl homoserine lactone (AHL) activatable luxI promoter and the activator gene is a LuxI. In some embodiments, the activatable promoter is a RpaR- N-acyl homoserine lactone (AHL) activatable RpaI promoter and the activator gene is a RpaI.
Also provided herein are pharmaceutical composition that include any of the bacterial strains described herein. In some embodiments, the pharmaceutical composition is formulated for in situ drug delivery. A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include oral or parenteral, e.g., intravenous, intradermal, subcutaneous, inhalation, transdermal (topical), transmucosal, and rectal administration. As used herein the language “pharmaceutically acceptable carrier” includes solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.
Provided herein are systems including: a co-culture of at least two bacterial strains (e.g., at least a first bacterial strain and a second bacterial strain), wherein the at least two bacterial strains comprise a first bacterial strain having at least a portion of a first synchronized lysis circuit, wherein the first synchronized lysis circuit comprises a first lysis plasmid and a first activator plasmid and wherein the first lysis plasmid is activated by the first activator plasmid. In some embodiments, the first bacterial strain comprises the first lysis plasmid. In some embodiments, the first bacterial strain comprises the first activator plasmid. In some embodiments, the at least two bacterial strains further comprise a second bacterial strain. In some embodiments, the second bacterial strain comprises the first activator plasmid. In some embodiments, each of the first bacterial strain and the second bacterial strain comprise the first activator plasmid.
In some embodiments, the first lysis plasmid of the first bacterial strain operates independent of at least one other bacterial strain in the co-culture. In some embodiments, the first lysis plasmid of the first bacterial strain responds to a signal generated by at least one other bacterial strain in the co-culture.
In some embodiments, the second bacterial strain has at least a portion of a second synchronized lysis circuit, wherein the second synchronized lysis circuit comprises a second lysis plasmid and a second activator plasmid. In some embodiments, the second bacterial strain comprises the second lysis plasmid. In some embodiments, the second bacterial strain comprises the second activator plasmid. In some embodiments, the first bacterial strain comprises the second activator plasmid. In some embodiments, the second lysis plasmid of the second bacterial strain operates independent of at least the first bacterial strain. In some embodiments, the second lysis plasmid of the second bacterial strain responds to a signal generated by the first bacterial strain.
In some embodiments of any of the systems described herein, the signal is a quorum sensing signal. In some embodiments, the first activator plasmid encodes a quorum sensing signal. In some embodiments, the second activator plasmid encodes a quorum sensing signal.
In some embodiments, at least one of the at least two bacterial strains (e.g., at least one of a first bacterial strain and a second bacterial strain) has a growth advantage compared to at least one other bacterial strain. In some embodiments, the first bacterial strain is competitive with at least one other bacterial strain in the co-culture. In some embodiments, the co-culture is stable for at least 48 hours.
In some embodiments, the at least two bacterial strains (e.g., at least a first bacterial strain and a second bacterial strain) do not comprise engineered positive or negative interactions between each other. In some embodiments, at least one of the at least two bacterial strains (e.g., at least one of a first bacterial strain and a second bacterial strain) dynamically controls its population without exogenous input. In some embodiments, each of at least two of the at least two bacterial strains (e.g., each of at least a first bacterial strain and a second bacterial strain) dynamically controls its own population without exogenous input. In some embodiments, the system further comprises one or more plasmid stabilizing elements.
In some embodiments, the plasmid stabilizing element is selected from a toxin/antitoxin system and an actin-like protein partitioning system.
In some embodiments, the first activator plasmid encodes a degradation tagging sequence. In some embodiments, the second activator plasmid encodes a degradation tagging sequence. In some embodiments, the first activator plasmid encodes an N-acyl homoserine lactone.
Provided herein are drug delivery systems including any of the systems described herein. Provided herein are periodic drug delivery systems including any of the systems described herein.
Provided herein are microfluidic sample traps including any of the systems described herein.
Provided herein are microfluidic devices including one or more microfluidic sample traps. In some embodiments, the microfluidic system further includes at least one channel in fluid communication with the microfluidic sample trap.
In one aspect, provided herein is a method of maintaining a co-culture by quorum sensing, the method comprising co-culturing at least a first bacterial strain and a second bacterial strain during a period of time of at least 12 hours; wherein at least one of the first and second bacterial strains has a growth advantage compared to at least one other bacterial strain; and each of the first and second bacterial strains comprises: a lysis plasmid having a lysis gene under the control of an activatable promoter; and an activator plasmid having an activator gene, the expression of which promotes the accumulation of a quorum sensing molecule, wherein both the activatable promoter of the lysis gene and the expression of the activator gene is activated by the quorum sensing molecule, wherein the quorum-sensing molecule of the first strain is different from the quorum-sensing molecule of the second strain, and wherein each quorum-sensing molecule of the first and second strains has no or substantially no effect on the activatable promoter of the lysis gene of the other strain.
In another aspect, a bacterial strain is provided comprising a lysis plasmid and an activator plasmid; wherein the lysis plasmid comprises a lysis gene, an activatable promoter, and optionally a reporter gene; and the activator plasmid comprises an activator gene, a degradation tag, and optionally a reporter gene.
In another aspect, a pharmaceutical composition is provided comprising any of the bacterial strains described herein.
In another aspect, a system is provided comprising a co-culture of at least a first bacterial strain and a second bacterial strain, wherein the first bacterial strain has at least a portion of a first synchronized lysis circuit, wherein the first synchronized lysis circuit comprises a first lysis plasmid, a first activator plasmid, and a first plasmid stabilizing element, and wherein the first lysis plasmid is activated by the first activator plasmid, and wherein the second bacterial strain has at least a portion of a second synchronized lysis circuit, wherein the second synchronized lysis circuit comprises a second lysis plasmid, a second activator plasmid, and a second plasmid stabilizing element, and wherein the second lysis plasmid is activated by the second activator plasmid, and wherein the first and second synchronized lysis circuits are orthogonal in that each has no or substantially no effect upon the other.
In another aspect, a drug delivery system is provided comprising the system of any one of the systems described herein.
In another aspect, a periodic drug delivery system is provided comprising any one of the systems described herein.
In another aspect, a method of treating a disease in a subject is provided, the method comprising administering to a subject in need therapeutically effective amounts of any of the bacterial strains described herein or any of the pharmaceutical compositions described herein, to thereby treat the disease in the subject.
The systems, methods, and compositions described herein provide several advantages. Synthetic biologists have used lysis to control populations before12, but not until recently have populations been engineered to dynamically control their own population without exogenous input16. Since the lysis systems rely on DNA parts carried on plasmids, undesired mutations may arise which can hinder the function of the circuit. Bacteria may mutate toxic or burdensome genes, and any possible mutants may gain a selective advantage over non-mutated members of the population. In this regard, strategies to enhance stability of the circuit components inside the host cells could help ensure long term robustness of the synthetic ecosystem24. Additionally, in the absence of antibiotics, bacteria may encounter a selective pressure to lose the circuit plasmids. This may result in difficulties when introducing the synthetic ecosystem to an environment without any selective agents. Some ways to address these challenges include integrating circuit components within the genome or using plasmid-stabilizing elements in the circuit. Elements such as toxin/antitoxin systems and actin-like protein partitioning systems have previously been shown to stabilize plasmids in environments without antibiotics25. The emergence of escapees is a direct consequence of strong selection imposed by periodic lysis, and recent evidence also suggests that repeated pruning of a population suppresses beneficial mutations that confer growth advantages unrelated to the lysis circuit26. Therefore, the strategies described herein (e.g., ortholysis strategy, or orthogonal co-lysis) are attractive methodologies to impose certain population dynamics or types of selection in evolution experiments. The challenge in maintaining a population of metabolically competitive microbial organisms has long been recognized21. Strategies to maintain the long-term stability of engineered microbial ecosystems that have thus far been investigated mainly consist of mutualistic interactions, such as metabolic interdependencies, or predator-prey type interactions27, 28. Recent evidence suggests, however, that competition is likely the dominant interaction in microbial communities29. In this vein, the systems, methods, and compositions described herein (e.g., “ortholysis” or orthogonal co-lysis systems, methods, and compositions) can be viewed as a strategy to stabilize competitive strains without engineering positive and negative interactions between members of the population. Moreover, recent evidence has identified quorum-sensing controlled self-lysis as a naturally occurring phenomenon in Pseudomonas aeruginosa30, which is a relevant example of how the interests of synthetic biologists and microbial ecologists are merging in the field of engineered microbial ecosystems. With the additional modeling of the circuit it becomes clear that the transition from monoculture synthetic biology to synthetic engineered ecosystems will be marked by an explosion of possibilities. A circuit designed for monocultures, such as the SLC, can have drastically broadened use-cases when expanded into the setting of a community. The systems, methods, and compositions described herein (e.g., “ortholysis” or orthogonal co-lysis systems, methods, and compositions) are immediately applicable for further expansion on the periodic in situ drug delivery system'. Additionally, this phenomenon of stably co-culturing two metabolically competitive strains through orthogonal self-lysing offers the possibility of many unique applications beyond drug delivery where the use of synthetic microbial ecosystems is advantageous.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
Microbial ecologists are increasingly turning to small, synthesized ecosystems1-5 as a reductionist tool to probe the complexity of native microbiomes6, 7. Concurrently, synthetic biologists have gone from single-cell gene circuits8-11 to controlling whole populations using intercellular signaling12-16. The intersection of these fields is giving rise to new approaches in waste recycling17, industrial fermentation18, bioremediation19, and human health16, 20. These applications share a common challenge7 well known in classical ecology21, 22; stability of an ecosystem cannot arise without mechanisms that prohibit the faster growing species from eliminating the slower. Here, orthogonal quorum sensing systems and a population control circuit with diverse self-limiting growth dynamics are combined in order to engineer two ‘ortholysis’ circuits capable of maintaining a stable co-culture of metabolically competitive strains in microfluidic devices. While no successful co-cultures are observed in a two-strain ecology without synthetic population control, the ‘ortholysis’ design dramatically increases the co-culture rate from 0% to approximately 80%. Agent-based and deterministic modeling reveal that the system can be adjusted to yield different dynamics, including phase-shifted, anti-phase or synchronized oscillations as well as stable steady-state population densities. The ‘ortholysis’ approach establishes a paradigm for constructing synthetic ecologies by developing stable communities of competitive microbes without the need for engineered codependency.
