Compositions and methods comprising a synthetic cell generating ATP and / or nadh

Abstract

Provided herein are compositions and methods comprising a synthetic cell that generates adenosine triphosphate (ATP) and / or nicotinamide adenine dinucleotide (NADH). In particular, the present invention relates to the compositions and methods comprising synthetic cells that generate ATP and / or NADH from the uptake of glucose.

Description

[0001] COMPOSITIONS AND METHODS COMPRISING A SYNTHETIC CELL GENERATING ATP AND / OR NADH

[0002] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under HL170510 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0004] FIELD

[0005] Provided herein are compositions and methods comprising a synthetic cell that generates adenosine triphosphate (ATP) and / or nicotinamide adenine dinucleotide (NADH). In particular, the present invention relates to the compositions and methods comprising synthetic cells that generate ATP and / or NADH from the uptake of glucose.

[0006] BACKGROUND

[0007] ATP, or adenosine triphosphate, serves as the fundamental "energy currency" of the cell, playing a pivotal role in cellular energy metabolism. This energy-rich molecule drives a multitude of essential biochemical processes within cells, including muscle contraction, active transport of molecules across cell membranes, and DNA synthesis. Consequently, ATP is an indispensable resource for the survival and proper functioning of cells, tissues, and organisms.

[0008] The absence or insufficiency of ATP in cells can severely impair cellular function and lead to various physiological consequences, such as muscle weakness, fatigue, and reduced exercise tolerance, highlighting its critical role in preserving cellular function. The importance of sustaining ATP production and function is underscored by the compl exity of ATP synthesis, involving intricate biochemical pathways, and coordinated enzymatic reactions, emphasizing the necessity of precise regulation and sufficient availability of substrates and cofactors. Disruptions in ATP production can hinder critical cellular processes, compromising overall cellular function and metabolism. Moreover, severe ATP depletion can trigger cellular distress signals and ultimately lead to programmed cell death, contributing to tissue damage and organ dysfunction. Thus, maintaining adequate levels of ATP is imperative for sustaining cellular homeostasis and preserving organi smal health amidst the intricate and interconnected pathways involved in ATP production and utilization.

[0009] In view of the importance of ATP, the current medical interventions available for situations where ATP production is impaired or insufficient are limited. In cases of ATP deficiency, treatment strategies often involve supportive therapy, which may include nutritional management, exercise regimens, and supplementation with vitamins or amino acids. However, while these interventions aim to bolster ATP production indirectly, there is no guarantee of efficacy due to the complexity of the ATP synthesis. Additionally, these therapies are known to be encumbered with a number of deleterious side effects. These limitations have a profound impact on the efficacy of treatment and overall patient health. To date, there are no medical interventions that can independently produce ATP with the surrounding by-products in the environment.

[0010] Emerging research suggests that building more complex synthetic cells from the bottom up could offer a promising solution as synthetic cells can hold a variety of functional modules. Nevertheless, while synthetic cells hold great potential across various applications, they are currently limited by factors such as short lifetimes, resource availability for ATP production, functional complexity, and integration with living cells. Hence, there is a pressing need for the development of synthetic cells capable of sustaining ATP production with a minimal design that can seamlessly integrate with living cells, offering new avenues for therapeutic interventions, and advancing the understanding of cellular and synthetic biology.

[0011] SUMMARY

[0012] Provided herein are compositions and methods comprising a synthetic cell that generates adenosine triphosphate (ATP) and / or nicotinamide adenine dinucleotide (NADH). In particular, the present invention relates to the compositions and methods comprising synthetic cells that generate ATP and / or NADH from the uptake of glucose.

[0013] In some embodiments, the present disclosure provides a synthetic cell comprising an outer structure, wherein the outer structure comprises at least one glucose transporter, and an internal structure, wherein the internal structure is contained within the outer structure, the internal structure comprising at least one glucose transporter, a glucose oxidase (GOx), a catalase (CAT) and an adenosine triphosphate (ATP) synthase.

[0014] In some embodiments, the outer structure and / or internal structure comprise a dioleoyl- sn-glycero-3-phosphocholine / polybutadiene-poly ethylene oxide (DOPC / PBD-PEO) hybrid vesicle.

