RESUMEN
Baroplastic diblock copolymers exhibit order-disorder transitions and melt upon compression at low temperatures, in some cases even at ambient temperatures. Their unique low-temperature processability makes them promising candidates for sustainable polymeric materials. Despite their potential, however, the molecular mechanisms governing the pressure-induced phase transitions of these copolymers remain largely unexplored. This study develops a compressible self-consistent field theory for baroplastic copolymers based on a simple lattice vacancy model that explicitly incorporates voids to account for compressibility. The theory shows that the selective presence of voids in compressible domains stabilizes the ordered phase, while a reduction of voids under compression leads to the order-disorder transition. In addition, this work demonstrates for the first time the critical role of gas absorption rates in each segment in the pressure-induced order-disorder transition of baroplastic diblock copolymers. These findings have significant implications for the rational design of baroplastic polymers with tailored low-temperature processability.
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The ocean constitutes approximately 70% of Earth's surface. Its average depth is 3688 m, of which depths beyond 200 m are classified as the deep sea. The deep sea is distinct from the surface of the ocean in terms of pressure, temperature, and sunlight. The unique physicochemical processes under the extreme environment of the deep sea and the specialized biochemical mechanisms developed by organisms to survive in the deep sea can serve as a vast source of inspiration for scientific and technological advancements. In this Perspective, we discuss three examples of deep-sea-inspired chemistry: (1) soft materials that respond to high pressures such as those observed in the deep sea; (2) molecular self-assembly inspired by the chemistry of hot and compressed water in deep-sea hydrothermal vents; and (3) nanobiotechnology and biomimetics inspired by survival strategies of deep-sea organisms. Finally, we provide an outlook on deep-sea-inspired chemistry. This Perspective aims to promote the sustainable utilization of the ocean based on knowledge, as opposed to the conventional utilization of the ocean solely based on resources. We hope that this Perspective will encourage chemists to harness their inspiration gleaned from the deep sea.
RESUMEN
Cells from strain GE09T, isolated from an artificially immersed nanofibrous cellulose plate in the deep sea, were Gram-stain-negative, motile, aerobic cells that could grow with cellulose as their only nutrient. Strain GE09T was placed among members of Cellvibrionaceae, in the Gammaproteobacteria, with Marinagarivorans algicola Z1T, a marine degrader of agar, as the closest relative (97.4â% similarity). The average nucleotide identity and digital DNA-DNA hybridization values between GE09T and M. algicola Z1T were 72.5 and 21.2â%, respectively. Strain GE09T degraded cellulose, xylan and pectin, but not starch, chitin and agar. The different carbohydrate-active enzymes encoded in the genomes of strain GE09T and M. algicola Z1T highlights their differences in terms of target energy sources and reflects their isolation environments. The major cellular fatty acids of strain GE09T were C18â:â1 ω7c, C16â:â0 and C16â:â1 ω7c. The polar lipid profile showed phosphatidylglycerol and phosphatidylethanolamine. The major respiratory quinone was Q-8. Based on these distinct taxonomic characteristics, strain GE09T represents a new species in the genus Marinagarivorans, for which we propose the name Marinagarivorans cellulosilyticus sp. nov. (type strain GE09T=DSM 113420T=JCM 35003T).
Asunto(s)
Gammaproteobacteria , Noma , Humanos , Japón , Agar , Ácidos Grasos/química , Filogenia , Análisis de Secuencia de ADN , ARN Ribosómico 16S/genética , ADN Bacteriano/genética , Técnicas de Tipificación Bacteriana , Composición de Base , Bacterias , CelulosaRESUMEN
Biomineralization in animals exclusively features oxygen-based minerals with a single exception of the scaly-foot gastropod Chrysomallon squamiferum, the only metazoan with an iron sulfide skeleton. This unique snail inhabits deep-sea hot vents and possesses scales infused with iron sulfide nanoparticles, including pyrite, giving it a characteristic metallic black sheen. Since the scaly-foot is capable of making iron sulfide nanoparticles in its natural habitat at a relatively low temperature (â¼15 °C) and in a chemically dynamic vent environment, elucidating its biomineralization pathways is expected to have significant industrial applications for the production of metal chalcogenide nanoparticles. Nevertheless, this biomineralization has remained a mystery for decades since the snail's discovery, except that it requires the environment to be rich in iron, with a white population lacking in iron sulfide known from a naturally iron-poor locality. Here, we reveal a biologically controlled mineralization mechanism employed by the scaly-foot snail to achieve this nanoparticle biomineralization, through δ34 S measurements and detailed electron-microscopic investigations of both natural scales and scales from the white population artificially incubated in an iron-rich environment. We show that the scaly-foot snail mediates biomineralization in its scales by supplying sulfur through channel-like columns in which reaction with iron ions diffusing inward from the surrounding vent fluid mineralizes iron sulfides.