As used herein the term “co-culture” or “co-culturing” refers to growing or culturing two or more distinct cell types (e.g., at least two distinct bacterial strains) within a single recipient (e.g., a single cell culture vessel, a single cell culture plate, a single bioreactor, a single microfluidic device). Under optimal conditions of co-culturing, each of the at least two bacterial strains (e.g., each of at least a first bacterial strain and a second bacterial strain) has a positive growth rate.
A variety of different methods known in the art can be used to introduce any of the plasmids disclosed herein into a bacterial cell (e.g., a Gram negative bacterial cell, a Gram positive bacterial cell). Non-limiting examples of methods for introducing nucleic acid into a cell include: transformation, microinjection, electroporation, cell squeezing, sonoporation. Skilled practitioners will appreciate that the plasmids described herein can be introduced into any cell provided herein.
The term “treat(ment),” is used herein to denote delaying the onset of, inhibiting, alleviating the effects of, or prolonging the life of a subject suffering from disease, e.g., a cancer, an infection.
The terms “effective amount” and “amount effective to treat” as used herein, refer to an amount or concentration of a composition or treatment described herein, at least two bacterial strains (e.g., at least a first bacterial strain and a second bacterial strain), utilized for a period of time (including acute or chronic administration and periodic or continuous administration) that is effective within the context of its administration for causing an intended effect or physiological outcome. For example, effective amounts of at least two bacterial strains (e.g., at least a first bacterial strain and a second bacterial strain) that express and/or secrete a therapeutic agent (e.g., any of the therapeutic agents described herein) for use in the present disclosure include, for example, amounts that inhibit the growth of a cancer, e.g., tumor cells and/or tumor-associated immune cells, improve or delay tumor growth, improve survival for a subject suffering from or at risk of developing cancer, and improving the outcome of other cancer treatments. As another example, effective amounts of at least two bacterial strains (e.g., at least a first bacterial strain and a second bacterial strain) that express and/or secrete a therapeutic agent (e.g., any of the therapeutic agents described herein) can include amounts that advantageously affect a tumor microenvironment.
The term “subject” is used throughout the specification to describe an animal, human or non-human, to whom treatment according to the methods of the present disclosure is provided. Veterinary applications are clearly anticipated by the present disclosure. The term includes but is not limited to birds, reptiles, amphibians, and mammals, e.g., humans, other primates, pigs, rodents, such as mice and rats, rabbits, guinea pigs, hamsters, horses, cows, cats, dogs, sheep and goats. Preferred subjects are humans, farm animals, and domestic pets such as cats and dogs. In some embodiments, the subject is a human. For example, in any of the methods described herein, the subject can be at least 2 years or older (e.g., 4 years or older, 6 years or older, 10 years or older, 13 years or older, 16 years or older, 18 years or older, 21 years or older, 25 years or older, 30 years or older, 35 years or older, 40 years or older, 45 years or older, 50 years or older, 60 years or older, 65 years or older, 70 years or older, 75 years or older, 80 years or older, 85 years or older, 90 years or older, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16 ,18, 20, 21, 24, 25, 27, 28, 30, 33, 35, 37, 39, 40, 42, 44, 45, 48, 50, 52, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100, 101, 102, 103, or 104 years old).
The term “population” when used before a noun means two more of the specific noun. For example, the phrase “a population of bacterial strains” means two or more bacterial strains.
The term “cancer” refers to cells having the capacity for autonomous growth. Examples of such cells includes cells having an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include cancerous growths, e.g., tumors, oncogenic processes, metastatic tissues, malignantly transformed cells.
A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast, bone and liver origin. Metastases develop, e.g., when tumor cells shed, detach or migrate from a primary tumor, enter the vascular system, penetrate into surrounding tissues, and grow to form tumors at distinct anatomical sites, e.g., sites separate from a primary tumor.
Individuals considered at risk for developing cancer may benefit from the present disclosure, e.g., because prophylactic treatment can begin before there is any evidence and/or diagnosis of the disorder. Individuals “at risk” include, e.g., individuals exposed to carcinogens, e.g., by consumption (e.g., by inhalation and/or ingestion), at levels that have been shown statistically to promote cancer in susceptible individuals. Also included are individuals at risk due to exposure to ultraviolet radiation, or their environment, occupation, and/or heredity, as well as those who show signs of a precancerous condition such as polyps. Similarly, individuals in very early stages of cancer or development of metastases (i.e., only one or a few aberrant cells are present in the individual's body or at a particular site in an individual's tissue) may benefit from such prophylactic treatment.
Skilled practitioners will appreciate that a patient can be diagnosed, e.g., by a medical professional, e.g., a physician or nurse (or veterinarian, as appropriate for the patient being diagnosed), as suffering from or at risk for a condition described herein, e.g., cancer, using any method known in the art, e.g., by assessing a patient's medical history, performing diagnostic tests, and/or by employing imaging techniques.
Skilled practitioners will also appreciate that treatment need not be administered to a patient by the same individual who diagnosed the patient (or the same individual who prescribed the treatment for the patient). Treatment can be administered (and/or administration can be supervised), e.g., by the diagnosing and/or prescribing individual, and/or any other individual, including the patient her/himself (e.g., where the patient is capable of self-administration).
Provided herein are methods of maintaining a co-culture by quorum sensing. In some embodiments, the methods can include co-culturing at least a first bacterial strain and a second bacterial strain during a period of time of at least 12 hours; wherein at least one of the first and second bacterial strains has a growth advantage compared to at least one other bacterial strain. In some embodiments, each of the first and second bacterial strains comprises a lysis plasmid having a lysis gene under the control of an activatable promoter; and an activator plasmid having an activator gene, the expression of which promotes the accumulation of a quorum sensing molecule, wherein both the activatable promoter of the lysis gene and the expression of the activator gene is activated by the quorum sensing molecule, wherein the quorum-sensing molecule of the first strain is different from the quorum-sensing molecule of the second strain, and wherein each quorum-sensing molecule of the first and second strains has no or substantially no effect on the activatable promoter of the lysis gene of the other strain.
As used herein, “substantially no effect” means no measurable effect on the activatable promoter, as measured by the expression of the activatable promotor of a fluorescent protein.
In some embodiments, the methods can include co-culturing multiple co-cultures such that the method can include, e.g., co-culturing, along with the first and second strains, a third bacterial strain and a fourth bacterial strain that can be described similarly to the first and second bacterial strains. In some embodiments, the co-culturing can include co-culturing one or more sets of two bacterial strains described similarly to the first and second bacterial strains, such that a first set includes the first and second strain, a second set includes a third and fourth strain, and so on. In some embodiments, each set can comprise a co-lysis (e.g., orthogonal co-lysis) circuit.
In some aspects, the lysis plasmid and activator plasmid of at least one of the first and second strains can be the same plasmid. In some aspects, the lysis plasmid and activator plasmid of at least one of the first and second strains can be separate plasmids.
In some aspects, the at least the first and second strains can be metabolically competitive.
In some aspects, the at least the first and second strains can be selected from E. coli, S. typhimurium, or a bacterial variant thereof.