[0015] In some embodiments, the synthetic cell is in vivo. In some embodiments, synthetic cell is in vitro.

[0016] Embodiments of the present disclosure also include a method comprising uptaking a glucose from a surrounding environment through a glucose transporter into the outer structure of a synthetic cell; converting the glucose into gluconic acid via the glucose oxidase in an internal structure of the synthetic cell; and producing hydrogen peroxide. In some embodiments, the glucose is produced through enzymatic conversion. In some embodiments, the glucose is from blood.

[0017] In some embodiments, ADP is converted to an ATP with the ATP synthase contained in the internal structure.

[0018] In some embodiments, the synthetic cell is supplemented with a hexokinase and a glucose-6-PO4 dehydrogenase.

[0019] In some embodiments, NADH is generated using said ATP.

[0020] In some embodiments, the synthetic cell is used for diagnostic research and / or testing. In some embodiments, the synthetic cell is used for basic research applications. In some embodiments, the synthetic cell is used for therapeutic research and / or testing. In other embodiments, the synthetic cell is used for diagnostic research and / or testing as an in vivo diagnostic tool. In further embodiments, the synthetic cell is used for basic research application by integrating the synthetic cell with a natural living cell.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1: Western blot with anti -His tag antibody labeling GLUTl-Hise.

[0023] FIG. 2A-B: Vesicle generation by hydration followed by freeze-thaw. (A) A representative image of DOPC / PBD-PEO 50 / 50 hybrid vesicles after the vesicles were rehydrated with an encapsulating solution and vortex and (B) a representative image of DOPC / PBD-PEO 50 / 50 hybrid vesicles after the vesicles were generated by freeze-thaw cycles (to breakdown multilamellar structures).

[0024] FIG. 3A-B: Assay for detecting pH change using 8-Hydroxypyrene-l,3,6-trisulfonic acid (HPTS). (A) A representative image of DOPC / PBD-PEO 50 / 50 hybrid vesicles and (B) 100 pM of HPTS, 100 pg / ml glucose oxidase, and 100 pg / ml catalase in 100 mM NaCl in different pH solutions as indicated are encapsulated in DOPC / PBD-PEO GUVs (labeled with trace amount of rhodamine-PE). Emission with Ex488 increases as Ex405 decreases with increasing pH.

[0025] FIG. 4A-B: Generation of pH drop to activate ATP synthase for ATP production. (A) A graphical representation of pH titrations of the relative fluorescence intensities of HPTS at 514 nm, excited by 406 nm (Boe) and 460 nm (I460), with 1 pM HPTS in 10 mM HEPES and 50 mM KC1 at pH levels 4, 5, 6, 7, 7.4, and 8 (B) decrease in pH using 100 pg / ml glucose oxidase, 50 pg / ml catalase, and varying glucose concentrations (0, 10, 50, 100 mM) with 1 pM HPTS in 10 mM HEPES and 50 mM KC1 at initial pH 7.4. pH was determined using the standard curve shown in (A). FIG. 5A-B: Couple ATP-generation to polymerization of actin filaments in synthetic cells. (A) A GUV encapsulating F-actin labeled with fluorescent phalloidin (B) synthesized ATP that drives the polymerization of G-actin (globular-actin) to F-actin (fibrous-actin).

[0026] FIG. 6: Exemplary design of ATP- and NADH-generating synthetic cells. The exemplary design shows a synthetic cell that uptakes glucose through glucose transporters (GLUT) and converts it to gluconic acid using glucose oxidase (GOx) and catalase (CAT). This process activates the ATP synthase using a proton gradient to generate ATP. Additionally, the synthetic cell can utilize the circulating glucose and the generated ATP to produce NADH by employing hexokinase and glucose-6-PO4 dehydrogenase enzymes.

[0027] FIG. 7A-B: Synthetic organelle that generates ATP from glucose uptake. (A) Synthetic organelle uptakes glucose through glucose transporter (GLUT), converts glucose to gluconic acid using glucose oxidase (GOx) and catalase (CAT), and activates ATP synthase using proton gradient to generate ATP. (B) Encapsulation of ATP-generating organelle into GLUT- containing synthetic cell enables the synthetic cell to carry out various cellular activities that require ATP. Moreover, electron carrier metabolite NADH can also be generated by adding two different enzymes, hexokinase and glucose-6-PO4 dehydrogenase.

