Patent Grant
11649447
This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62/518,075 filed Jun. 12, 2017, which is incorporated herein by reference in its entirety for all purposes.
The present disclosure provides compositions, systems, and methods relating to the production of biomolecules using microorganisms. In particular, the present disclosure provides biomolecule production platforms that include genetically engineered microorganisms with genetic circuits functionally coupled to microcapsules formed from materials that are responsive to culture conditions. The biomolecule production platforms disclosed herein facilitate the efficient and robust production, purification, and/or analysis of any biomolecule-of-interest.
Stimulus-sensitive biomaterials have found diverse applications including controlled drug release and injectable implants due to their phase transition capability. In parallel, engineered bacteria programmed with synthetic gene circuits have shown tremendous potential as “smart” sensing and actuating agents. To date, few efforts have been made to integrate these two aspects. Yet, the function of many natural systems emerges from the interplay between cells and their changing environment.
“Smart” hydrogels, or stimuli-responsive hydrogels, can undergo reversible volume phase transitions upon minute changes in physical (e.g., temperature, light) or chemical (e.g., pH, ions) environments. Due in part to their unique properties, smart hydrogels have been entailed for transducing chemical or physical energy into mechanical work as sensors; controlled release of the drug at proper condition for therapeutic drug delivery; and sol-gel transition in situ in tissue engineering. In parallel, the past two decades have seen enormous progress in synthetic biology, leading to engineering of cell-based systems for sensing and responding to diverse signals and also to their physical confinement. This allows the engineered systems not only to function as biosensors or biomarkers, but also be applied in targeted drug delivery and on-demand protein synthesis.
Embodiments of the present disclosure include a system for biomolecule production. In accordance with these embodiments, the system includes a plurality of genetically engineered microorganisms comprising a genetic circuit and means for producing a biomolecule-of-interest, wherein the genetic circuit facilitates autonomous production of the biomolecule-of-interest; and an encapsulation material, wherein the encapsulation material is capable of forming microcapsules to contain the plurality of genetically engineered microorganisms, and wherein the microcapsules are functionally coupled to the genetic circuit.
In some embodiments, the plurality of genetically engineered organisms includes bacteria or yeast. In some embodiments, the plurality of genetically engineered organisms includes one or more strains of E. coli. In some embodiments, the one or more strains of E. coli include MC4100, MG1655, BL21, NISSLE1917, and any derivatives or variations thereof. In some embodiments, the genetic circuit includes a cell lysis module and a cell density sensing module. In some embodiments, the cell lysis module includes a gene encoding E protein from phage phiX174, and the cell density sensing module includes a mutated luxR gene and a ColE1 origin of replication lacking the Rom/Rop protein. In some embodiments, the biomolecule-of-interest is at least one of a peptide, a polypeptide, a protein, a nucleic acid, a polynucleic acid, a DNA molecule, a RNA molecule, or any derivatives or combinations thereof. In some embodiments, the biomolecule-of-interest is a polypeptide, and the means for producing the biomolecule-of-interest includes a plasmid comprising a gene encoding the polypeptide under the control of an inducible promoter. In some embodiments, the biomolecule-of-interest is a cytoplasmic polypeptide. In some embodiments, the biomolecule-of-interest is a polypeptide that includes a tag to facilitate binding to a substrate. In some embodiments, the encapsulation material includes chitosan, alginate, hyaluronic acid, polyethylene glycol polymers, polyethylene oxide polymers, elastin-like polypeptides (ELPs), resilin-like polypeptides (RLPs), or any derivatives or combinations thereof. In some embodiments, the encapsulation material is responsive to one or more conditions in the microcapsules. In some embodiments, the functional coupling of the microcapsules to the genetic circuit includes the plurality of microorganisms altering the one or more conditions in the microcapsules. In some embodiments, the one or more conditions in the microcapsules include pH or ionic strength. In some embodiments, the microorganisms are present in the microcapsules at a concentration from about 102 to about 1010. In some embodiments, the system further includes a microfluidics platform to facilitate the production, purification, and/or analysis of the biomolecule-of-interest. In some embodiments, the microfluidics platform includes an input channel, a production chamber, and an assay chamber.
Embodiments of the present disclosure also include a microfluidics platform for biomolecule production. In accordance with these embodiments, the platform includes a microfluidic device comprising an input channel, a production chamber, and an assay chamber, a plurality of genetically engineered microorganisms comprising a genetic circuit and means for producing a biomolecule-of-interest, wherein the genetic circuit facilitates autonomous production of the biomolecule-of-interest; and an encapsulation material, wherein the encapsulation material is capable of forming microcapsules to contain the plurality of genetically engineered microorganisms, and wherein the microcapsules are functionally coupled to the genetic circuit.
In some embodiments, the production chamber is fluidly coupled to the input channel and the assay chamber. In some embodiments, the plurality of genetically engineered microorganisms are contained in the production chamber. In some embodiments, the device further includes an output channel fluidly coupled to the assay chamber or the production chamber. In some embodiments, the device includes a second input channel fluidly coupled to the assay chamber. In some embodiments, the production chamber is fluidly coupled to a purification chip. In some embodiments, the inner surface of the purification chip is modified to bind the biomolecule-of-interest.
Embodiments of the present disclosure also include a method for producing a biomolecule-of-interest. In accordance with these embodiments, the method includes loading a plurality of genetically engineered microorganisms contained in microcapsules into a microfluidics device, wherein the plurality of genetically engineered microorganisms include a genetic circuit and means for producing a biomolecule-of-interest; providing nutrients to the plurality of microorganisms; and producing the biomolecule-of-interest.
In some embodiments, the plurality of genetically engineered organisms includes bacteria or yeast. In some embodiments, the biomolecule-of-interest is at least one of a peptide, a polypeptide, a protein, a nucleic acid, a polynucleic acid, a DNA molecule, a RNA molecule, or any derivatives or combinations thereof. In some embodiments, the microcapsules include an encapsulation material comprising chitosan, alginate, or any derivatives or combinations thereof. In some embodiments, the microfluidics device includes an input channel, a production chamber, and an assay chamber.
The present disclosure provides compositions, systems, and methods relating to the production of biomolecules using microorganisms. In particular, the present disclosure provides biomolecule production platforms that include genetically engineered microorganisms with genetic circuits functionally coupled to microcapsules formed from materials that are responsive to culture conditions. The biomolecule production platforms disclosed herein facilitate the efficient and robust production, purification, and/or analysis of any biomolecule-of-interest.
Small-scale production of biologics has great potential in facilitating individualized medicine and enhancing the accessibility of biomanufacturing. By exploiting cell-material feedback, a concise platform to achieve versatile production, analysis, and purification of diverse proteins was developed. The core of the technology described in the present disclosure is a microbial swarmbot, which includes a stimulus-sensitive polymeric microcapsule encapsulating engineered bacteria. By sensing the confinement, the bacteria undergo programmed partial lysis at a high local density. Conversely, the encapsulating material shrinks in response to the changing chemical environment caused by cell growth and death, squeezing out the protein products released from bacterial lysis. This platform was then integrated with downstream quantification of enzymatic kinetics and diverse modes of protein purification. Embodiments presented herein demonstrate the use of the feedback between living cells and materials to engineer a modular and flexible platform with a sophisticated programmed function.