In some aspects, the first strain does not have a growth advantage compared to at least one other bacterial strain. In some embodiments, the first strain does not have a growth advantage compared to at least the second bacterial strain. In some embodiments, the first strain does not have a growth advantage compared to at least one other bacterial strain in the co-culture that is not the second strain. In one aspect, in each of the first and second strains the lysis plasmid comprises a lysis gene, an activatable promoter, and optionally a reporter gene; and the activator plasmid comprises an activator gene, a degradation tag, and optionally a reporter gene. In some embodiments, at least one reporter gene is selected from a gene encoding a green fluorescent protein (GFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), or a variant thereof. In some embodiments, the degradation tag can be an ssrA-LAA degradation tag.
In some aspects, the lysis gene in at least one of the first and second strains can be E from a bacteriophage ΦX174.
In some aspects, in the first strain the activatable promoter is a LuxR-AHL activatable luxI promoter and the activator gene is a LuxI.
In some aspects, in the second strain the activatable promoter is a RpaR-AHL activatable RpaI promoter and the activator gene is a RpaI.
In some aspects, the co-culture is inoculated at a ratio of 1:100 of the bacterial strain having the growth advantage compared to the other bacterial strain to the other bacterial strain.
In some aspects, at least one of the plasmids is integrated into a genome of at least one of the first and second strains.
In some aspects, at least one of the plasmids can further comprises a plasmid-stabilizing element. In some embodiments, the plasmid-stabilizing element is a toxin/antitoxin system or an actin-like protein partitioning system.
In some aspects, the culturing can occur in a microfluidic device.
In some embodiments, the period of time can be from about 12 to about 72 hours. In some embodiments, the period of time is selected from at least 24 hours, at least 48 hours, at least 72 hours, and at least 96 hours. In some embodiments, the period of time is selected from 12 hours, 24 hours, 48 hours, 72 hours, and 96 hours.
In some aspects, the co-culturing of the first and second strains is in a constant lysis state; wherein the constant lysis state is characterized by a steady-state balance of growth and lysis of the at least two bacterial strains.
In some aspects, the co-culturing of the at least two bacterial strains is oscillatory; wherein the oscillatory co-culturing indicates a high level of activator degradation in at least one of the two bacterial strains.
Also provided herein are methods of generating a recombinant bacterial cell that can express and/or secrete a therapeutic agent (e.g., any of the therapeutic agents described herein) that include: introducing into a bacterial cell a lysis plasmid, an activator plasmid, a nucleic acid encoding the therapeutic agent to be produced in the recombinant bacterial cell, and a plasmid-stabilizing element; and culturing the recombinant bacterial cell under conditions sufficient for the expression and/or secretion of the toxin, antitoxin and therapeutic agent. In some embodiments, the introducing step can include introducing into a recombinant bacterial cell an expression vector including a nucleic acid encoding the therapeutic agent to be produced into a recombinant bacterial cell. In some embodiments, the bacterial cell is an E. coli cell, a S. typhimurium cell, or a bacterial variant thereof. In some embodiments, the bacterial strain is a Gram-negative bacterial strains, e.g., a Salmonella strain, an Acetobacter strain, an Enterobacter strain, a Fusobacterium strain, a Helicobacter strain, a Klebsiella strain, or an E. coli strain. In some embodiments, the bacterial strain is a Gram-positive bacterial strain, e.g., an Actinomyces strain, a Bacillus strain, a Clostridium strain, an Enterococcus strain, or a Lactobacillus strain. In some embodiments, the at least two bacterial strains (e.g., at least a first bacterial strain and a second bacterial strain) are all Gram negative bacterial strains or all Gram positive strains. In some embodiments, at least one of the at least two bacterial strains (e.g., at least one of a first bacterial strain and a second bacterial strain) is a Gram negative bacterial strain. In some embodiments, at least one of the at least two bacterial strains (e.g., at least one of a first bacterial strain and a second bacterial strain) is a Gram positive bacterial strain.
Methods of culturing bacterial cells are well known in the art, and examples of such methods are provided in the Examples. Bacterial cells can be maintained in vitro under conditions that favor proliferation and growth. Briefly, bacterial cells can be cultured by contacting a bacterial cell (e.g., any bacterial cell described herein) with a cell culture medium that includes the necessary growth factors and supplements to support cell viability and growth.
Methods of introducing nucleic acids and expression vectors into a bacterial cell are known in the art. For example, transformation can be used to introduce a nucleic acid into a bacterial cell.
Provided herein are bacterial strain that include a lysis plasmid, a plasmid-stabilizing element, and an activator plasmid; wherein the lysis plasmid comprises a lysis gene, an activatable promoter, a therapeutic agent, and optionally a reporter gene; and the activator plasmid comprises an activator gene, a degradation tag, and optionally a reporter gene.
In some embodiments of any of the bacterial strains described herein, the lysis gene is E from a bacteriophage ΦX174.
In some embodiments of any of the bacterial strains described herein, the activatable promoter is a LuxR-AHL activatable luxI promoter and the activator gene is a LuxI.
In some embodiments of any of the bacterial strains described herein, the activatable promoter is a RpaR-AHL activatable RpaI promoter and the activator gene is a RpaI.
In some embodiments, the plasmid-stabilizing element is a toxin/antitoxin system or an actin-like protein partitioning system. In some embodiments, the plasmid-stabilizing element is a toxin/antitoxin system (e.g., type I toxin/antitoxin system, type II toxin/antitoxin system, type II toxin/antitoxin system, type IV toxin/antitoxin system, type V toxin/antitoxin system, or type VI toxin/antitoxin system). Non-limiting examples of type I toxin/antitoxins include Hok and Sok, Fst and RNAII, TisB and IstR, LdrB and RdlD, FlmA and FlmB, Ibs and Sib, TxpA/BrnT and RatA, SymE and SymR, and XXCV2162 and ptaRNA1. Non-limiting examples of type II toxin/antitoxins include CcdB and CcdA; ParE and ParD; MaxF and MazE; yafO and yafN; HicA and HicB; Kid and Kis; Zeta and Epsilon; DarT and DarG. For example, type III toxin/antitoxin systems include interactions between a toxic protein and an RNA antitoxin, e.g., ToxN and ToxI. For example, type IV toxin/antitoxin systems include toxin/antitoxin systems that counteract the activity of the toxin and the two proteins do not directly interact. An example of a type V toxin/antitoxin system is GoT and GoS. An example of a type VI toxin/antitoxin system is SocA and SocB.
In some embodiments, the plasmid-stabilizing element is a bacteriocin. Bacteriocins are ribosomally-synthesized peptides that are produced by bacteria. Bacteriocins are non-toxic to bacteria that produce the bacteriocins and are toxic to other bacteria. Most bacteriocins are extremely potent, and exhibit antimicrobial activity at nanomolecular concentrations. By way of example, eukaryotic produced microbials have 102 to 103 lower activities (Kaur and Kaur (2015) Front. Pharmacol. doi: 10.3389).
Non-limiting examples of bacteriocins that can be included in any of the bacteria strains, systems and methods described herein include: acidocin, actagardine, agrocin, alveicin, aureocin, aureocin A53, aureocin A70, bisin, carnocin, carnocyclin, caseicin, cerein, circularin A, colicin, curvaticin, divercin, duramycin, enterocin, enterolysin, epidermin/gallidermin, erwiniocin, gardimycin, gassericin A, glycinecin, halocin, klebicin, lactosin S, lactococcin, lacticin, leucoccin, lysostaphin, macedocin, mersacidin, mesentericin, microbisporicin, microcin S, mutacin, nisin, paenibacillin, planosporicin, pediocin, pentocin, plantaricin, pneumocyclicin, pyocin, reutericin 6, sakacin, salivaricin, sublancin, subtilin, sulfolobicin, tasmancin, thuricin 17, trifolitoxin, variacin, vibriocin, warnericin, cytolisin, pyocyn S2, colicin A, colicin E1, microcin MccE492, and warnerin.
In some embodiments, the bacteriocin is obtained from a Gram negative bacteria (e.g., microcins (e.g., microcin V of E coli, subtilosin A from B. subtillis), colicins (E.g., colicin produced by and toxic to certain strains of E. coli (e.g., colicin A, colicin B, colicin E1, colicin E3, colicin E5, and colicin E7), tailocins (e.g., R-type pyocins, F-type pyocins)).
In some embodiments, the bacteriocin is obtained from a Gram positive bacteria (e.g., class I bacteriocins (e.g., Nisin, lantibiotics), class II bacteriocins (e.g., IIa pediocin-like bacteriocins, IIb bacteriocins (e.g., lactococcin G), IIc cyclic peptides (e.g., enterocin AS-48), IId single peptide bacteriocins (e.g., aureocin A53), class III bacteriocins (e.g., IIIa (e.g., bacteriolysins), and Mb (which kill the target by disrupting the membrane potential), or class IV bacteriocins (e.g., complex bacteriocins containing lipid or carbohydrate moieties)),
In some embodiments of any of the bacterial strains described herein, the therapeutic agent is selected from the group consisting of: an inhibitory nucleic acid, a cytokine, a fusion protein, and an antibody or antigen-binding fragment thereof.