[0028] FIG. 8: Exemplary design of ATP-generating synthetic organelle. The exemplary design shows a synthetic organelle that utilizes glucose transporters (GLUT) to uptake glucose, which is then converted to gluconic acid through the use of glucose oxidase (GOx) and catalase (CAT), resulting in the production of gluconic acid and hydrogen ions (H+). The circulating H+ ions energize the ATP synthase, catalyzing the conversion of ADP (adenosine diphosphate) to ATP (adenosine triphosphate). To visualize the ATP synthesis, luciferin, a light-emitting molecule, is employed, producing light upon ATP generation.

[0029] FIG. 9A-D: Traction force assay for quantifying ATP-driven actomyosin contraction. (A) Reconstitution of actomyosin network on 2D elastic substrate with biotinylated F-actin and neutravidin-coated fluorescent beads. (B) The traction stress field determined from bead displacements at 3 different times after the start of imaging: 4 min (left, right before adding 0.15 pM myosin), 9 min (right before adding an additional 2.2 mM ATP), and 11 min. (C) Time evolution of mean traction stress over time (red and black traces are two independent experiments). (D) Divergence of bead displacement field in the region at the two time points (before and after addition of myosin) are shown.

[0030] FIG. 10: Time course images of cell-free expression of GFP in HeLa lysate in a DOPC / cholesterol vesicle. DETAILED DESCRIPTION

[0031] Provided herein are compositions and methods comprising a synthetic cell that generates adenosine triphosphate (ATP) and / or nicotinamide adenine dinucleotide (NADH). In particular, the present invention relates to the compositions and methods comprising synthetic cells that generate ATP and / or NADH from the uptake of glucose.

[0032] Existing ATP -generating methods have yet to achieve a methodology that emulates a minimalist approach to ATP generation, one that seamlessly integrates with the surrounding living environment and utilizes sugars available in the environment creating an alternative synthetic pathway. Consequently, the ability to select the most suitable method for each scenario or explore alternative strategies to overcome challenges becomes paramount. The development of novel synthetic pathways, capable of sustaining and producing ATP and / or NADH, represents an important approach to address current limitations of energy production in living and synthetic organisms. Harnessing the ability to combine diverse pathways or enzymes sourced from various organisms stands as a significant advantage of synthetic cell platforms. Ultimately, there is a need in the field for the creation of a sustainable, multifunctional synthetic cell capable of meeting diverse biological needs for research, diagnostic, and therapeutics applications.

[0033] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.

[0034] 1. DEFINITIONS

[0035] 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. The phrase “in one embodiment” as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention. 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.

[0036] 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.

[0037] 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. 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.

[0038] As used herein, the term "deconstruct" refers to the process of breaking down vesicles or vesicle-like structures into their constituent components or functional units. This process may involve disassembling the vesicle membrane and releasing its contents, such as proteins, lipids, nucleic acids, or other biomolecules, for further analysis, manipulation, or application

[0039] As used herein, "in vivo" refers to processes, experiments, or phenomena that occur within a living organism (e.g., a human, animal, or plant) in its natural environment or within its tissues or organs. In further embodiments, the context of this patent application, "in vivo" may encompass various applications and methodologies, including but not limited to studying the effects of pharmaceutical compounds or therapeutic interventions within living organisms, evaluating biological responses to external stimuli or treatments, and assessing the efficacy of medical devices or procedures in vivo. Additionally, "in vivo" investigations may involve a wide range of biological models, including animal models such as rodents, primates, and other vertebrates, as well as cellular or tissue-based systems designed to mimic physiological conditions within living organisms.

[0040] As used herein, the terms "synthetic cell" or “synthetic organelle” refers to an artificially engineered cell or organelle created through synthetic biology techniques. Such synthetic cells may incorporate non-genetic messages and modifications to mimic or enhance natural cellular functions for various purposes. Examples of applications include, but are not limited to, the development of recombinant organisms, cell lines, or strains for biotechnological, biomedical, agricultural, or industrial uses. Synthetic cells may also encompass modified viruses or strains designed for specific applications, as well as synthetic organisms tailored to perform specialized functions in research, medicine, or other fields of science and technology. Synthetic cells may comprise natural cells modified to include a synthetic organelle or other synthetic structure.