Bacteria are a common host to produce diverse biologics, accounting for about 30% of biopharmaceuticals. Synthesis of recombinant proteins using bacterial hosts entails multiple steps including culturing, disruption of bacteria by physical or chemical means, and subsequent isolation and purification of the desired product. For industrial operations, these steps are usually carried out on a large scale; consequently, each step requires a sophisticated and delicate infrastructure to ensure efficiency and product quality. While this may be important for producing molecules in large amounts, this format is not flexible or economically suited for producing diverse biologics when only a small amount is needed for each. Moreover, standard biomanufacturing is not accessible in remote or underdeveloped areas that lack the basic infrastructure (e.g., transportation, equipment, or electricity) and personnel with necessary technical training. Conventional manufacturing of certain products in advance could also result in wasted labor and resources due to the high cost of production, the need for a cold chain for transportation and storage, and the short shelf-life of biologics. Therefore, there is a need to develop technologies for versatile and scalable production of diverse biologics on demand, as well as subsequent analysis and purification.
This need has motivated the development and adoption of single-use technologies (SUTs) for biomanufacturing. To date, however, the SUTs have focused on replacing traditional stainless steel-based fixed reactors with flexible and disposable parts or the direct miniaturization of reactors, without changing the fundamental structure of the manufacturing process. In particular, distributing production of biologics into multiple steps is a standard practice and important for implementing the production and purification as unit operations, where each unit operation can be individually optimized for large-scale biomanufacturing. However, each step can lead to an opportunity for product loss and thus reduced efficiency, particularly for small-scale operations. During biopharmaceutical manufacturing operating costs, 75%-80% of the total cost is spent on the downstream processing, including the disruption of cells, subsequent separation, clarification and purification of target products. For certain proteins, engineered secretion can facilitate the extraction process. Yet, the diverse secretion mechanism of bacteria makes the selection of the appropriate secretion pathway for each recombinant protein complicated and time consuming. Besides, the efficiency of secretion is often limited in bacterial hosts, and some large cytoplasmic proteins may be physically impossible to translocate. Induced lysis can allow effective release of diverse proteins, but it does not simplify the process of cell debris separation and downstream purification.
To overcome these limitations, we combine aspects of synthetic biology and stimulus-responsive biomaterials to integrate the multiple steps of production, disruption and separation into a concise format. Embodiments of the present disclosure couple programmed lysis of engineered bacteria and controlled periodic capsule phase transition. When the local cell density inside the capsule is sufficiently high, autonomous partial lysis will occur and allows the cells to release their contents, including the protein product of interest. The bacterial growth can alter the local environmental conditions (e.g., pH and ionic strength), driving the phase transition of the encapsulating material. Consequently, in some embodiments, the released protein is transported from the interior to exterior with shrinking of the growth-sensitive capsule, while cells and large debris are trapped inside the capsules. In accordance with these embodiments, nutrient replenishment can be used as a cue to allow the capsule to swell again, while it resets the capsule environment, and also allows the cell density to begin increasing after it drops to a certain level. The interplay between sustained partially lysis of engineered bacteria and periodic phase transition of the encapsulating material enables sustained product synthesis and separation.
The biomolecule production systems of the present disclosure demonstrate the use of active feedback control mediated by the interplay between engineered bacteria and stimulus-responsive materials, and facilitate integrated protein synthesis, separation, and downstream processing. This design enables the development of a completely novel format of SUT for distributed biomanufacturing. By design, the biomolecule production systems of the present disclosure are modular and scalable: the engineered gene circuit, the host strain, the target product, the encapsulating material and downstream processing (e.g.,
Polymeric capsules provide physical support to the living cells and restrain the free diffusion of cells and large debris. Chitosan was used in this study in part due to its well-established stimulus responsiveness. Its stability facilitates the preparation of MSBs in advance and enables long-term storage with negligible decrease in performance. In general, other stimulus-responsive materials can be adopted to fine-tune biocompatibility, durability, and separation capability. For example, capsules with different pore sizes or charge properties can enable more selective purification of macromolecules. Also, the separation and clarification function could be further enhanced by surface modification on the polymeric capsules (
Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.
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. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.
For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
“Correlated to” as used herein refers to compared to.
“Identical” or “identity,” as used herein in the context of two or more polypeptide or polynucleotide sequences, can mean that the amino acid or nucleotide sequences have a specified percentage of residues that are the same over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of the single sequence are included in the denominator but not the numerator of the calculation.
“Isolated polynucleotide” as used herein may mean a polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or a combination thereof) that, by virtue of its origin, the isolated polynucleotide is not associated with all or a portion of a polynucleotide with which the “isolated polynucleotide” is found in nature; is operably linked to a polynucleotide that it is not linked to in nature; or does not occur in nature as part of a larger sequence.
“Peptide” refers an oligomer to short polymer of amino acids linked together by peptide bonds. In contrast to other amino acid polymers (e.g., proteins, polypeptides, etc.), peptides are of about 50 amino acids or less in length. A peptide may comprise natural amino acids, non-natural amino acids, amino acid analogs, and/or modified amino acids. A peptide may be a subsequence of naturally occurring protein or a non-natural (artificial) sequence.
“Polypeptide” refers to a polymer of amino acids linked together by peptide bonds that is greater than about 50 amino acids in length. Polypeptides may comprise natural amino acids, non-natural amino acids, amino acid analogs and/or modified amino acids, and may be a naturally occurring sequence, or a non-natural (artificial) sequence, or a subsequence of naturally occurring protein or a non-natural (artificial) sequence. “Artificial” refers to compositions and systems that are designed or prepared by man, and are not naturally occurring. For example, an artificial sequence refers to an amino acid or nucleotide sequence that does not occur in nature (e.g., a polypeptide without 100% identity with a naturally-occurring protein or a fragment thereof). “Conservative” amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid having similar chemical properties, such as size or charge. For purposes of the present disclosure, each of the following eight groups contains amino acids that are conservative substitutions for one another:
1) Alanine (A) and Glycine (G);
2) Aspartic acid (D) and Glutamic acid (E);
3) Asparagine (N) and Glutamine (Q);
4) Arginine (R) and Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V);
6) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W);
7) Serine (S) and Threonine (T); and
8) Cysteine (C) and Methionine (M).
Naturally occurring residues may be divided into classes based on common side chain properties, for example: polar positive (or basic) (histidine (H), lysine (K), and arginine (R)); polar negative (or acidic) (aspartic acid (D), glutamic acid (E)); polar neutral (serine (S), threonine (T), asparagine (N), glutamine (Q)); non-polar aliphatic (alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M)); non-polar aromatic (phenylalanine (F), tyrosine (Y), tryptophan (W)); proline and glycine; and cysteine. As used herein, a “semi-conservative”amino acid substitution refers to the substitution of an amino acid in a peptide or polypeptide with another amino acid within the same class.
“Variant” is used herein to describe a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. “SNP” refers to a variant that is a single nucleotide polymorphism. Representative examples of “biological activity” include the ability to be bound by a specific antibody or to promote an immune response. Variant is also used herein to describe a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree, and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101, incorporated fully herein by reference. Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. Substitutions may be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.
“Vector” is used herein to describe a nucleic acid molecule that can transport another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double-stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors can replicate autonomously in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. “Plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions, can be used. In this regard, RNA versions of vectors (including RNA viral vectors) may also find use in the context of the present disclosure.
Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
Embodiments of the present disclosure include the use of microorganisms for biomolecule production. In accordance with these embodiments, systems and platforms for biomolecule production include a plurality of genetically engineered microorganisms comprising a genetic circuit and means for producing a biomolecule-of-interest. In some embodiments, microorganisms include various strains of bacteria and yeast (including both probiotic and non-probiotic strains). In some embodiments, various E. coli strains are used, given their genetic diversity and robust use in biomolecule production. Bacterial strains that can be used in the biomolecule platforms and systems of the present disclosure include, but are not limited to, E. coli strains such as MC4100 (e.g., MC4100Z1), MG1655, BL21 (e.g., BL21(DE3)), NISSLE1917, and any variant or derivative strains thereof. Other bacterial strains that can be used are included in Table 1 below.