Asunto(s)
Compuestos Ferrosos/metabolismo , Respiraderos Hidrotermales , Nanopartículas del Metal/química , Caracoles/fisiología , Animales , Compuestos Ferrosos/química , Océano ÍndicoRESUMEN
Some aphids that live in the leaf galls of the host plant are known to fabricate liquid marbles consisting of honeydew and wax particles as an inner liquid and a stabilizer, respectively. In this study, the liquid marbles fabricated by the galling aphids, Eriosoma moriokense, were extensively characterized with respect to size and size distribution, shape, nanomorphology, liquid/solid weight ratio, and chemical compositions. The stereo microscopy studies confirmed that the liquid marbles have a near-spherical morphology and that the number-average diameter was 368 ± 152 µm, which is 1 order of magnitude smaller than the capillary length of the honeydew. The field emission scanning electron microscopy studies indicated that micrometer-sized wax particles with fiber- and dumpling-like shapes coated the honeydew droplets, which rendered the liquid marbles hydrophobic and nonwetting. Furthermore, the highly magnified scanning electron microscopy images confirmed that the wax particles were formed with assemblage of submicrometer-sized daughter fibers. The contact angle measurements indicated that the wax was intrinsically hydrophobic and that the liquid marbles were stabilized by the wax particles in the Cassie-Baxter model. The weight ratio of the honeydew and the wax particles was determined to be 96/4, and the honeydew consisted of 19 wt % nonvolatile components and 81 wt % water. The 1H nuclear magnetic resonance, Fourier transform infrared spectroscopy, and mass spectroscopy studies confirmed that the wax mainly consisted of triglycerides and that the honeydew mainly consisted of saccharides (glucose and fructose) and ribitol. The atomic force microscopy studies confirmed that honeydew is sticky in nature.
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Áfidos/metabolismo , Carbonato de Calcio/metabolismo , Nanoestructuras , Ceras/metabolismo , Animales , Áfidos/química , Carbonato de Calcio/química , Interacciones Hidrofóbicas e Hidrofílicas , Nanoestructuras/química , Tamaño de la Partícula , Ceras/químicaRESUMEN
Two distinct bacterial and eukaryotic serine racemases (SRs) have been identified based on phylogenetic and biochemical characteristics. Although some reports have suggested that marine heterotrophic bacteria have the potential to produce d-serine, the gene encoding bacterial SRs is not found in those bacterial genomes. In this study, using in-depth genomic analysis, we found that eukaryotic SR homologues were distributed widely in various bacterial genomes. Additionally, we selected a eukaryotic SR homologue from a marine heterotrophic bacterium, Roseobacter litoralis Och 149 (RiSR), and constructed an RiSR gene expression system in Escherichia coli for studying the properties of the enzyme. Among the tested amino acids, the recombinant RiSR exhibited both racemization and dehydration activities only towards serine, similar to many eukaryotic SRs. Mg2+ and MgATP enhanced both activities of RiSR, whereas EDTA abolished these enzymatic activities. The enzymatic properties and domain structure of RiSR were similar to those of eukaryotic SRs, particularly mammalian SRs. However, RiSR showed lower catalytic efficiency for L-serine dehydration (kcat/Km=0.094 min(-1) mM(-1)) than those of eukaryotic SRs reported to date (kcat/Km=0.6-21 min(-1) mM(-1)). In contrast, the catalytic efficiency for L-serine racemization of RiSR (kcat/Km=3.14 min(-1) mM(-1)) was 34-fold higher than that of l-serine dehydration. These data suggested that RiSR primarily catalysed serine racemization rather than dehydration.
Asunto(s)
Proteínas Bacterianas/química , Eucariontes/enzimología , Racemasas y Epimerasas/química , Roseobacter/enzimología , Secuencia de Aminoácidos , Bacterias/química , Bacterias/clasificación , Bacterias/enzimología , Bacterias/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Eucariontes/química , Eucariontes/clasificación , Eucariontes/genética , Expresión Génica , Humanos , Cinética , Datos de Secuencia Molecular , Filogenia , Estructura Terciaria de Proteína , Transporte de Proteínas , Racemasas y Epimerasas/genética , Racemasas y Epimerasas/metabolismo , Roseobacter/química , Roseobacter/genética , Alineación de Secuencia , Especificidad por SustratoRESUMEN
A ß-1,3-glucanase (LpGluA) of deep subseafloor Laceyella putida JAM FM3001 was purified to homogeneity from culture broth. The molecular mass of the enzyme was around 36 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). LpGluA hydrolyzed curdlan optimally at pH 4.2 and 80 °C. In spite of the high optimum temperature, LpGluA showed relatively low thermostability, which was stabilized by adding laminarin, xylan, colloidal chitin, pectin, and its related polysaccharides. The gene for LpGluA cloned by using degenerate primers was composed of 1236 bp encoding 411 amino acids. Production of both LpGluA and a chitinase (LpChiA; Shibasaki et al. Appl Microbiol Biotechnol 98, 7845-7853, 2014) was induced by adding N-acetylglucosamine (GluNAc) to a culture medium of strain JAM FM3001. Construction of expression vectors containing the gene for LpGluA and its flanking regions showed the existence of a putative repressor protein.