[0041] As used herein a “hybrid vesicle” is the assembly of two or more vesicles (e.g., DOPC and PBD-PEO). In addition, hybrid vesicles include but are not limited to, hydrogels (polymeric nanogels or macromolecular micelles), materials made of complex compositions (metals, oxides, chalcogenides, etc.) and coordination polymers.

[0042] Exemplary methods and materials are described below. 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.

[0043] 2. SYNTHETIC CELLS

[0044] Provided herein are compositions and methods comprising a synthetic cell that generates adenosine triphosphate (ATP) and / or nicotinamide adenine dinucleotide (NADH). In particular, the present invention relates to the compositions and methods comprising synthetic cells that generate ATP and / or NADH from the uptake of glucose.

[0045] Synthetic cells generally comprise membrane-bound micro-compartments that mimic, replace, or enhance one or more functions of natural cells. Synthetic cells may comprise giant vesicles with a single-layer boundary, giant unilamellar vesicles, proteinosomes, giant capsules with a multi-layer boundary, membrane-less microcompartments (e.g., coacervates), and / or nanoassemblies that mimic natural organelles (e.g., polymersomes, micelles, photosynthetic organelles, polymer nanoparticles, nano-sized hydrogels, clustered nano-assemblies, etc.). The synthetic structures can be manufactured by any suitable approach, including, but not limited to, self-assembly of amphiphilic copolymers or mixtures of lipids, proteins, or peptides. In some embodiments, the synthetic cells comprise one or more compartments within another compartment. The present disclosure provides a synthetic cell comprising an outer structure, wherein the outer structure comprises at least one glucose transporter, and an internal structure, wherein the internal structure is contained within the outer structure, the internal structure comprising at least one glucose transporter, a glucose oxidase (GOx), a catalase (CAT) and an adenosine triphosphate (ATP) synthase.

[0046] In some embodiments, the types of glucose transporter include, but are not limited to, GLUT1 (SLC2A1), GLUT2 (SLC2A2), GLUT3 (SLC2A3), GLUT4 (SLC2A4), GLUT5 (SLC2A5), GLUT6 (SLC2A6), GLUT7 (SLC2A7), GLUT8 (SLC2A8), GLUT9 (SLC2A9), GLUT10 (SLC2A10), GLUT11 (SLC2A11), GLUT12 (SLC2A12), as well as SGLT (Sodium- Glucose Transporter), for example, SGLT 1 (Sodium-Glucose Transporterl), SGLT 2 (Sodium-Glucose Transported), SGLT 3 (Sodium-Glucose Transporters), SGLT 4 (Sodium- Glucose Transported), SGLT 5 (Sodium-Glucose Transporters), and the like. In some embodiments, the glucose transporters can uptake substances including but not limited to, glucose, fructose, galactose, mannose, maltose, sorbitol, xylose, myoinositol, 3-0- methylglucose, 2-deoxyglucose, 2-deoxyfructose, glucosamine, mannitol, fucose, arabinose, ribose, trehalose, rhamnose, glucuronic acid, lactose, sucrose, dehydroascorbic acid, thiamine and the like. In some embodiments, the glucose transporters can be naturally occurring or synthetically produced.

[0047] In various embodiments, the glucose oxidase (GOx) may encompass different enzyme types, such as glucose dehydrogenase (GDH), including variants like pyrroloquinoline quinone (PQQ) dependent GDH, flavin adenine dinucleotide (FAD) dependent GDH, or nicotinamide adenine dinucleotide (NAD) dependent GDH. Additionally, enzymes like pyrroloquinoline quinone glucose dehydrogenase (PQQGDH) or flavin adenine dinucleotide glucose dehydrogenase (FADGDH) could be utilized for glucose analysis, while lactate oxidase may serve this function for lactate analysis. For other analytes, alternative enzymes may be employed. These enzymes facilitate analyte electrolysis by transferring electrons between the analyte and the electrode through the redox mediator.