E. coli expression strains.
E. coli. Expression vector cannot
In some embodiments, various strains of yeast can be used with the biomolecule production platforms and systems of the present disclosure. These include, for sample, strains from the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula and Yarrowia.
Other microorganisms can also be used, as would be recognized by one of ordinary skill in the art based on the present disclosure.
Embodiments of the present disclosure include the use of microorganisms for biomolecule production that include one or more genetic circuits. Genetic circuits can include one or more modules for carrying out various functions, such as biomolecule production. For example, as described herein, the ePop genetic circuit comprises a cell lysis module (e.g., based on a gene encoding E protein from phage phiX174) and a cell density sensing module (e.g., based on a mutated luxR gene and a ColE1 origin of replication lacking the Rom/Rop protein), which facilitate autonomous biomolecule production. In accordance with these embodiments, engineered ePop circuits can be used to program autonomous lysis. The circuits can be configured to exploit the cell-density dependent copy number control in ColE1-type plasmids that is coupled to leaky expression of a toxin (see, e.g.,
Genetic circuits can include any number of modules required to carry out a desired function, including but not limited to, sensing of cell density, and cell density-dependent cell lysis (e.g., triggered expression of any cell lysis mechanism, including expression of any gene/protein that can lead to cell lysis), as described herein. In some embodiments, cell density sensing and related functions can be based on quorum sensing systems, and can be used to generate one or more modules of a genetic circuit. Quorum sensing systems are well known in the art and can be the basis for generating genetic circuits in accordance with the methods and systems described herein (see, e.g., You, L. et al., Nature, 428: pp. 868-871 (22 Apr. 2004)).
As described herein, many other genetic circuits can be used as part of the biomolecule production platforms and systems of the present disclosure. As would be recognized by one of ordinary skill in the art based on the present disclosure, various genetic circuits can be created by exploiting the various biological processes in microorganisms, as summarized in Table 2 below (see, e.g., Bradley et al., Tools and Principles for Microbial Gene Circuit Engineering, J. of Mol. Biol. (2016): 428(5B), pp. 862-888).
M. florum Lon
M. florum Lon protease is orthogonal
Embodiments of the present disclosure provide systems and platforms for biomolecule production using genetically engineered microorganisms (e.g., MSBs), as described herein. In accordance with these embodiments, biomolecules that can be produced using the methods, systems, and platforms described herein include, but are not limited to, peptides, polypeptides, proteins, nucleic acids, polynucleic acids, DNA molecules (double or single-stranded), RNA molecules (double or single-stranded), small molecule compounds, and any derivatives or combinations thereof.
In some embodiments, the biomolecule-of-interest is a protein-based molecule made of amino acids. Such proteins/peptides include, but are not limited to, enzymes, antibodies, peptide hormones, therapeutic biologics/biosimilars, immunomodulatory proteins, antigenic proteins, immunogenic proteins, cytoplasmic proteins, nuclear proteins, fusion proteins, tagged proteins, chimeric antigen receptor proteins, transcription factors, receptors, receptor binding ligands, viral proteins, proteins comprising cancerous epitopes, antimicrobial proteins, glycoproteins, synthetic proteins, natural proteins, biopolymers, scaffolding proteins, and the like.
In some embodiments, proteins that can be produced using the methods, systems, and platforms described herein include, ELPs (some of them are fused with spytag and spycatcher), RLPs, ligand-binding proteins, pigment proteins, and enzyme (see, e.g.,
In addition to genetic circuits, microorganisms described herein can include a means for producing a biomolecule-of-interest, such as a protein/peptide. In accordance with these embodiments, a means for producing a biomolecule-of-interest can include a plasmid comprising a gene that encodes a protein/peptide. In some embodiments, such plasmids include regulatory elements, such as promoters, that control their expression. In other embodiments, such plasmids are inserted into the genome of a microorganism such that its expression can be controlled by adjacent regulatory elements. As described herein, E. coli strain MC4100Z1 included an ePop circuit along with a plasmid expressing BlaM under control of an IPTG-inducible promoter; E. coli strain BL21(DE3) included an ePop circuit and plasmids expressing ELPs-GFP-His or RLPs-GFP-His under control of a T7 promoter; and E. coli strain MC4100Z1 included an ePop circuit and other circuits with T5 promoter regulation (see, e.g., Table 3).
Embodiments of the present disclosure include platforms and systems for biomolecule production using microorganisms that are contained within an encapsulation material (e.g., form microcapsules containing the microorganisms). In accordance with these embodiments, encapsulation materials can be functionally coupled to a genetic circuit within the microorganisms, such that the encapsulation materials are responsive to one or more environmental (e.g., culture) conditions capable of being altered by the microorganisms.
For example, as described herein, microorganism growth can affect both the local pH and ionic strength of culture conditions, which can then cause phase transitions in an encapsulation material (e.g., chitosan network). As microorganisms grew within the microcapsules, the pH and ionic strength of the culture conditions changed, which caused shrinking of the size of the microcapsules, thus releasing a biomolecule-of-interest into the culture media, which can be analyzed/collected/purified. Other means of functionally coupling encapsulation materials to genetic circuit(s) in a microorganism can be based on the various properties of the genetic circuit(s), as would be recognized by one of ordinary skill in the art based on the present disclosure. For example, encapsulating material can be engineered to respond to other culture conditions, such as but not limited to, temperature and light, or any other conditions that microorganisms can be engineered to alter in a manner that can be sensed by the encapsulation material.
As described herein, various encapsulation materials can be used, including but not limited to, any natural and/or synthetic materials capable of forming polymers, such as chitosan, alginate, hyaluronic acid, polyethylene glycol polymers, polyethylene oxide polymers, elastin-like polypeptides (ELPs), resilin-like polypeptides (RLPs), or any derivatives or combinations thereof. Other encapsulation materials can also be used, as would be recognized by one of ordinary skill in the art based on the present disclosure.
Encapsulation materials generally include the capability and properties to form microcapsules in order to contain the microorganisms described herein. In some embodiments, a microcapsule is configured to have a certain size or diameter, for example, to carry out a given function or to mitigate the development of mutant microorganisms. In some embodiments, microcapsules can range from about 50 μm to about 5000 μm in diameter, from about 50 μm to about 4000 μm in diameter, from about 50 μm to about 3000 μm in diameter, from about 50 μm to about 2000 μm in diameter, from about 50 μm to about 1000 μm in diameter, from about 50 μm to about 500 μm in diameter, or from about 50 μm to about 250 μm in diameter. In some embodiments, microcapsules can range from about 100 μm to about 5000 μm in diameter, from about 250 μm to about 4000 μm in diameter, from about 500 μm to about 3000 μm in diameter, from about 500 μm to about 2000 μm in diameter, or from about 1000 μm to about 1500 μm in diameter. In some embodiments, microcapsules can range from about 100 μm to about 500 μm in diameter, from about 200 μm to about 500 μm in diameter, from about 300 μm to about 500 μm in diameter, or from about 300 μm to about 400 μm in diameter. Accordingly, microorganism concentration within microcapsules can range from about 102 to about 1010, from about 102 to about 108, or from about 102 to about 106, depending on the stain of microorganism, available nutrients, and various culture conditions.