Also provided herein are methods of co-culturing at least two (e.g., at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) bacterial strains (e.g., any of the bacterial strains described herein). In some embodiments, a ratio of inoculation of at least one bacterial strain to at least one other bacterial strain is between 100 000:1 and 1:100 000 (e.g., 100 000:1, 95 000:1, 90 000:1, 85 000:1, 80 000:1, 75 000:1, 70 000:1, 65 000:1, 60 000:1, 55 000:1, 50 000:1, 45 000:1, 40 000:1, 35 000:1, 30 000:1, 25 000:1, 20 000:1, 15 000:1, 10 000:1, 9000:1, 8500:1, 8000:1, 7500:1, 7000:1, 6500:1, 6000:1, 5500:1, 5000:1, 4500:1, 4000:1, 3500:1, 3000:1, 2500:1, 2000:1, 1500:1, 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 150:1, 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 25:1, 20:1, 18:1, 16:1, 15:1, 14:1, 12:1, 10:1, 8:1, 6:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:6, 1:8, 1:10, 1:12, 1:14, 1:15, 1:16, 1:18, 1:20, 1:25, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, 1:1000, 1:1500, 1:2000, 1:2500, 1:3000, 1:3500, 1:4000, 1:4500, 1:5000, 1:5500, 1:6000, 1:6500, 1:7000, 1: 7500, 1:8000, 1:8500, 1:9000, 1:9500, 1:10 000, 1:15 000, 1: 20 000, 1:25 000, 1:30 000, 1:35 000, 1:40 000, 1:45 000, 1:50 000, 1:55 000, 1:60 000, 1:65 000, 1:70 000, 1: 75 000, 1:80 000, 1:85 000, 1:90 000, 1:95 0000, 1:100 000).
In some embodiments, a ratio of inoculation of the first bacterial strain to the second bacterial strain is between 100 000:1 and 1:100 000 (e.g., 100 000:1, 95 000:1, 90 000:1, 85 000:1, 80 000:1, 75 000:1, 70 000:1, 65 000:1, 60 000:1, 55 000:1, 50 000:1, 45 000:1, 40 000:1, 35 000:1, 30 000:1, 25 000:1, 20 000:1, 15 000:1, 10 000:1, 9000:1, 8500:1, 8000:1, 7500:1, 7000:1, 6500:1, 6000:1, 5500:1, 5000:1, 4500:1, 4000:1, 3500:1, 3000:1, 2500:1, 2000:1, 1500:1, 1000:1, 950:1, 900:1, 850:1, 800:1, 750:1, 700:1, 650:1, 600:1, 550:1, 500:1, 450:1, 400:1, 350:1, 300:1, 250:1, 200:1, 150:1, 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 25:1, 20:1, 18:1, 16:1, 15:1, 14:1, 12:1, 10:1, 8:1, 6:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:6, 1:8, 1:10, 1:12, 1:14, 1:15, 1:16, 1:18, 1:20, 1:25, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:950, 1:1000, 1:1500, 1:2000, 1:2500, 1:3000, 1:3500, 1:4000, 1:4500, 1:5000, 1:5500, 1:6000, 1:6500, 1:7000, 1:7500, 1:8000, 1:8500, 1:9000, 1:9500, 1:10 000, 1:15 000, 1: 20 000, 1:25 000, 1:30 000, 1: 35 000, 1: 40 000, 1: 45 000, 1:50 000, 1: 55 000, 1:60 000, 1:65 000, 1:70 000, 1: 75 000, 1:80 000, 1:85 000, 1:90 000, 1:95 0000, 1:100 000).
In some embodiments, a cycle of lysis of any of the bacterial strains described herein can be between 1 hour to 35 days (e.g., 1 hour to 30 days, 1 hour to 28 days, 1 hour to 26 days, 1 hour to 25 days, 1 hour to 24 days, 1 hour to 22 days, 1 hour to 20 days, 1 hour to 18 days, 1 hour to 16 days, 1 hour to 14 days, 1 hour to 12 days, 1 hour to 10 days, 1 hour to 8 days, 1 hour to 7 days, 1 hour to 6 days, 1 hour to 5 days, 1 hour to 4 days, 1 hour to 72 hours, 1 hour to 70 hours, 1 hour to 68 hours, 1 hour to 66 hours 1 hour to 64 hours, 1 hour to 62 hours, 1 hour to 60 hours, 1 hour to 58 hours, 1 hour to 56 hours, 1 hour to 54 hours, 1 hour to 52 hours, 1 hour to 50 hours, 1 hour to 48 hours, 1 hour to 46 hours, 1 hour to 44 hours, 1 hour to 40 hours, 1 hour to 38 hours, 1 hour to 36 hours, 1 hour to 34 hours, 1 hour to 32 hours, 1 hour to 30 hours, 1 hour to 28 hours, 1 hour to 26 hours, 1 hour to 24 hours, 1 hour to 22 hours, 1 hour to 20 hours, 1 hour to 18 hours, 1 hour to 16 hours, 1 hour to 14 hours, 1 hour to 12 hours, 1 hour to 10 hours, 1 hour to 8 hours, 1 hour to 6 hours, 1 hour to 4 hours, 1 hour to 2 hours, 2 hours to 35 days, 2 hours to 30 days, 2 hours to 28 days, 2 hours to 26 days, 2 hours to 25 days, 2 hours to 24 days, 2 hours to 22 days, 2 hours to 20 days, 2 hours to 18 days, 2 hours to 16 days, 2 hours to 14 days, 2 hours to 12 days, 2 hours to 10 days, 2 hours to 8 days, 2 hours to 7 days, 2 hours to 6 days, 2 hours to 5 days, 2 hours to 4 days, 2 hours to 72 hours, 2 hours to 70 hours, 2 hours to 68 hours, 2 hours to 66 hours 2 hours to 64 hours, 2 hours to 62 hours, 2 hours to 60 hours, 2 hours to 58 hours, 2 hours to 56 hours, 2 hours to 54 hours, 2 hours to 52 hours, 2 hours to 50 hours, 2 hours to 48 hours, 2 hours to 46 hours, 2 hours to 44 hours, 2 hours to 40 hours, 2 hours to 38 hours, 2 hours to 36 hours, 2 hours to 34 hours, 2 hours to 32 hours, 2 hours to 30 hours, 2 hours to 28 hours, 2 hours to 26 hours, 2 hours to 24 hours, 2 hours to 22 hours, 2 hours to 20 hours, 2 hours to 18 hours, 2 hours to 16 hours, 2 hours to 14 hours, 2 hours to 12 hours, 2 hours to 10 hours, 2 hours to 8 hours, 2 hours to 6 hours, 2 hours to 4 hours, 4 hours to 35 days, 4 hours to 30 days, 4 hours to 28 days, 4 hours to 26 days, 4 hours to 25 days, 4 hours to 24 days, 4 hours to 22 days, 4 hours to 20 days, 4 hours to 18 days, 4 hours to 16 days, 4 hours to 14 days, 4 hours to 12 days, 4 hours to 10 days, 4 hours to 8 days, 4 hours to 7 days, 4 hours to 6 days, 4 hours to 5 days, 4 hours to 4 days, 4 hours to 74 hours, 4 hours to 70 hours, 4 hours to 68 hours, 4 hours to 66 hours 4 hours to 64 hours, 4 hours to 64 hours, 4 hours to 60 hours, 4 hours to 58 hours, 4 hours to 56 hours, 4 hours to 54 hours, 4 hours to 54 hours, 4 hours to 50 hours, 4 hours to 48 hours, 4 hours to 46 hours, 4 hours to 44 hours, 4 hours to 40 hours, 4 hours to 38 hours, 4 hours to 36 hours, 4 hours to 34 hours, 4 hours to 34 hours, 4 hours to 30 hours, 4 hours to 28 hours, 4 hours to 26 hours, 4 hours to 24 hours, 4 hours to 24 hours, 4 hours to 20 hours, 4 hours to 18 hours, 4 hours to 16 hours, 4 hours to 14 hours, 4 hours to 14 hours, 4 hours to 10 hours, 4 hours to 8 hours, 4 hours to 6 hours, 6 hours to 35 days, 6 hours to 30 days, 6 hours to 28 days, 6 hours to 26 days, 6 hours to 25 days, 6 hours to 24 days, 6 hours to 22 days, 6 hours to 20 days, 6 hours to 18 days, 6 hours to 16 days, 6 hours to 14 days, 6 hours to 12 days, 6 hours to 10 days, 6 hours to 8 days, 6 hours to 7 days, 6 hours to 6 days, 6 hours to 5 days, 6 hours to 4 days, 6 hours to 76 hours, 6 hours to 70 hours, 6 hours to 68 hours, 6 hours to 66 hours 6 hours to 64 hours, 6 hours to 66 hours, 6 hours to 60 hours, 6 hours to 58 hours, 6 hours to 56 hours, 6 hours to 54 hours, 6 hours to 56 hours, 6 hours to 50 hours, 6 hours to 48 hours, 6 hours to 46 hours, 6 hours to 44 hours, 6 hours to 40 hours, 6 hours to 38 hours, 6 hours to 36 hours, 6 hours to 34 hours, 6 hours to 36 hours, 6 hours to 30 hours, 6 hours to 28 hours, 6 hours to 26 hours, 6 hours to 24 hours, 6 hours to 26 hours, 6 hours to 20 hours, 6 hours to 18 hours, 6 hours to 16 hours, 6 hours to 14 hours, 6 hours to 16 hours, 6 hours to 10 hours, 6 hours to 8 hours, 12 hours to 35 days, 12 hours to 30 days, 12 hours to 28 days, 12 hours to 26 days, 12 hours to 25 days, 12 hours to 24 days, 12 hours to 22 days, 12 hours to 20 days, 12 hours to 18 days, 12 hours to 16 days, 12 hours to 14 days, 12 hours to 12 days, 12 hours to 10 days, 12 hours to 8 days, 12 hours to 7 days, 12 hours to 6 days, 12 hours to 5 days, 12 hours to 4 days, 12 hours to 72 hours, 12 hours to 70 hours, 12 hours to 68 hours, 12 hours to 66 hours 12 hours to 64 hours, 12 hours to 62 hours, 12 hours to 60 hours, 12 hours to 58 hours, 12 hours to 56 hours, 12 hours to 54 hours, 12 hours to 512 hours, 12 hours to 50 hours, 12 hours to 48 hours, 12 hours to 46 hours, 12 hours to 44 hours, 12 hours to 40 hours, 12 hours to 38 hours, 12 hours