[0048] In various embodiments, catalase (CAT) may encompass different enzyme types, such as peroxidases, including variants like heme-containing peroxidases, manganese-containing peroxidases, or iron-containing peroxidases. For example, heme-containing catalase, manganese-containing catalase, iron-containing catalase, nickel-containing catalase, lignin peroxidase, manganese peroxidase, haloalkaline peroxidase, cytochrome c peroxidase, chloroperoxidase, pseudomonas catalase and the like could be utilized for specific catalase- related applications. For other reactions requiring catalytic decomposition of hydrogen peroxide, alternative enzymes may be employed. These enzymes facilitate the breakdown of hydrogen peroxide into water and oxygen by accelerating the reaction rate.

[0049] In further embodiments, the ATP synthase may involve different enzyme types, including but not limited to, those utilizing proton gradients for ATP production for example, thermophilic Bacillus PS3 F0F1-ATP synthase from E. coll DK8 strainl3,75 (TR19ASDSsAc). Such enzymes could include F-type ATP synthase, V-type ATP synthase, and A-type ATP synthase, each utilizing distinct mechanisms for ATP synthesis. Additionally, ATP synthase variants like mitochondrial ATP synthase or bacterial ATP synthase may be employed depending on the application. These enzymes harness proton gradients across membranes to catalyze the conversion of ADP to ATP, facilitating cellular energy production.

[0050] In some embodiments, the outer structure and / or internal structure comprise a dioleoyl- sn-glycero-3-phosphocholine / polybutadiene-poly ethylene oxide (DOPC / PBD-PEO) hybrid vesicle. In addition, any vesicle useful as an effective carrier system for delivering therapeutic payloads in a targeted and controlled manner and / or to enhance stability may be used. Vesicles include, but are not limited to, for example, unilamellar vesicles, multilamellar vesicles, giant unilamellar vesicles (GUVs), janus vesicles, polymer-lipid hybrid vesicles, pH-sensitive vesicles, temperature-sensitive vesicles, targeted vesicles and the like.

[0051] In some embodiments, the synthetic cell is in vivo. In some embodiments, synthetic cell is in vitro.

[0052] 3. METHODS OF USE

[0053] Embodiments of the present disclosure also include a method comprising uptaking a glucose from a surrounding environment through a glucose transporter into the outer structure of a synthetic cell; converting the glucose into gluconic acid via the glucose oxidase in the internal structure of the synthetic cell; and producing hydrogen peroxide.

[0054] In some embodiments, the glucose is produced through chemical synthesis. In some embodiments, glucose is produced through enzymatic conversion. In some embodiments, the glucose is from blood. In some embodiments, the glucose is d-glucose, 1-glucose, a-glucose, P-glucose, a glucose polymer and the like.

[0055] In some embodiments, ADP is converted ATP with an ATP synthase contained in the internal structure.

[0056] In some embodiments, the ATP is released into the outer structure.

[0057] In some embodiments, the synthetic cell is supplemented with a hexokinase and a glucose-6-PO4 dehydrogenase.

[0058] In some embodiments, NADH is generated using said ATP. In some embodiments, the synthetic cell is used for diagnostic research and / or testing. In some embodiments, the synthetic cell is used for basic research applications. In some embodiments, the synthetic cell is used for therapeutic research and / or testing. In other embodiments, the synthetic cell is used for diagnostic research and / or testing as an in vivo diagnostic tool. In further embodiments, the synthetic cell is used for basic research application by integrating the synthetic cell with a natural living cell.

[0059] 4. EXAMPLES

[0060] One of the key functions essential for all life forms is the ability to generate universal “energy currency,” which is adenosine triphosphate (ATP). ATP powers most cellular activities and biochemical reactions, including DNA / RNA / protein synthesis, cell division, signal transduction, and motility. Most bottom-up synthetic cell designs that require ATP start by supplementing ATP at the beginning when synthetic cells are made, and the desired function only lasts until ATP is used up. Thus, to successfully construct synthetic cells that can sustain out-of-equilibrium functions like in living cells, it is vital to develop a mechanism for ATP regeneration or synthetic organelles that allow synthetic cells to generate ATP on their own. The technology provided herein addresses this important and unmet challenge and provides minimal metabolism for generating ATP and / or nicotinamide adenine dinucleotide (NADH), another high-energy molecule, in synthetic cells from a basic energy source.