Methods of producing, manufacturing, or fabricating microcapsules containing microorganisms (e.g., MSBs) are generally known in the art, and include, but are not limited to electrospraying, electrohydrodynamic spraying, PDMS stamping, and methods using droplet-generating microfluidic devices, among others.
Embodiments of the present disclosure also include microfluidics devices and platforms for biomolecule production using microorganisms (e.g., MSBs) as described herein. In accordance with these embodiments, microfluidics devices and platforms can be used to facilitate the production, isolation, collection, purification, and analysis of biomolecules being produced. In some embodiments, microfluidic devices and platforms include an input channel, a production channel, and an assay chamber. The production chamber generally contains the microorganisms contained within the microcapsules, and it can be fluidly coupled to an input channel such that culture media and nutrients flow from a source, into the input channel, and then into the production chamber to reach the microorganisms. More than one input channel can be included, depending on the desired nutrient flow and culture conditions. In some embodiments, the assay chamber is fluidly coupled downstream of the production chamber, in order to facilitate the collection and analysis of a biomolecule-of-interest produced and released by the microorganisms in the production chamber.
In some embodiments, the microfluidics device or platform includes an output channel that is fluidly coupled to the assay chamber or production chamber, such a biomolecule-of-interest produced and released by the microorganisms in the production chamber will flow to the output channel for collection and/or purification. In some embodiments, the microfluidics device or platform includes a purification chip that is fluidly coupled to the assay chamber or production chamber, such a biomolecule-of-interest produced and released by the microorganisms in the production chamber will flow into the purification chip for collection and/or purification. In some embodiments, the purification chip can be modified to facilitate the binding of the biomolecule-of-interest, including coating the inner surface of the purification chip to facilitate binding of the biomolecule-of-interest to a substrate, which can subsequently be treated in order to elute the biomolecule-of-interest (e.g., PEG-coated chelated Cu2+).
As described herein, to facilitate isolation, collection, and/or purification of a biomolecule-of-interest from other components, microfluidics devices and platforms of the present disclosure can involve configuring a biomolecule-of-interest to include a tag or other means for attachment or binding to a substrate or to part of the microfluidics device. In conjunction with a particular tag, the microfluidics device can include means for binding the tag such that the biomolecule-of-interest can be separated from other components (e.g., His-tag purification).
Embodiments of the present disclosure include methods for producing a biomolecule-of-interest using the genetically engineered microorganisms (e.g., MSBs), as described herein. In accordance with these embodiments, the method includes loading a plurality of genetically engineered microorganisms contained in microcapsules into a microfluidics device. The plurality of genetically engineered microorganisms can include a genetic circuit and means for producing a biomolecule-of-interest. The method also includes providing nutrients to the plurality of microorganisms, and allowing for the production of the biomolecule-of-interest. In some embodiments, the method also includes isolating, collecting, purifying, and/or analyzing the biomolecules being produced.
The methods described herein enable the production of a biomolecule-of-interest at various yields. In some embodiments, when the biomolecule-of-interest is a protein/peptide, the yield can be from about 10 mg/L to about 100 mg/L, from about 10 mg/L to about 90 mg/L, from about 10 mg/L to about 80 mg/L, from about 10 mg/L to about 70 mg/L, from about 10 mg/L to about 60 mg/L, from about 10 mg/L to about 50 mg/L, from about 10 mg/L to about 40 mg/L, from about 10 mg/L to about 30 mg/L, or from about 10 mg/L to about 20 mg/L.
As described herein, autonomous biomolecule production can be based on the interaction between a genetic circuit(s) that is functionally coupled to a stimulus-responsive encapsulation material, such as microorganism growth/lysis coupled to pH/ionic strength alterations that cause microcapsules to shrink, thus releasing the biomolecule into the culture. In addition, biomolecule production can be independent of (or decoupled from) microorganism growth/lysis. In some embodiments, biomolecule production may occur, but the biomolecule is actively or passively transported out of the microcapsules without lysis. In some embodiments, genetic circuits may be developed or designed to facilitate growth-independent biomolecule production.
Bacterial Strains. Escherichia coli strain MC4100Z1 was used for carrying the ePop circuit alone, or with a plasmid expressing BlaM under control of an IPTG-inducible promoter. E. coli strain BL21(DE3) was used for carrying the ePop circuit and plasmids expressing ELPs-GFP-His or RLPs-GFP-His under control of a T7 promoter. E. coli strain MC4100Z1 was used for carrying ePop circuit and other circuits with T5 promoter. The constructs used herein are provided in Table 3 below.
Circuit and Plasmids. ePop (ColE1 origin) was published previously. Briefly, it was constructed using the luxbox region (140 bp upstream of luxI in V. fischeri) from pluxGFPuv and E gene coding sequence from φX174 (NEB). Each region was PCR-amplified and then joined together in an overlap PCR reaction. The ‘lux box-E gene’ fragment was inserted into the Aatll site of host vector pLuxRI2.
BlaM (p15A origin) was published previously. Briefly, it was constructed by PCR-amplifying bla gene from pSND-1 without first 66 base pairs and inserting it under Plac/ara-1 of pPROLar.A122 (Clonetech).
ELPs-GFP-His (model protein), RLPs-GFP-His, GLP-1-ELPs-His and other ELPs/RLPs variants, were previously constructed. The fragments were cloned into the vector with T7 or T5 promoter and p15A origin.
His-AAA, His-BBB, His-AA-mcherry and His-BB were constructed using plasmids previously described. The fragments were cloned into the vector with T5 promoter and p15A origin.
3A-SUP was constructed using plasmids previously described. The fragment was cloned into the vector with T5 promoter and p15A origin.
3A-PrancerPurple was constructed using a plasmid from ATUM (CPB-37-441) and His-AAA. The PrancerPurple was cloned into the downstream of His-AAA.
3A-BlaM was constructed using the synthesized fragment (3A-BlaM) from GENEWIZ. The fragment was cloned into the vector with T5 promoter and p15A origin.
Griffithsin-His was constructed using plasmids previously described. The fragment was cloned into the vector with T7 promoter and p15A origin.
Growth Media. The LB medium: 25 g LB Broth Powder (MO BIO Laboratories, Inc) was added into 1 L deionized H2O. After autoclaving for 45 mins, the LB medium was stored at room temperature. The medium was supplemented with appropriate antibiotics (100 μg/mL chloramphenicol, 50 μg/mL kanamycin, 100 μg/mL ampicillin) when applicable.
The M9 medium: 1×M9 salts (48 mM Na2HPO4, 22 mM KH2PO4, 862 mM NaCl, 19 mM NH4Cl), 0.4% glucose, 0.2% casamino acids (Teknova), 0.5% thiamine (Sigma), 2 mM MgSO4, 0.1 mM CaCl2 were added into 1 L deionized H2O. M9 medium was adjusted with to pH=7 by VWR Symphony SB70P pH Meter and filtrated through 0.22 μm filter.
Liquid Cultures. Cells carrying the ePop circuit were streaked onto an agar plate supplemented with 2% (w/v) glucose, and incubated at 37° C. for 16 h. Then, a single colony was picked and inoculated in 3 mL LB medium. The agar and the liquid culture medium were supplemented with appropriate antibiotics when applicable. For cells carrying ePop circuit, 2% (w/v) glucose was supplemented in the overnight cultures. Bacteria carrying all protein-expression circuits were induced with 1 mM IPTG.