to 36 hours, 12 hours to 34 hours, 12 hours to 312 hours, 12 hours to 30 hours, 12 hours to 28 hours, 12 hours to 26 hours, 12 hours to 24 hours, 12 hours to 22 hours, 12 hours to 20 hours, 12 hours to 18 hours, 12 hours to 16 hours, 12 hours to 14 hours, 1 day to 35 days, 1 day to 30 days, 1 day to 28 days, 1 day to 26 days, 1 day to 25 days, 1 day to 24 days, 1 day to 22 days, 1 day to 20 days, 1 day to 18 days, 1 day to 16 days, 1 day to 14 days, 1 day to 12 days, 1 day to 10 days, 1 day to 8 days, 1 day to 6 days, 1 day to 5 days, 1 day to 4 days, 1 day to 3 days, 1 day to 2 days, 2 days to 35 days, 2 days to 30 days, 2 days to 28 days, 2 days to 26 days, 2 days to 25 days, 2 days to 24 days, 2 days to 22 days, 2 days to 20 days, 2 days to 18 days, 2 days to 16 days, 2 days to 15 days, 2 days to 14 days, 2 days to 12 days, 2 days to 10 days, 2 days to 8 days, 2 days to 6 days, 2 days to 4 days, 2 days to 3 days, 4 days to 35 days, 4 days to 30 days, 4 days to 28 days, 4 days to 26 days, 4 days to 25 days, 4 days to 24 days, 4 days to 22 days, 4 days to 20 days, 4 days to 18 days, 4 days to 16 days, 4 days to 15 days, 4 days to 14 days, 4 days to 12 days, 4 days to 10 days, 4 days to 8 days, 4 days to 6 days, 7 days to 35 days, 7 days to 30 days, 7 days to 28 days, 7 days to 26 days, 7 days to 25 days, 7 days to 24 days, 7 days to 22 days, 7 days to 20 days, 7 days to 18 days, 7 days to 16 days, 7 days to 15 days, 7 days to 14 days, 7 days to 12 days, 7 days to 10 days, 7 days to 8 days, 14 days to 35 days, 14 days to 30 days, 14 days to 28 days, 14 days to 26 days, 14 days to 25 days, 14 days to 24 days, 14 days to 22 days, 14 days to 20 days, 14 days to 18 days, 14 days to 16 days, 14 days to 15 days, 21 days to 35 days, 21 days to 30 days, 21 days to 28 days, 21 days to 26 days, 21 days to 25 days, 21 days to 24 days, 21 days to 22 days, 28 days to 35 days, or 28 days to 30 days; 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, 21 days, 22 days, 24 days, 25 days, 26 days, 28 days, 30 days, 32 days, 34 days or 35 days).
The length of a cycle can be regulated by using strains that lyse at different ODs. Cell lysis can also be regulated by tuning the internal circuitry of the quorum sensing components, e.g., tuning of AHL degradation, tuning lysis of protein degradation, tuning of promoters to increase or decrease expression of molecules involved in the quorum sensing circuitry.
Various methods known in the art can be used to determine whether the quorum threshold is reached. For example, the quorum threshold can be measured using traditional protein quantification methods to measure the level of AHL expression in the culture medium. The quorum threshold can also be measured using reporter proteins driven by the luxI promoter. In some embodiments, the reporter protein is a fluorescent protein, a bioluminescent luciferase reporter, a secreted blood/serum or urine reporter (e.g., secreted alkaline phosphatase, soluble peptides, Gaussian luciferase).
Various methods are known in the art to determine and/or measure cell lysis. For example, cell lysis can be determined phenotypically using microscopy by the change in intensity of transmitted light and/or absorbance at various wavelengths including 600 nm light. In some embodiments, bacterial cell lysis is synchronized. In other embodiments, bacterial cell lysis is not synchronized. Synchronized lysis can be measured via optical density at 600 nm absorbance (OD600) in a plate reader or other quantitative instruments.
Provided herein are systems that can include a co-culture of at least two bacterial strains (e.g., at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12), wherein the at least two bacterial strains can include a first bacterial strain having at least a portion of a first synchronized lysis circuit, wherein the first synchronized lysis circuit comprises a first lysis plasmid and a first activator plasmid and wherein the first lysis plasmid is activated by the first activator plasmid.
In some embodiments described herein, a system comprises a co-culture of at least a first bacterial strain and a second bacterial strain, wherein the first bacterial strain has at least a portion of a first synchronized lysis circuit, wherein the first synchronized lysis circuit comprises a first lysis plasmid, a first activator plasmid, and a first plasmid stabilizing element, and wherein the first lysis plasmid is activated by the first activator plasmid, and wherein the second bacterial strain has at least a portion of a second synchronized lysis circuit, wherein the second synchronized lysis circuit comprises a second lysis plasmid, a second activator plasmid, and a second plasmid stabilizing element, and wherein the second lysis plasmid is activated by the second activator plasmid, and wherein the first and second synchronized lysis circuits are orthogonal in that each has no or substantially no effect upon the other.
As used herein, “substantially no effect” means no measurable effect on the activatable promoter, as measured by the expression of the activatable promotor of a fluorescent protein.
In some aspects, the first bacterial strain can include the first lysis plasmid.
In some aspects of any of the systems described herein, the first bacterial strain can include the first activator plasmid.
In some aspects of any of the systems described herein, the at least two bacterial strains further can include a second bacterial strain.
In some aspects of any of the systems described herein, the second bacterial strain can include the first activator plasmid.
In some aspects of any of the systems described herein, each of the first bacterial strain and the second bacterial strain can include the first activator plasmid.
In some aspects of any of the systems described herein, the first lysis plasmid of the first bacterial strain operates independent of at least one other bacterial strain in the co-culture. In some embodiments, the first lysis plasmid of the first bacterial strain operates independent of at least the second bacterial strain. In some embodiments, the first lysis plasmid of the first bacterial strain operates independent of at least one bacterial strain in the system that is not the second bacterial strain.
In some aspects of any of the systems described herein, the first lysis plasmid of the first bacterial strain responds to a signal generated by at least one other bacterial strain in the co-culture. In some embodiments, the first lysis plasmid of the first bacterial strain responds to a signal generated by at least the second bacterial strain. In some embodiments, the first lysis plasmid of the first bacterial strain responds to a signal generated by at least one bacterial strain in the system that is not the second bacterial strain.
In some aspects of any of the systems described herein, the signal is a quorum sensing signal. In some aspects of any of the systems described herein, the first activator plasmid encodes a quorum sensing signal. In some aspects of any of the systems described herein, the second activator plasmid encodes a quorum sensing signal. In some embodiments, the quorum sensing signal can be a quorum sensing signaling molecule. In some embodiments, one or more of the bacterial strains respond to a quorum sensing signal. In some embodiments, the quorum sensing signals for two or more of the bacterial strains are different quorum sensing signals. In some embodiments, the quorum sensing signals for two or more of the bacterial strains are the same quorum sensing signals.