[0061] There have been major endeavors in recent years to build, from the bottom up, synthetic cells that replicate cellular behaviors such as motility, cell division, and cell communication, with the goal that these functional modules can be combined to construct synthetic cells that exhibit complex life-like functionality similar to living cells. Unlike engineering existing organisms, building synthetic cells from scratch allows researchers to have control over their functions and constituent materials. Thus, there is great potential to utilize custom-designed synthetic cells in applied science, such as in biosensing and drug delivery applications. Moreover, from a basic biology perspective, it serves as an experimental platform to understand and interrogate the origin of life.

[0062] Given the central role metabolism plays in the information-metabolism-compartment global picture of unicellular life, building minimal metabolism in the context of energy generation and coupling it to cellular activities such as in vitro transcription-translation and cytoskeletal network assembly provides a profound impact on synthetic cell research and development. Example 1

[0063] Expression and optimization of glucose transporter membrane reconstitution.

[0064] Glucose transporters (GLUT) are highly conserved amongst species. Although GLUT2 has been used in artificial beta cells, GLUT1 has a Km (the Michaelis constant) of 2 mM for glucose compared to 20 mM for GLUT272. An aspect of these experiments is that synthetic cells can operate in human bodies with a blood glucose concentration of ~5 mM. Therefore, using GLUT1 is selected over GLUT2 for ATP -generating synthetic organelle . Mouse (Mus musculus) GLUT1 is 492 amino acids long (~54 kDa) and can transport glucose only from high to low concentration. GLUT1 is cloned into pET28a expression vector with a C- terminal 6xHis tag and expressed in the Rosetta 2 E. coli strain. GLUT1 is purified in the presence of detergent DDM using affinity chromatography by using a Ni-NTA column to capture GLUTl-His. In a western blot, GLUT1 is purified (Fig. 1). Expression induction parameters (e.g., IPTG concentration, temperature and duration of induction, and using an autoinduction media) and expression strain (e.g., Rosetta 2, C41(DE3), and C43(DE3)) may be selected to provide desired expression levels.

[0065] Example 2 Optimization of ATP generation from synthetic ATP-generating organelle.

[0066] To reconstitute GLUT1 into an ATP-generating synthetic organelle, a method for generating vesicles via hydration of dried lipid film on glass beads followed by freeze-thaw is utilized (Fig. 2A-B). Hybrid vesicles of lipids and block copolymers (i.e., PBD-PEO) have been shown to significantly enhance robustness and their ability to successfully support cell- free expressed reconstitution of membrane channels. DOPC / PBD-PEO hybrid vesicles have been utilized for constructing ATP-generating synthetic organelles. DOPC / PBD-PEO vesicles encapsulating GOx and CAT stably maintained pH over a range of pH using an HPTS-based assay (Fig. 3A-B). 8-Hydroxypyrene-l,3,6-trisulfonic acid (HPTS) is used as a cell- impermeable fluorescent pH probe. HPTS exhibits a pH dependent absorption shift in 450 nm and 403 nm excitation, the intensities of which decrease and increase, respectively, as the pH increased from 5 to 9, with emission at 510 nm. HPTS can be used in the encapsulated system to report pH changes.

[0067] To generate a pH drop for activating ATP synthase in ATP production, GOx and CAT with glucose as the substrate were used (Fig. 4A-B). A pH difference of 2 was shown to be sufficient to activate ATP synthase to generate ATP when ATP synthase was reconstituted in a vesicle membrane, where the pH inside the vesicle was lower than outside. GOx, CAT, and varying glucose concentrations were shown to reduce the pH from 7.4 to below 5 (Fig. 4B), which was a sufficient pH difference to activate ATP synthase.

[0068] To reconstitute GLUT1 into hybrid vesicles containing GOx and CAT, a freeze-thaw cycle is preformed followed by washing, similar to what has been shown previously for reconstituting GLUT2 into GOx and CAT-containing vesicles. The efficiency of GLUT1 incorporation can be tested by immunofluorescence (via anti-His6 antibody staining) or GLUT1 ELISA assay. GLUT1 :lipid ratio from 1 : 100 to 1 : 10000 (with no GLUT1 as a negative control) may be selected. GLUT1 for glucose transport is tested for specificity by adding cytochalasin B, a GLUT inhibitor.