Evaluation of Programmed Lysis. Engineered bacteria carrying the ePop circuit (MC4100Z1(ePop)) were inoculated in 3 mL LB medium containing antibiotics and 2% glucose, and cultured at 37° C. with a shaking speed at 250 rpm for overnight. Then the calibrated cultures were diluted into LB medium, which was supplemented with antibiotics and various concentration of glucose. 200 μL cultures were aliquoted into 96-well plates, which were then sealed with 50 μL mineral oil to prevent evaporation. Optical density of cultures (OD600) was measured using a plate reader (Tecan infinite M200 pro). For data analysis, the measured OD600 values were background corrected. Lysis circuit is considered functional when multiple cycles of population oscillations can be observed during long-term batch cultures.
For observing the oscillatory behavior of cells on the microfluidic device, the 20 μL overnight culture (MC4100Z1(ePop/GFP)) was inoculated into 2 mL LB medium and cultured for 8 hours. Then, the cells were re-suspended in M9 medium and loaded them into the culturing chamber. M9 medium was supplemented continuously at 120 μL/hour. The design of the microfluidic device was published previously, while with the height of device is about 1.5 μm. The whole device was cultured at 37° C., and Nikon Ti-E microscope with 100× objective was employed for the long-term monitoring.
Production of Alginate and Chitosan Microcapsules. Chitosan microcapsules containing bacterial cells were fabricated by electrospray (
Efficiency of Protein Export. A high molecular weight (about 70000 g/mol) dextran conjugated with Rhodamine (Sigma-Aldrich) was used as a model protein. About 30 μL dextran-Rhodamine (20 mg/mL) and 30 μL overnight culture of MG1655 cells (sensitive to Ampicillin) were mixed and encapsulated them using 300 μL chitosan or alginate solutions. The chitosan/alginate capsules were first washed three times by PBS/CaCl2 (1.5% w/v), and then cultured in 3 mL LB or M9 medium. As a control, the capsules were cultured in LB or M9 medium with ampicillin eliminating the cell growth. 100 μL of the culturing medium was taken at different time points and the fluorescence signal from medium was measured by a plate reader (Tecan infinite M200 pro). Measurements were done in triplicate and background corrected.
Quantifying Beta-lactamase Activity. Bacteria MC4100Z1 carrying the ePop circuit and a BlaM-expressing plasmid (MC4100Z1 (ePop/BlaM)) were induced with 1 mM IPTG. About 20 μL supernatant was diluted into 200 μL using PBS and mixed with Fluorocillin™ Green (Thermo Fisher) to attain a final substrate concentration of 2 μM. Upon cleavage by BlaM, Fluorocillin™ Green reagent can be converted to a fluorescent product. The resulting fluorescence was measured using a plate reader (Tecan infinite M200 pro). Measurements were done in triplicate and background corrected.
Genetic Stability. Kinetic models were developed to examine the genetic stability of the ePop circuit under different conditions, and the impact on the overall efficiency of protein production. Table 4 and Table 5 (below) provide details pertaining to model development, parameter choices, and initial condition for simulation.
To determine the dependence of gene stability on time experimentally, 30 μL overnight culture of MC4100Z1 (ePop) cells was inoculated into 3 mL M9 medium, or encapsulated into capsules using 300 μL chitosan solution, and supplemented with additional 3 mL M9 medium. After 24, 48 and 72 hours, bacteria were collected from the bulk culture or from MSBs (see detailed releasing procedure below) and plated onto agar plates. To release bacteria from MSBs, 2.5 M NaCl was used to destabilize the capsules by repeated pipetting (
To determine the dependence of the circuit stability on the lysis rate, 30 μL overnight culture of MC4100Z1 (ePop) cells was inoculated into 3 mL M9 medium, or encapsulated into capsules using 300 μL chitosan solution, and supplemented with the 3 mL M9 medium. Each culture was supplemented with 0%, 0.05% or 0.1% (w/v) glucose to adjust lysis rate, which decreases with the glucose concentration (
Beta-lactamase Activity. Equal amount (30 μL) of overnight culture of MC4100Z1(ePop/BlaM) cells were inoculated into 3 mL M9 medium, or encapsulated into capsules using 300 μL chitosan solution, and supplemented with 3 mL M9 medium. After cultured for 72 hours at 37° C. in an incubator, 20 μL supernatant was diluted into 200 μL using PBS and mixed with Fluorocillin™ Green (Thermal fishier technology) to attain a final substrate concentration of 2 μM. The resulting fluorescence was measured using a plate reader (Tecan infinite M200 pro) as a function of time. Each measurement was done in triplicate and background corrected.
The conversion of the Fluorocillin into a fluorescent product can be described by the Michaelis-Menten kinetics:
where v is the initial rate of increase in the fluorescence (F), k is the catalytic constant, K is the Michaelis-Menten constant, E0 is the concentration of BlaM, and [S] is the concentration of the substrate (Fluorocillin). When the substrate is saturating ([S]>>K), v=kE0, thus v provides a direct measurement of the enzyme activity. Experimentally, v is determined by taking the time derivative of F in the initial time window, when F increases linearly with time (
Microfluidic Device Fabrication. The photoresist master mold was fabricated by spin-coating two layers of SU8 3050 (MicroChem, 1000 rpm for 30 seconds) onto a silicon wafer, which was then pre-baked for 60 mins at 95° C., and UV crosslinked with a pre-designed photomask (AutoCAD). The UV exposed wafer was post-baked for 20 mins at 95° C., developed with SU8 developer, and finally hard-baked for overnight at 95° C. The height of the SU8 mold for the microfluidic device was about 450 μm. Next, the PDMS microfluidic device containing the designed structure was fabricated by pouring a mixed PDMS solution (Dow Corning SYLGARD® 184, 10:1 as monomer: curing agent) onto the master mold, degassing the solution under vacuum, and baking it at 80° C. for 30 mins. After the solution solidified, holes were drilled for input and output flow with tissue punches. The PDMS device was bonded to a cover slip by plasma treatment at 40 watts for 30 seconds.
Two different microfluidic devices were fabricated, depending on its purpose. The first device was designed to monitor and quantify BlaM production. The device includes a main channel (width=500 μm, length=5.3 mm), a circular culturing chamber (diameter=1 mm), two trapping channels (width=60 μm, length=720 μm), a substrate delivery channel (width=150 μm, length=2.2 mm), an assay chamber (Diameter=400 μm), and an output channel (width=240 μm, length=2.2 mm). The capsules were captured in the culturing chamber since their diameters (200-400 μm) were larger than that of the trapping channel. The substrate delivery channel was used to flow in a fluorocillin solution to assay the activity of BlaM released from the culturing chamber.
The second device was designed to demonstrate integrated protein production and purification. It includes two separate chips. The first, a production chip is similar to that described above for the first device. The production chip includes a main channel (width=1200 μm, length=8 mm), a culturing chamber (diameter=3 mm), and three trapping channels (width=90 μm, length=4.5 mm). The three trapping channels merge into a single output, connected, by a Teflon tube, to the second chip (a purification chip). The purification chip includes repetitive serpentine channels (width=1 mm, length=17 mm, diameter out=1.75 mm, diameter in=0.75 mm). The PDMS device was glued to a Cu-NTA (copper-nitrilotriacetic acid) coated glass-slide (MicroSurfaces Inc) for binding His-tagged proteins.
Culturing and Monitoring MSBs. Before loading microcapsules into the culturing chamber, the microfluidic device was vacuumed for 15 mins to prevent generation of air bubbles. Unless indicated otherwise, a programmable syringe pump (Harvard Apparatus, Pump 11 Elite) was used to control the input flow patterns. For periodic pulsing, the flow rate of each perfusion was set as 0.5 μL/min for 30 mins and then delayed (0 μL/min) 90 mins before the next pulse. The flow pattern for nutrients and substrate (10 μM Fluorocillin in PBS) is synchronized. For binding of the crude into the purification chip, the flow rate was set as 0.5 μL/min. The same flow rate was used for subsequent washing (PBS) and elution steps (100 mM EDTA). The microfluidic device was installed in the microscopy hood and cultured at 37° C. An inverted fluorescence microscope (Nikon Eclipse Ti) with 10× objective was employed to monitor the dynamics of capsule size and the fluorescence signal generated by the enzymatic reactions. The fluorescence signal intensity was quantified by averaging the gray level of whole assay chamber using ImageJ.