In some embodiments, the quorum sensing signaling molecule for the first and second synchronized lysis circuits are orthogonal in that each has no measurable effect upon the other.
In some aspects of any of the systems described herein, the second bacterial strain has at least a portion of a second synchronized lysis circuit, wherein the second synchronized lysis circuit comprises a second lysis plasmid and a second activator plasmid.
In some aspects of any of the systems described herein, the second bacterial strain comprises the second lysis plasmid.
In some aspects of any of the systems described herein, second bacterial strain comprises the second activator plasmid.
In some aspects of any of the systems described herein, the first bacterial strain comprises the second activator plasmid.
In some aspects of any of the systems described herein, the second lysis plasmid of the second bacterial strain operates independent of at least the first bacterial strain.
In some aspects of any of the systems described herein, the second lysis plasmid of the second bacterial strain responds to a signal generated by the first bacterial strain.
In some aspects of any of the systems described herein, at least one of the at least two bacterial strains (e.g., at least one of a first bacterial strain and a second bacterial strain) has a growth advantage compared to at least one other bacterial strain. In some embodiments, at least the first bacterial strain has a growth advantage compared to at least the second bacterial strain. In some embodiments, at least the second bacterial strain has a growth advantage compared to at least the first bacterial strain. In some embodiments, at least the first bacterial strain has a growth advantage compared to a bacterial strain present in the system that is not the second bacterial strain. In some embodiments, at least the second bacterial strain has a growth advantage compared to a bacterial strain present in the system that is not the first bacterial strain.
In some embodiments, the system can contain multiple orthogonal co-lysis circuits. For example, a system described herein could include a first co-lysis circuit comprising a first bacterial strain and a second bacterial strain as described herein, as well as a second co-lysis circuit comprising a third bacterial strain and a fourth bacterial strain. In some embodiments, the third and fourth bacterial strains each comprise a lysis plasmid having a lysis gene under the control of an activatable promoter; and an activator plasmid having an activator gene, the expression of which promotes the accumulation of a quorum sensing molecule, wherein both the activatable promoter of the lysis gene and the expression of the activator gene is activated by the quorum sensing molecule, wherein the quorum-sensing molecule of the third strain is different from the quorum-sensing molecule of the fourth strain, and wherein each quorum-sensing molecule of the third and fourth strains has no or substantially no effect on the activatable promoter of the lysis gene of the other strain. In some embodiments, the third and fourth bacterial strains can be described in the same manner that the first and second bacterial strains have been described herein. In some embodiments, a system described herein can contain 3, 4, 5, 6, 7, 8, 9, 10, or more co-lysis circuits.
In some aspects of any of the systems described herein, the first bacterial strain is competitive with at least one other bacterial strain in the co-culture. In some embodiments, the first bacterial strain is competitive with at least the second bacterial strain in the co-culture. In some embodiments, the first bacterial strain is competitive with at least one other bacterial strain in the co-culture that is not the second bacterial strain.
In some aspects of any of the systems described herein, the co-culture is stable for at least 48 hours.
In some aspects of any of the systems described herein, the at least two bacterial strains (e.g., at least a first bacterial strain and a second bacterial strain) do not comprise engineered positive or negative interactions between each other.
In some aspects of any of the systems described herein, at least one of the at least two bacterial strains (e.g., at least one of a first bacterial strain and a second bacterial strain) dynamically controls its population without exogenous input.
In some aspects of any of the systems described herein, each of at least two of the at least two bacterial strains (e.g., each of at least a first bacterial strain and a second bacterial strain) dynamically controls its own population without exogenous input.
In some aspects of any of the systems described herein, the system can further include one or more plasmid stabilizing elements. In some aspects of any of the systems described herein, the plasmid stabilizing element is selected from a toxin/antitoxin system and an actin-like protein partitioning system.
In some aspects of any of the systems described herein, the first activator plasmid encodes a degradation tagging sequence.
In some aspects of any of the systems described herein, the second activator plasmid encodes a degradation tagging sequence.
In some aspects of any of the systems described herein, the first activator plasmid encodes an N-acyl homoserine lactone.
Provided herein are methods of treating a disease in a subject (e.g., a cancer, an infectious disease). Exemplary methods include administering to a subject in need of treatment therapeutically effective amounts of any of the bacterial strains of described herein or any pharmaceutical composition described herein, to thereby treat the disease in the subject.
In methods described herein, administering includes administering at least two bacterial strains (e.g., at least a first bacterial strain and a second bacterial strain) to the subject.
In some embodiments of methods described herein, the at least two bacterial strains include a first bacterial strain and a second bacterial strain, wherein the first bacterial strain has at least a portion of a first synchronized lysis circuit, wherein the first synchronized lysis circuit comprises a first lysis plasmid, a first activator plasmid, and a first plasmid stabilizing element, and wherein the first lysis plasmid is activated by the first activator plasmid, wherein the second bacterial strain has at least a portion of a second synchronized lysis circuit, wherein the second synchronized lysis circuit comprises a second lysis plasmid, a second activator plasmid, and a second plasmid stabilizing element, and wherein the second lysis plasmid is activated by the second activator plasmid, and wherein the first and second synchronized lysis circuits are essentially orthogonal in that each has no or substantially no effect upon the other.
In some embodiments of any of the methods described herein, the first and the second bacterial strains are different bacterial strains that each express and/or secrete a different therapeutic agent (e.g., any of the therapeutic agents described herein).
In some embodiments of any of the methods described herein, the first and the second bacterial strain do not express or secrete a therapeutic agent (e.g., any of the therapeutic agents described herein). In some embodiments of any of the methods described herein, the first and/or the second bacterial strain produce a bacteriocin (e.g., any of the bacteriocins described herein).
In some embodiments of any of the methods described herein, the subject has a cancer or an infection.
In some embodiments wherein the subject has a cancer, the cancer can be, e.g., a primary tumor, or a metastatic tumor.
In some embodiments, the cancer is a non-T-cell-infiltrating tumor.
In some embodiments of any of the methods described herein, the cancer is selected from the group consisting of: glioblastoma, squamous cell carcinoma, breast cancer, colon cancer, hepatocellular cancer, melanoma, neuroblastoma, pancreatic cancer, and prostate cancer. Treatment of multiple cancer types at the same time is contemplated by and within the present disclosure.
In some instances, the subject having the cancer may have previously received cancer treatment (e.g., any of the cancer treatments described herein).
In some embodiments of any of the methods described herein, the subject has an infection (e.g., an infectious disease). In some embodiments of any of the methods described herein, the infection is caused by an infectious agent selected from the group consisting of: Camphylobacter jejuni, Clostridium botulinium, Escherichia coli, Listeria monocytogenes and Salmonella.
Administering may be performed, e.g., at least once (e.g., at least 2-times, at least 3-times, at least 4-times, at least 5-times, at least 6-times, at least 7-times, at least 8-times, at least 9-times, at least 10-times, at least 11-times, at least 12-times, at least 13-times, or at least 14-times) a week. Also contemplated are monthly treatments, e.g., administering at least once per month for at least 1 month (e.g., at least two, three, four, five, or six or more months, e.g., 12 or more months), and yearly treatments (e.g., administration once a year for one or more years). Administration can be via any art-known means, e.g., intravenous, subcutaneous, intraperitoneal, oral, and/or rectal administration, or any combination of known administration methods.
As used herein, treating includes “prophylactic treatment”, which means reducing the incidence of or preventing (or reducing the risk of) a sign or symptom of a disease (e.g., a cancer, an infection) in a subject at risk of developing a disease (e.g., a cancer, an infection). The term “therapeutic treatment” refers to reducing signs or symptoms of a disease, e.g., reducing cancer progression, reducing severity of a cancer, and/or re-occurrence in a subject having cancer, reducing inflammation in a subject, reducing the spread of an infection in a subject.
The methods described herein can be used in cancer treatments. Non-limiting examples of cancer include: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, Burkitt Lymphoma, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative neoplasm, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymotna, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, gliotna, head and neck cancer, hairy cell leukemia, hepatocellular cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ. lung cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, midline tract carcinoma involving NUT gene, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis tungoides, myelodysplastic syndrome, myelodysplastic/myeloproliferative neoplasm, nasal cavity and para-nasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytomas, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphoma, teratoid tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms' tumor.
For example, any of the methods described herein can be used to treat a cancer selected from the group consisting of: glioblastoma, squamous cell carcinoma, breast cancer, colon cancer, hepatocellular cancer, melanoma, neuroblastoma, pancreatic cancer, and prostate cancer.
The term “therapeutic agent” refers to a therapeutic treatment that involves administering to a subject a therapeutic agent that is known to be useful in the treatment of a disease, e.g., a cancer, an infection. For example, a cancer therapeutic agent can decrease the size or rate of tumor growth. In other instances, a cancer therapeutic agent can affect the tumor microenvironment.