[0069] For reconstitution of ATP synthase, thermophilic Bacillus PS3 F0F1-ATP synthase from E. coli DK8 strainl3,75 (TR19ASDSsAc, hereafter referred to as ATP synthase) is expressed and purified. ATP synthase and GLUT1 is co-reconstituted, along with glucose oxidase and catalase, in vesicles by lipid solubilization in 6-octyl-b-D-glucopyranoside followed by removal of the detergent by SM2 Bio-Beads to generate proteoliposomes. ATP generation by ATP -generating synthetic organelles in the presence of glucose is quantified by using luciferase and measuring bioluminescence as illustrated in Fig. 8.

[0070] Example 3

[0071] Incorporate ATP-generating organelle into cell-free expression systems.

[0072] Cell-free protein synthesis via in vitro transcription-translation reactions over long periods is hampered by energy constraints. ATP regeneration system using creatine phosphate and creatine kinase can prolong cell-free reactions to some extent and also successfully increased protein production by supplementing mitochondria in cell-free lysates in AliCE (Almost Living Cell-free Expression). ATP-generating organelles are supplied to various CFE (cell-free expression) systems, including bacterial, mammalian, and PURE (protein synthesis using recombinant protein components) CFE systems in bulk reactions expressing fluorescence proteins to enhance protein yield and reaction lifetime to reactions without added synthetic organelles.

[0073] Example 4

[0074] Demonstrate actomyosin contraction driven by ATP synthesis using synthetic ATP- generating organelle.

[0075] Synthetic ATP-generating organelles are quantified for traction stresses exerted by actomyosin networks by using Fourier transform traction cytometry. To do this, biotinylated actin filaments are coupled to a soft Matrigel with 200 nm yellow-green neutravidin-coated beads embedded on the top surface of the gel. This substrate is prepared in an open chamber, allowing the addition of actin and other components (e.g., ATP -generating synthetic organelle). Polymerized actin filaments with 10% labeled monomeric actin and 10% biotinylated actin is deposited on the substrate. The coupling of biotinylated actin to fluorescence neutravidin beads permits the transmission of contraction forces to the substrate and the addition of myosin and ATP which has allowed for the contraction and extraction of exerted traction force via the displacement field of the embedded fluorescent beads (Fig. 9A). The addition of myosin (with residual ATP in solution) and extra ATP generated traction stress is shown (Fig. 9B and Fig.9C). Local divergence of the bead displacement field upon the addition of myosin revealed a convergent contraction toward the actin enriched on the 2D actin network template is shown (Fig. 9D). Using this assay, ATP generated by the synthetic organelle can manifest in the traction stress characteristics. Different amounts of ATP-generating organelle and glucose are added to drive varying amounts of ATP synthesis and compared to the addition of known amounts of ATP.

[0076] Example 5

[0077] Construction of synthetic cells capable of ATP generation from glucose.

[0078] To make synthetic cells capable of autonomous ATP generation, ATP-generating synthetic organelles are encapsulated in synthetic cells made of DOPC / block copolymers using cDICE (continuous droplet interface crossing encapsulation). cDICE has been previously used for generating hybrid vesicles. Glucose, ADP, luciferase, and luciferin, along with the synthetic organelle are encapsulated in synthetic cells and tested to determine the level of bioluminescence following ATP synthesis. GLUT1 is reconstituted in the membrane of synthetic cells and tested to determine the degree to which externally added glucose leads to the generation of ATP inside synthetic cells, as measured by luciferase activity. GLUT1 and glucose concentrations are varied to characterize and optimize ATP generation for any particular system.

[0079] Example 6

[0080] Demonstrate ATP-generating synthetic cells have prolonged cell-free protein synthesis.

[0081] ATP-generating organelles and CFE reactions in synthetic cells is encapsulated to couple ATP generation with a cell-like function. In the absence of ATP generation, GFP (green fluorescence protein) expression typically reaches a maximum level in ~4 hour (Fig. 10). Glucose is added to synthetic cells with or without ATP-generating organelles. GFP expression is monitored over time. Example 7

[0082] Actin assembly induced by ATP generation in synthetic cells.