Protein Purification. For His-tag affinity purification, cOmplete™ His-Tag Purification Column (Sigma-Aldrich) was used for on-bench continuous purification using the protocol suggested by Sigma-Aldrich. cOmplete™ His-Tag Purification Column was used due to its minimized nickel ion leakage, which can be important for therapeutic related protein purification.
For purification of ELPs-tagged proteins by microfiltration, 10 mL crude from a MSB culture was first filtrated with a 0.45 μm HT Tuffryn® Membrane and then mixed with the exogenous ELPs (5 μM) and NaCl (2 M) to trigger the LCST phase transition. The mixture containing the aggregates (consisting of the trigger ELPs and ELPs-tagged target protein) was loaded and filtrated through a syringe coupled with a 0.2 μm HT Tuffryn® Membrane. To wash the aggregates deposited onto the filter, 5 mL of 2 M NaCl was injected through the filter to remove proteins in the solution while retaining the aggregates. To elute the aggregation, 1 ml of D.I. water was passed through the filter, causing re-solubilization of the retained aggregates. The eluted solution was collected for the next step His-tag purification. For all protein samples, SDS-PAGE, using Coomassie Blue stain, was used to verify the purity of the protein-of-interest.
Production of ELPs/RLPs tagged proteins, therapeutic proteins, and other proteins using MSB. For on bench production of ELPs/RLPs tagged, therapeutic and other proteins, 375 μL of overnight culture of cells carrying both the lysis circuit and protein expression circuit were centrifuged and re-suspended in 37.5 μL LB medium, and encapsulated into 375 μL chitosan solution for the MSB culture. The capsules were loaded into 20 mL syringe supplemented with 10 mL LB medium with appropriate antibiotics, and cultured in 37° C. for 24 hours. A 0.45 μm HT Tuffryn® Membrane with low protein binding property was attached at the tip of the syringe to avoid leaking and evaporation of the culture medium. The concentration of purified protein was measured by absorbance of 280 nm by using Nanodrop Spectrophotometer ND-1000.
Long term monitoring of MSB platform performance. To evaluate long-term performance of MSBs, the MSB was first made using the same protocol described above. Briefly, 375 μL of overnight culture of cells BL21(DE3) (ePop/ELPs-GFP-His) were centrifuged and re-suspended in 37.5 μL LB medium, and encapsulated into 375 μL chitosan solution for the MSB culture. The MSB was centrifuge at 7000 rpm for 5 mins and then stored at either 4° C. or −80° C. After various time windows, the MSB was taken out and then recovered with 3 mL LB medium and cultured at 37° C. for 48 hours. The amount of purified protein was quantified by measuring absorbance at 280 nm. All the data is normalized with the amount of protein produced using MSBs made fresh and cultured with 3 mL LB at 37° C. for 48 hours. The experiments are done in triplicate.
Modification on the surface of the capsules by layer-by-layer coating technique. About 375 μL of overnight culture of cells (MC4100Z1 (ePop/BlaM)) were centrifuged and re-suspended in 37.5 μL LB medium, and encapsulated into 375 μL chitosan solution. The capsules were washed first with PBS, and then incubated in 1.5 mL alginate solution (0.1%/(w/v)) for 15 mins with gentle shaking. The resultant capsules were centrifuged, collected, washed again with PBS and then incubated in 1.5 mL chitosan solution (0.4%/(w/v)) with gentle shaking for another 15 mins. The resultant capsules were washed again with PBS and collected.
To calculate the percentage of escape cells and compare the yield, equal amount of normal and surface modified capsules were inoculated into 3 mL LB with appropriate antibiosis and 1 mM IPTG, and cultured in 37° C. for 24 hours. To calculate the percentage of escape cells, the CFU in the surrounding medium is counted for both cases (normal and modified) in MSB culture, and divided by the CFU of bulk culture condition. To compare the yield, 20 μL supernatant was diluted into 200 μL using PBS and mixed with Fluorocillin™ Green (Thermo Fisher) to attain a final substrate concentration of 2 μM. Upon cleavage by BlaM, Fluorocillin™ Green reagent can be converted to a fluorescent product. The resulting fluorescence was measured using a plate reader (Tecan infinite M200 pro). Measurements were done in triplicate and background corrected.
Mathematical Derivation. The population is defined as N, which stands for the density of the population. The unit for the density is defined as cells·mL−1. Here, the total number of population would be N·V, where Vis the volume of the culturing environment. Because spatial segregation by encapsulation provides smaller volume for each population, the total number of cells Nt for each swarmbot capsule would be smaller than that of the bulk liquid culturing environment. The scale for the total cell number is assumed as below.
MSB capsule: Nt∈[0, 103]
Liquid culture: Nt∈[0, 109]
Then, how segregation increases the genetic stability against rise in number of mutants can be mathematically derived. First, λ is the average number of mutants in a population calculated by frequency of mutants, ρ, and the change in density defined with growth rate, G, cell density, N, and volume, V.
λ=ρVGNΔt
Following the equation for Poisson distribution, we can derive the following. Probability of no mutants with Poisson distribution:
Probability of no mutants for MSB capsule (The subscript S stands for small volume).
Defining.
Probability of no mutants for bulk liquid culture (The subscript L stands for large volume).
P(O)L=αV
Thus, the benefit of MSB platform defined as η can be written as
Defining
m=αV
Then
The derivative of η(m)
The function is monotonic decaying.
η(1)=1
Therefore,
η(m)>1
Especially, the benefit increases with the degree of segregation degree increases (m increases).
Model equations and justifications. A kinetic model was developed to examine how spatial segregation (via encapsulation of bacteria in microcapsules) modulates the genetic stability of the ePop circuit and the yield of a protein-of-interest. The model comprises a set of algebraic equations and ordinary differential equations that describe the temporal dynamics of five components: wild-type cell density (N), nutrient concentration (S), E protein (E), protein-of-interest (P) and mutant density (M).
Model equations for one compartment (e.g., the bulk culture). Growth rate (G) is modeled by the Monod equation that accounts for availability of nutrients. Also, it includes a logistic term that accounts for the carrying capacity of the system. The carrying capacity can be modulated depending on the environment (MSB versus culturing chamber):
Lysis rate (L) is modeled as depending on the E protein concentration following a Hill function, with a Hill exponent (p):
Cell density changes with respect to growth and lysis rates. Decrease in density due to mutant generation is assumed to be negligible:
Nutrient level changes by consumption or recovery from cells upon lysis:
The rate of E protein synthesis is assumed to be proportional to the cell density. The degradation of E protein is assumed to follow a first-order kinetics. The effective degradation rate constant accounts for a basal rate constant γ2 and dilution due to cell growth. A weighing factor r, modulates the decay rate of E protein:
The rate of release of a protein-of-interest (POI) into the environment is proportional to the lysis rate (for simplicity, it was assumed that mutant cells do not lyse or contribute to the production of the POI):
After generation (see below), the mutant population is assumed to grow following the same dynamics as the wild type but do not undergo lysis:
Generation of a mutant cell is implemented by the following logic. First, it was assumed that the average number of potential mutants arising from a population is proportional to the increase in cell density for a certain time window (i.e., mutants only arise if there is active cell growth): λ=α2GNVlΔt, where Vl is a conversion factor to convert density N into total number of cells in the specified environment. Whether a mutant actually arises is assumed to follow a Poisson distribution, where the probability of no mutant is P(O)=e−λ. That is, the probability of maintaining mutant-free decreases exponentially with λ, which increases with the population size, the effective growth rate, and the time window of interest (Δt).