Non-limiting examples of therapeutic agents that can be expressed and/or secreted in any of the bacterial strains described herein include: an inhibitory nucleic acid (e.g., a microRNA, a short hairpin RNA, a small interfering RNA, an antisense), a cytokine, a chemokine, a toxin (e.g., a diphtheria toxin, a gelonin toxin, anthrax toxin), an antimicrobial peptide, a fusion protein, and an antibody or antigen-binding fragment thereof.
In some instances, the therapeutic agent is a therapeutic polypeptide. In some instances, the therapeutic polypeptide includes one or more polypeptides (e.g., 2, 3, 4, 5, or 6). In some instances, the therapeutic polypeptide is conjugated to a toxin, a radioisotope, or a drug via a linker (e.g., a cleavable linker, a non-cleavable linker).
In some instances, the therapeutic agent is cytotoxic or cytostatic to a target cell.
The phrase “cytotoxic to a target cell” refers to the inducement, directly or indirectly, in the death (e.g., necrosis or apoptosis) of the target cell. For example, a target cell can be a cancer cell (e.g., a cancerous cell or a tumor-associated immune cell (e.g., macrophage) or an infected cell.
The phrase “cytostatic to a target cell” refers to direct or indirect decrease in the proliferation (cell division) of a target cell in vivo or in vitro. When a therapeutic agent is cytostatic to a target cell, the therapeutic agent can, e.g., directly or indirectly result in cell cycle arrest of the target cell. In some examples, the therapeutic agent that is cytostatic can reduce the number of target cells in a population of cells that are in S phase (as compared to the number of target cells in a population of cells that are in S phase prior to contact with the therapeutic agent). In some instances, the therapeutic agent that is cytostatic can reduce the percentage of target cells in S phase by at least 20% (e.g., at least 40%, at least 60%, at least 80%) as compared to the percentage of target cells in a population of cells that in S phase prior to contact with the therapeutic agent.
Also provided herein are pharmaceutical compositions that include at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) of any of the bacterial strains described herein that express and/or secrete at least one of any of the therapeutic agents described herein.
The pharmaceutical compositions can be formulated in any matter known in the art. The pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, subcutaneous, intraperitoneal, rectal, or oral). In some embodiments, the pharmaceutical composition is administered directly into the site of disease or diseased tissue, e.g., administered into the tumor, administered into the infected tissue. In some embodiments, administering is targeting, e.g., the pharmaceutical composition includes a targeting moiety (e.g., a targeting protein or peptide).
In some embodiments, the pharmaceutical compositions can include a pharmaceutically acceptable carrier (e.g., phosphate buffered saline). Single or multiple administrations of formulations can be given depending on for example: the dosage (i.e., number of bacterial cells per mL) and the frequency as required and tolerated by the subject. The dosage, frequency and timing required to effectively treat a subject may be influenced by the age of the subject, the general health of the subject, the severity of the disease, previous treatments, and the presence of comorbidities (e.g., diabetes, cardiovascular disease). The formulation should provide a sufficient quantity of active agent to effectively treat, prevent or ameliorate conditions, diseases or symptoms. Toxicity and therapeutic efficacy of compositions can be determined using conventional procedures in cell cultures, pre-clinical models (e.g., mice, rats, or monkeys), and humans. Data obtained from in vitro assays and pre-clinical studies can be used to formulate the appropriate dosage of any compositions described herein (e.g., pharmaceutical compositions described herein).
Efficacy of any of the compositions described herein can be determined using methods known in the art, such as by the observation of the clinical signs of a disease (e.g., tumor size, presence of metastasis).
Also provided herein are kits that include at least three of any of the bacterial strains described herein that express and/or secrete at least one of any of the therapeutic agents described herein. In some instances, the kits can include instructions for performing any of the methods described herein. In some embodiments, the kits can include at least one dose of any of the pharmaceutical compositions described herein. The kits described herein are not so limited; other variations will be apparent to one of ordinary skill in the art.
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
Plasmids and Strains
The circuit strains without the lysis plasmid were cultured in LBmedia with 50 μmml−1 kanamycin, in a 37° C. incubator. The circuit strains with the lysis plasmid were cultured in the same way but with 34 μg ml−1 of chloramphenicol as well along with 0.2% glucose. For microscopy and plate reader experiments 1 nM of 3-oxo-C6-HSL was added to all media. Plasmids were constructed using the CPEC method of cloning or using standard restriction digest/ligation cloning. The lux activator plasmid (Kan, ColE1) and lux-lysis plasmid (Chlor, p15A) were used in previous work16, 31. The RpaR and RpaI genes were obtained via PCR off the Rhodopseudomonas palustris genome obtained through ATCC to create the Rpa-activator and Rpa-lysis plasmids. The lux-sfGFP lysis circuit alone was characterized in E. coli. Co-culturing was performed with nonmotile S. typhimurium, SL1344. The SLC, in both the Lux and Rpa case, is composed of an activator plasmid and a lysis plasmid. For the circuit characterization experiments, there were three variations of the activator plasmid. The first is pTD103LuxI-sfGFP which was used in previous work31. This plasmid contains a LuxI with the ssrA-LAA degradation tag (amino-acid sequence of AANDENYALAA) and sfGFP, a superfolding green fluorescent protein variant32. pTD103LuxI (TS) sfGFP was constructed by adding the TS-linker (amino acid sequence of TS) between the ssrA-LAA tag and LuxI. pTD103LuxI(-LAA) sfGFP was constructed by removing the ssrA-LAA tag from LuxI. For the dual lysis experiments, the Lux-CFP strain used the activator plasmid with the ssrA-LAA tagged LuxI instead with a CFP in place of the sfGFP. The Rpa-GFP strain's activator plasmid was created by replacing LuxR with RpaR, and the LuxI with an ssrA-LAA tagged Rpat The lysis plasmids have a p15a origin of replication and a chloramphenicol resistance marker33 and have been previously described16. The lysis gene, E from the bacteriophage ΦX174, was kindly provided by Lingchong You and was taken from the previously reported ePop plasmid via PCR34. The E gene was placed under the expression of the LuxR-AHL activatable luxI promoter for both the Lux-CFP and Rpa-GFP strains. Most of the construction was done using the CPEC method of cloning35. See
The microscopy and microfluidics techniques used in Example 1 have been previously described35. In short, micron-scale features are baked onto silicon wafers using cross-linked photoresist. The microfluidic device resin, PDMS (polydimethysiloxane), is then poured over the wafers and solidified by baking. The PDMS, which contains numerous devices, is peeled off and individual devices are cut out from the whole. Holes are then punched into the device at their input and output where the fluid lines will eventually plug in. After puncturing, the devices are bonded onto glass coverslips via plasma-activation. The devices were then put in a vacuum and the outlet was loaded with cells and the inlet with media. Vacuum pressure loads cells into the traps and media lines are plugged in before the cells can contaminate the upstream section of the device. The flow was then adjusted by changing the relative heights of the syringes, which for all experiments the meniscus of the media was set to one inch above the meniscus of the waste, resulting in a low, constant hydrostatic pressure driven flow.
All microfluidic experiments were done in a side-trap array device as previously described14, and in all cases 0.075% Tween20 was added to the media to deter cells from sticking to the channels and the ports of the device. The bacteria growth chambers were 100 μm wide 85 μm deep and approximately 1.6 μm in height. For lysis characterization (
For dual lysis and co-culturing experiments (
For dual lysis and co-culturing experiments: Phase-contrast images were taken at 20× magnification with 50-200 ms exposure times. Fluorescent imaging at 20× was performed at 300 ms for GFP, 30% setting on the Lumencor SOLA light source, and 300 ms and 35% for CFP. Images were taken every 3 minutes for the course of the experiment (˜2 days). Co-culture was determined to be lost if the fluorescence of either CFP or GFP went below background fluorescence, and then was checked manually in cases of the oscillatory lysing CFP strain which can go below threshold between lysis events. For the lysis characterization (
For the well-plate experiments the strains were grown in a standard Falcon tissue culture 96-well flat bottom plate with appropriate antibiotics (kanamycin only for non-lysis and kanamycin and chloramphenicol for lysis strains). For consistency with microfluidic experiments, 1 nM of 3OC6-HSL was added to all media. The bacterial strains used in
PopulationLux is the population estimate of the Lux-CFP strain in a co-culture. Area(CFPmix) is the area of the CFP fluorescence time-series curve of a given co-culture. Area(BGCFP) is the area of the background CFP fluorescence time-series line. Area(CFPLux) is the average area of the CFP fluorescence time-series curve in the wells with only the Lux-CFP strain. Area(GFPLux) is the average area of the GFP fluorescence time-series curve in the wells with only the Lux-CFP strain (For this strain the GFP fluorescence should technically be at background, further normalization is done because the Tecan's GFP sensor reads into the CFP emission profile). Area(BGGFP) is the area of the background GFP fluorescence time-series line. η is the calculated fluorescence emission cross-talk scalar, and is only needed to scale GFP values as the CFP sensor does not read any GFP. The normalized, filtered, GFP value is thus given by GFPReal. Area(GFPmix) is the area of the GFP fluorescence time-series curve of a given co-culture. Area(GFPRpa) is the average area of the GFP fluorescence time-series curve in the wells with only the Rpa-GFP strain. Finally, PopulationRpa is the population estimate of the Lux-CFP strain in a co-culture.