[0083] Photosynthetic artificial organelles have been utilized to power ATP-dependent actin polymerization. ATP -generating synthetic cells is tested to evaluate their capabilities to produce ATP from glucose to drive F-actin assembly (Fig. 5A-B). Globular-actin (G-actin), labeled actin, and ATP -generating synthetic organelles in actin polymerization buffer devoid of ATP is encapsulated in GUVs. The time course of actin assembly following glucose addition is monitored. Following the demonstration of ATP-dependent actin assembly, muscle myosin II is included. Experiments in GUVs with an actin cortex are performed to determine contractile forces that lead to GUV deformation, similar to cellular shape changes.

[0084] Example 8

[0085] NADH generation in synthetic cells from glucose.

[0086] To generate NADH from glucose, synthetic cells are supplemented with hexokinase and glucose-6-PO4 dehydrogenase. The system is tested for a range of enzyme concentrations with varying amounts of glucose and ATP concentrations in bulk reactions in the presence of NAD+. This allows for the identification of the enzyme concentration. NADH is naturally fluorescent (excitation wavelength: 340 nm and emission wavelength: 460 nm) and can be readily detected on a fluorescence microscope. ATP -generating synthetic organelles with the NADH-generating reactions, again in bulk, are included for the examination of the kinetics of NADH generation in these coupled reactions initiated by glucose addition. Synthetic cells are produced encapsulating both the ATP -generating and the NADH-generating reactions. NADH production is monitored by NADH fluorescence following glucose addition.

[0087] Example 9

[0088] Conversion of acetaldehyde to ethanol in NADH-producing synthetic cells.

[0089] Acetaldehyde is encapsulated in synthetic cells and alcohol dehydrogenase which turns acetaldehyde into ethanol utilizing the NADH generated in the synthetic cells. Since ethanol is membrane-permeable, it can be analyzed from the outer solution of synthetic cells by mass spectrometry to detect the presence of ethanol as a measure of the synthetic cell design performance.

Claims

CLAIMS1. A synthetic cell comprising:A. an outer structure, wherein the outer structure comprises at least one glucose transporter; andB. an internal structure, wherein the internal structure is contained within the outer structure, the internal structure comprising at least one glucose transporter, a glucose oxidase (GOx), a catalase (CAT), and an adenosine triphosphate (ATP) synthase.

2. The synthetic cell of claim 1, wherein the outer structure and / or internal structure comprise a di oleoyl-sn-glycero-3-phosphocholine / polybutadiene-poly ethylene oxide (DOPC / PBD-PEO) hybrid vesicle.

3. The synthetic cell of claim 1 or 2, wherein the synthetic cell is in vivo.

4. The synthetic cell of claim 1 or 2, wherein the synthetic cell is in vitro.

5. A method comprising:A. uptaking a glucose from a surrounding environment through a glucose transporter into an outer structure of a synthetic cell;B. converting the glucose into gluconic acid via the glucose oxidase in an internal structure of the synthetic cell; andC. producing hydrogen peroxide.

6. The method of claim 5, wherein the glucose is produced through enzymatic conversion.

7. The method of claim 5, wherein the glucose is from blood.

8. The method of any of claims 5-7, wherein ADP is converted to an ATP with the ATP synthase contained in the internal structure.

9. The method of claim 8, wherein the ATP is released into the outer structure.

10. The methods of any of claims 5-9, wherein the synthetic cell is supplemented with a hexokinase and a glucose-6-PO4 dehydrogenase.

11. The method of claim 10, wherein NADH is generated using said ATP.

12. Use of a synthetic cell of any of the claims 1-4.

13. Use of a synthetic cell of any of claims 1-4 for diagnostic research and / or testing.

14. Use of a synthetic cell of any of claims 1-4 for basic research applications.

15. Use of a synthetic cell of any of claims 1-4 for therapeutic research and / or testing.

16. The use of claim 15, wherein the diagnostic research and / or testing is an in vivo diagnostic tool.

18. The use of claim 15 wherein the basic research application is integrating the synthetic cell with a natural living cell.

Images