In a numerical simulation, this scenario described above is implemented by generating a random number between 0 and 1 during each time step. If the random number is <P(O), a mutant is added in the simulation with conversion factor of Vl. This conversion factor is used to convert the number of mutants into cell density. This number sets the initial condition for the mutant population thereafter. Mutant generation is assumed to occur only once.
Two compartment model for the MSB dynamics. The equations are expanded and applied to describe the dynamics in two compartments, by accounting for transport between the compartments.
Inside the MSB (index=1).
Outside the MSB (index=2).
A unidirectional transport of cells from MSB capsule to the environment was assumed. The equations are developed for simulating both static and periodic dosing of fresh medium into the MSB platform assuming a microfluidic set-up. Is indicates concentration of nutrients in the fresh media. P(t) is a function of pulse duration, and period.
It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present disclosure described herein are readily applicable and appreciable, and may be made using suitable equivalents without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein. Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are merely intended only to illustrate some aspects and embodiments of the disclosure, and should not be viewed as limiting to the scope of the disclosure. The disclosures of all journal references, U.S. patents, and publications referred to herein are hereby incorporated by reference in their entireties.
The present disclosure has multiple aspects, illustrated by the following non-limiting examples.
Two basic modules, autonomous lysis cells and growth sensitive capsules, can be used to integrate function of the systems of the present disclosure, as shown in
The polysaccharide chitosan was used as the encapsulating material due to its biocompatibility and stimulus responsiveness. The resulting three-dimensional cross-linked networks inside the polymeric capsules trapped the living cells and large debris but allowed transport of small molecules and proteins. At low pH, protonation of the amine groups extends the chitosan chains due to electrostatic repulsion between the charged groups, therefore swelling the network. In contrast, a high ionic strength shields the electric charges, leading to shrinking of the network due to collapse of the polymer chains.
Bacterial growth affected both the local pH and ionic strength, which can then cause phase transition in the chitosan network. Therefore, the change in size of the capsule can both influence and provide a visual readout for the dynamics of encapsulated bacterial. Indeed, when the engineered bacteria (MC4100Z1 (ePop/GFP)) encapsulated capsules were cultured in M9 medium, their growth led to shrinking of the chitosan network (
The phase transition of chitosan capsule is a general property of the changed microenvironment due to bacterial growth with or without the ePop circuit (
The density-dependent lysis programmed by ePop circuit is a functional aspect of the systems disclosed herein, as it functionally couples protein production and release. Yet, the programmed lysis creates a selection pressure for mutants that can escape killing (
To demonstrate integrated protein synthesis and analysis, beta-lactamase was used as an exemplary protein. Briefly, the ePop bacteria was transformed with a plasmid encoding a modified beta-lactamase (BlaM) gene under an inducible promoter (MC4100Z1(ePop/BlaM)). In comparison with the wild type Bla, BlaM does not contain the periplasm-localization sequence. As a result, it is cytoplasmic and is functional only when the producing cell is lysed (
Fresh medium was periodically added (
The culture chamber containing MSBs can be integrated with a purification module without compromising continuity of operation. As an illustration, another exemplary protein was used, ELPs-GFP-His, which includes a His-tag and an elastin-like polypeptides (ELPs) tag (BL21(DE3) (ePop/ELPs-GFP-His)). His-tag allows purification by immobilized metal affinity chromatography (IMAC). ELPs are repetitive artificial polypeptides that undergo lower critical solution temperature phase transition behavior (
To demonstrate an online affinity-based purification, a purification chip was fabricated containing a serpentine channel. In this chip, the surface of the glass slide is modified with PEG-coated chelated Cu2+. As the extruded lysate is transported from the production chamber to the purification chip (
The MSB systems of the present disclosure are modular and scalable. On a larger scale, the protein production and release can be carried out on the bench top using a syringe loaded with MSBs. In particular, a 10-mL syringe was used as the production chamber and a syringe filter was attached to it. About 10,000 MSBs (containing a total of about 107 bacteria) were loaded in the syringe and incubated in LB medium at 37° C. for 24 hours. The syringe was then connected, together with the syringe filter, to a Ni-NTA column (
Protein production of ELPs-GFP-His and RLPs-GFP-His in the single-dose production using a syringe in 24 hours. The total culture volume was 10 mL LB medium. The yield was calculated by the total production divided by the total nutrients. Experiments were done in triplicate.
Protein production of ELPs-GFP-His by different dosing frequency with the same total nutrients. The total nutrients included 3 mL LB medium. The nutrients are dispensed in a periodic dosing way. For the sample 3 mL×1, 3 mL LB medium was supplemented at time 0 and collected on 72 hours. For the sample 1.5 mL×2, 1.5 mL LB medium was supplemented at time 0 and collected on 36 hours; and another 1.5 mL was supplemented at time 36 and collected on 72 hours. Experiments were done in triplicate
The yield was further improved when the same amount of LB medium was supplied in multiple batches (
ELPs and ELPs-tagged proteins can undergo LCST phase transition in an aqueous solution, leading to formation of micron-sized aggregates. This property is often exploited for protein purification by using inverse transition cycling, which involves multiple cycles of temperature shift and centrifugation (
Combining the LCST phase behavior of ELPs-tagged protein with affinity His-tag provides two orthogonal methods, but highly complementary for protein purification, which enables the isolation of proteins with higher purity than is possible by either method alone. Moreover, these methods enable separation and purification of multiple proteins from the same crude, increasing the throughput of the purification. To demonstrate, the crude from two MSB cultures containing ELPs-GFP-His and another His-tagged protein (His-AAA, in which A represents a SpyTag peptide) were mixed. The use of His-tag purification alone did not separate them. However, by first going through a round of ELPs-based purification (induced aggregation+microfiltration), ELPs-GFP-His (as aggregates) was retained in the membrane while the His-AAA was in the solution. The two proteins were then separately purified by IMAC (
The MSB biomolecule production platform of the present disclosure enables the production and analysis or separation of any protein that can be expressed at a sufficiently high level. The results provided herein have demonstrated the production of a wide range of proteins, including multiple ELPs (some of them are fused with spytag and spycatcher), RLPs, ligand-binding protein, pigment protein and enzyme (
Also, this approach was extended to express multiple therapeutic biologics, including glucagon-like peptide 1 (GLP-1), that is used to treat type-2 diabetes, and griffithsin, a potent inhibitor of HIV and Zika infections (
It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure, which is defined solely by the appended claims and their equivalents. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the disclosure, may be made without departing from the spirit and scope thereof.
This application is a national phase application under 35 U.S.C. § 371 of PCT International Application No. PCT/US2018/037111, filed Jun. 12, 2018, which claims priority to U.S. Provisional Application No. 62/518,075, filed Jun. 12, 2017, each of which are hereby incorporated by reference in its entirety.