For the agent-based model, to simulate bacterial motion, a mechanical agent-based model was adapted from previous work36, 37. Each cell is modeled as a spherocylinder of unit diameter that grows linearly along its axis and divides equally after reaching a critical length ld=4. It can also move along the plane due to forces and torques produced by interactions with other cells. The slightly inelastic cell-cell normal contact forces are computed via the standard spring-dashpot model, and the tangential forces are computed as velocity-dependent friction. To describe the intracellular dynamics of each cell, the ordinary differential equation model was adapted from another study16. Specifically, the intracellular dynamics are
Here the variables Plux; Hi; Ii and Li are the activity of luxI promoter, intracellular AHL, LuxI and lysis protein of the i-th cell. He(xi; t) is the extracellular concentration of AHL at the location of the i-th cell. luxI promoter is induced by AHL. b*(Ii/(KI+Ii)) is the production term for AHL. Dm(He(xi; t)−Hi) describes the exchange of intra- and extra-celluar AHL across the cell membrane. CIPlux and γIIi are the production and degradation terms for LuxI. CLPlux and γIIi are the production and degradation terms for lysis protein. The extracellular AHL concentration He(x; t) is governed by linear diffusion equation
In the simulation, 2D finite difference methods were used to describe the diffusion of AHL. The model in traps were implemented with different side lengths (20, 40 and 60). To simulate the lysis of each cell, we assume that when the concentration of lysis protein Li is above a threshold Lth, the cell has a probability of Pr=pL(Li−Lth) per unit of time to lyse and once a cell lyses, it is removed from the trap.
Model parameters were chosen to qualitatively fit the experimental results and the parameters H0; m; b; pL were chosen to account for the differences of experimental measurements and dynamic behaviors between Lux-CFP and Rpa-GFP strains. The parameter values for the Lux-CFP strain are α0=0:1 (Lux promoter basal production); αH=2 (Lux promoter AHL induced production); H0=1 (AHL binding affinity to Lux promoter); m=4 (Hill coefficient of AHL induced production of Lux promoter); b=1:5 (AHL production rate); KI=1 (Conc. of LuxI resulting half maximum production of AHL); Dm=10 (Diffusion constant of AHL across cell membrane); CI=1 (LuxI copy number); γI=1 (Degradation rate of LuxI); CL=1 (Lysis gene copy number); γL=0:5 (Degradation rate of lysis protein); dH=0:1 (Dilution rate of extracellular AHL); DH=65 (Diffusion constant of extracellular AHL); pL=0:3 (Probability of lysing); Lth=1:6 (Threshold of lysis protein for lysis).
To simulate the constant-lysis Rpa-GFP strain, these parameters have different values: H0=0:2; m=1; b=0:8; pL=0:03. Besides, Rpa-GFP strain's growth rate is 10% larger than Lux-CFP strain.
Deterministic Modeling
Single lysis oscillator strain: The population level mechanisms that lead to oscillations in population size as observed with the synchronized lysis circuit are described. To gain an intuitive understanding, a reduced model was used that aims to reproduce the observed population level behavior using only the fundamental ingredients of the circuit: Autocatalytic production of quorum sensing agent and quorum sensing agent-induced lysis of cells. The basic equations for a single strain equipped with the lysis circuit are as follows (see
The cell density is denoted by n. Cells divide with a rate α and die with a maximal rate γ due to lysis. 0≤f(q)≤1 characterizes the promoter under which the QS and lysis proteins are expressed, so it determines the dependence of the death rate on q and the auto-catalyzed production of the QS agent q. αq is the basal production rate of QS agent, which can be increased by the presence of q to a maximum production rate of αq+αq*. q is diluted in the environment with a rate γq. A standard Hill function for f(q) was used:
where qc is the concentration of q that results in the half-maximum death rate (and auto-catalyzed production of q) and m is the Hill coefficient.
A linear stability analysis shows that the system (1) has a stable fixed point when
The border of this stability region corresponds to the onset of oscillations. Basal parameters are ,unless otherwise mentioned: α=1, γ=4, αq=0:4, αq*=8, γq=1, qc=1, m=2. These parameters lead to oscillations according to (3). All simulations are carried out using the Runge-Kutta-Fehlberg (RKF45) method. An example trajectory is depicted in
While individual proteins or enzymes were not explicitly modeled, an understanding for the influence of LuxI degradation by ClpXP was gained with the model (1) using the following logic: When there is very little LuxI (i.e. the positive feedback loop has not been activated), fast degradation by ClpXP will have a strong influence on the steady-state level of LuxI. LuxI with a strong degradation tag will experience fast degradation by ClpXP leading to a low basal production rate of QS agent (αq), whereas LuxI with a weak degradation tag will have a higher steady-state level and therefore a higher basal procution rate aq. In contrast, once the positive feedback has been activated, the concentration of LuxI (and consequently the parameter αq* of the model) have a much weaker dependence on its degradation tag since an abundance of LuxI produced from a fully activated promoter saturates the limited enzymatic processing capacity of ClpXP and therefore the level of LuxI will be determined mainly by dilution due to cell growth. As seen from (3), decreasing aq by a larger factor than αq* generally brings the system closer to oscillations, which is consistent with the requirement of a strong degradation tag for sustained oscillations demonstrated in
Microfluidic traps and multiple strains: A microfluidic trap is clearly a finite environment, but because nutrients are constantly replenished by diffusion from fresh media in the channel, logistic growth (as is often assumed in other scenarios with finite carrying capacities) would be an unrealistic description of the population dynamics. Instead, it was assumed that growth is unaffected as long as the population density is below the carrying capacity c of the trap. The cell density was capped at c, corresponding to any extra cells being washed away by the flow in the main channel (“spillover”).
Numerically, the cell density was reset to c after every time step of the simulation if it exceeds c. In all simulations c=1.
where η1 and η2′ correspond to the population densities before the reset. More specifically, this way of limiting the total population density to the carrying capacity c corresponds to assuming a well-mixed environment, such that the relative population densities of the two strains remain unchanged upon spillover.
Consequently, two oscillating strains in one trap that use completely orthogonal quorum sensing systems only interact if the total population density hits the carrying capacity c. As below, the main text, the strains will eventually lock into an anti-phase pattern where they avoid reaching their peak density at the same time. In order to model cross-talk, the equation of the “receiver” strain (strain 2 in this case) was modified to read
where ε determines how much strain 2 responds to the QS agent of strain 1, i.e. the strength of the cross-talk.
Additional parameters used in the main text: For the parameter scan of a single strain in =0:6 and
=0:12, respectively. Weak lysis (strain 1,
In order to engineer a stable co-culture of two competitive bacterial strains, the dynamics of a small library of quorum sensing (QS) components were first characterized (
To understand how an ecosystem harboring the synchronized lysis circuit (SLC) can be altered, the range of possible self-limiting dynamics of the circuit was establised (
To build a synthetic ecosystem of two orthogonal SLC strains, the previously built circuit was used based on the Lux quorum sensing system and constructed a new circuit with the Rpa system. The Rpa system had RpaR in place of LuxR and an ssrA tagged RpaI in place of LuxI (
The strains were then grown in microfluidic devices, with a seeding ratio of 1:10 (Rpa-GFP to Lux-CFP) optimized for the new system, in order to examine the long-term dynamics of the co-culture. The microfluidic trap (growth chamber) harboring the two strains without the lysis gene quickly lost its co-culture and was taken over by the Rpa-GFP strain alone (
A reduced deterministic model was developed to explore a wider space of possible dynamics achieved through differences in growth rates, QS systems, and lysis circuit regimes. For each case, communication motifs are distinguished and suitable experimental candidate QS systems are chosen to achieve the displayed dynamic. For the two individual lysis circuits, either Non-lysing (no SLC), Lysing (SLC), or Weak Lysing (less effective SLC) were considered. With two non-lysing strains, the faster growing strain will eventually dominate the population (
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 62/508,801, filed on May 19, 2017. The entire contents of the foregoing are hereby incorporated by reference.
This invention was made with Government support under Grant Nos. RO1-GM069811 and P50-GM085764 awarded by the National Institutes of Health and National Institute of General Medical Sciences. The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/033555 | 5/18/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62508801 | May 2017 | US |