This invention was made with Government support under Federal Grant Nos. R 01 GM098642 and CBET-0953202 awarded by the NIH and NSF, respectively. The Federal Government has certain rights to the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/037111 | 6/12/2018 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/231834 | 12/20/2018 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 4554101 | Hopp | Nov 1985 | A |
| 4801529 | Perlman | Jan 1989 | A |
| 20090018195 | Balagadde et al. | Jan 2009 | A1 |
| 20090226984 | Nonaka et al. | Sep 2009 | A1 |
| 20110189743 | Yoshikuni et al. | Aug 2011 | A1 |
| 20140287489 | Lee et al. | Sep 2014 | A1 |
| 20150026840 | Kerfeld et al. | Jan 2015 | A1 |
| 20160015781 | Olschlager et al. | Jan 2016 | A1 |
| Number | Date | Country |
|---|---|---|
| WO 2000065030 | Nov 2000 | WO |
| WO 2018017845 | Jan 2018 | WO |
| Entry |
|---|
| Papi et al., “Encapsulated Escherichia coli in alginate beads capable of secreting a heterologous pectin lyase”, Microbial Cell Factories, 2005, 4:35, pp. 1-8. doi:10.1186/1475-2859-4-35. |
| International Search Report and Written Opinion for PCT/US18/37111. dated Sep. 11, 2018. 14 pages. |
| Bradley et al., Tools and Principles for Microbial Gene Circuit Engineering. J Mol Biol. Feb. 27, 2016;428(5 Pt B):862-88. |
| Kyte et al., A simple method for displaying the hydropathic character of a protein. J Mol Biol. May 5, 1982;157(1):105-32. |
| You et al., Programmed population control by cell-cell communication and regulated killing. Nature. Apr. 22, 2004;428(6985):868-71. |
| Balagaddé, et al., Long-term monitoring of bacteria undergoing programmed population control in a microchemostat. Science. Jul. 1, 2005;309(5731):137-40. |
| Carnes et al., Confinement-induced quorum sensing of individual Staphylococcus aureus bacteria. Nat Chem Biol. Jan. 2010;6(1):41-5. |
| Chang, Therapeutic applications of polymeric artificial cells. Nat Rev Drug Discov. Mar. 2005;4(3):221-35. |
| Dai et al., Microgel particles: The structure-property relationships and their biomedical applications. Journal of Polymer Science Part a-Polymer Chemistry 2013: 51, 2995-3003. |
| Din et al., Synchronized cycles of bacterial lysis for in vivo delivery. Nature. Aug. 4, 2016;536(7614):81-85. |
| Ebara et al., Smart Biomaterials. Springer, 2014. TOC only. 10 pages. |
| Ford et al., Synthetic biology expands chemical control of microorganisms. Curr Opin Chern Biol. Oct. 2015;28:20-8. |
| Ganji et al., Hydrogels in Controlled Drug Delivery Systems. Iranian Polymer Journal. 2009: 18, 63-88. |
| Ge et al., Purification of an elastin-like fusion protein by microfiltration. Biotechnol Bioeng. Oct. 20, 2006;95(3):424-32. |
| Gu et al., Glucose-responsive microgels integrated with enzyme nanocapsules for closed-loop insulin delivery. ACS Nano. Aug. 27, 2013;7(8):6758-66. |
| Hassouneh et al., Elastin-like polypeptides as a purification tag for recombinant proteins. Curr Protoc Protein Sci. Aug. 2010;Chapter 6:Unit6.11. 20 pages. |
| Hassouneh et al., Fusions of elastin-like polypeptides to pharmaceutical proteins. Methods Enzymol. 2012;502:215-37. |
| Hoffman, Hydrogels for biomedical applications. Adv Drug Deliv Rev. Jan. 17, 2002;54(1):3-12. |
| Huang et al., Coupling spatial segregation with synthetic circuits to control bacterial survival. Mol Syst Biol. Feb. 29, 2016;12(2):859. |
| Ihssen et al., Production of glycoprotein vaccines in Escherichia coli. Microb Cell Fact. Aug. 11, 2010 ;9:61. 13 pages. |
| Ionov, Hydrogel-based actuators: possibilities and limitations. Materials Today. 2014: 17, 494-503. |
| Khalil et al., Synthetic biology: applications come of age. Nat Rev Genet. May 2010;11(5):367-79. |
| Klouda et al., Thermoresponsive hydrogels in biomedical applications. Eur J Pharm Biopharm. Jan. 2008;68(1):34-45. |
| Kwon et al., High quality protein microarray using in situ protein purification. BMC Biotechnol. Aug. 23, 2009;9:72. |
| Lopez-Leon et al., Physicochemical characterization of chitosan nanoparticles: electrokinetic and stability behavior. J Colloid Interface Sci. Mar. 15, 2005;283(2):344-51. |
| Marguet et al., Oscillations by minimal bacterial suicide circuits reveal hidden facets of host-circuit physiology. PLoS One. Jul. 30, 2010;5(7):e11909. 11 pages. |
| Meyer et al., Purification of recombinant proteins by fusion with thermally-responsive polypeptides. Nat Biotechnol. Nov. 1999;17(11):1112-5. |
| Meyer et al., Quantification of the effects of chain length and concentration on the thermal behavior of elastin-like polypeptides. Biomacromolecules. May-Jun. 2004;5(3):846-51. |
| Naderi et al., Review paper: critical issues in tissue engineering: biomaterials, cell sources, angiogenesis, and drug delivery systems. J Biomater Appl. Nov. 2011;26(4):383-417. |
| Nguyen et al., Injectable biodegradable hydrogels. Macromol Biosci. Jun. 11, 2010;10(6):563-79. |
| Oh et al., The development of microgels/nanogels for drug delivery applications. Progress in Polymer Science 2008: 33, 448-477. |
| Osada et al., A polymer gel with electrically driven motility. Nature, 1992; 355, 242-244. |
| Pai et al., Optimal tuning of bacterial sensing potential. Mol Syst Biol. 2009;5:286. 11 pages. |
| Pardee et al., Portable, On-Demand Biomolecular Manufacturing. Cell. Sep. 22, 2016;167(1):248-259.e12. |
| Perez-Pinera et al., Synthetic biology and microbioreactor platforms for programmable production of biologies at the point-of-care. Nat Commun. Jul. 29, 2016;7:12211. 10 pages. |
| Santi et al., Biological nitrogen fixation in non-legume plants. Ann Bot. May 2013;111(5):743-67. |
| Slomovic et al., Synthetic biology devices for in vitro and in vivo diagnostics. Proc Natl Acad Sci USA. Nov. 24, 2015;112(47):14429-35. |
| Tanouchi et al., Programming stress-induced altruistic death in engineered bacteria. Mol Syst Biol. 2012;8:626. |
| Trongsatitkul et al., Temperature dependence of serum protein adsorption in PEGylated PNIPAm microgels. Colloids Surf B Biointerfaces. Mar. 1, 2013;103:244-52. |
| Vinogradov, Colloidal microgels in drug delivery applications. Curr Pharm Des. 2006;12(36):4703-12. |
| Bhatla, N. et al., “Light and hydrogen peroxide inhibit C. elegans feeding through gustatory receptor orthologs and pharyngeal neurons.” Neuron 85.4 (2015): 804-818. |
| Edwards S.L. et al., “A novel molecular solution for ultraviolet light detection in Caenorhabditis elegans.” PLoS biology 6.8 (2008): e198. |
| Huang, S. et al., “Dynamic control and quantification of bacterial population dynamics in droplets.” Biomaterials 61 (2015): 239-245. |
| Yu, W. et al., “Study on membrane characteristics of alginate-chitosan microcapsule with cell growth.” Journal of Membrane Science 377.1-2 (2011): 214-220. |
| Number | Date | Country | |
|---|---|---|---|
| 20200109393 A1 | Apr 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62518075 | Jun 2017 | US |