Asunto(s)
Bacillales/enzimología , Bacillales/genética , Glucano 1,3-beta-Glucosidasa/genética , Glucano 1,3-beta-Glucosidasa/metabolismo , Acetilglucosamina/metabolismo , Secuencia de Aminoácidos , Bacillales/aislamiento & purificación , Secuencia de Bases , Clonación Molecular , Medios de Cultivo/química , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Sedimentos Geológicos , Glucano 1,3-beta-Glucosidasa/química , Glucano 1,3-beta-Glucosidasa/aislamiento & purificación , Concentración de Iones de Hidrógeno , Hidrólisis , Datos de Secuencia Molecular , Peso Molecular , Temperatura , Activación Transcripcional , beta-Glucanos/metabolismoRESUMEN
BACKGROUND: Special features of the Japanese ocean include its ranges of latitude and depth. This study is the first to examine the diversity of Class I and II PHA synthases (PhaC) in DNA samples from pelagic seawater taken from the Japan Trench and Nankai Trough from a range of depths from 24 m to 5373 m. PhaC is the key enzyme in microorganisms that determines the types of monomer units that are polymerized into polyhydroxyalkanoate (PHA) and thus affects the physicochemical properties of this thermoplastic polymer. Complete putative PhaC sequences were determined via genome walking, and the activities of newly discovered PhaCs were evaluated in a heterologous host. RESULTS: A total of 76 putative phaC PCR fragments were amplified from the whole genome amplified seawater DNA. Of these 55 clones contained conserved PhaC domains and were classified into 20 genetic groups depending on their sequence similarity. Eleven genetic groups have undisclosed PhaC activity based on their distinct phylogenetic lineages from known PHA producers. Three complete DNA coding sequences were determined by IAN-PCR, and one PhaC was able to produce poly(3-hydroxybutyrate) in recombinant Cupriavidus necator PHB-4 (PHB-negative mutant). CONCLUSIONS: A new functional PhaC that has close identity to Marinobacter sp. was discovered in this study. Phylogenetic classification for all the phaC genes isolated from uncultured bacteria has revealed that seawater and other environmental resources harbor a great diversity of PhaCs with activities that have not yet been investigated. Functional evaluation of these in silico-based PhaCs via genome walking has provided new insights into the polymerizing ability of these enzymes.
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Aciltransferasas/genética , Aciltransferasas/aislamiento & purificación , Metagenómica , Agua de Mar/microbiología , Aciltransferasas/metabolismo , Clonación Molecular , Análisis por Conglomerados , Cupriavidus necator/genética , Cupriavidus necator/metabolismo , Expresión Génica , Variación Genética , Hidroxibutiratos/metabolismo , Japón , Marinobacter/genética , Datos de Secuencia Molecular , Filogenia , Poliésteres/metabolismo , Análisis de Secuencia de ADN , Homología de SecuenciaRESUMEN
Anomalous long-range repulsion, extending over several micrometres, emerged between silica surfaces around the ridge of density fluctuations in supercritical ethanol at temperatures and pressures near the gas/liquid critical point (T(c) = 241 °C, P(c) = 6.14 MPa). Analysis shows that augmentation of ethanol density around silica surfaces in the presence of density fluctuations facilitates dissociation of silanol groups, leading to long-range electrostatic repulsion in the nonpolar medium.
RESUMEN
It is well known that prokaryotic life can withstand extremes of temperature, pH, pressure, and radiation. Little is known about the proliferation of prokaryotic life under conditions of hyperacceleration attributable to extreme gravity, however. We found that living organisms can be surprisingly proliferative during hyperacceleration. In tests reported here, a variety of microorganisms, including Gram-negative Escherichia coli, Paracoccus denitrificans, and Shewanella amazonensis; Gram-positive Lactobacillus delbrueckii; and eukaryotic Saccharomyces cerevisiae, were cultured while being subjected to hyperaccelerative conditions. We observed and quantified robust cellular growth in these cultures across a wide range of hyperacceleration values. Most notably, the organisms P. denitrificans and E. coli were able to proliferate even at 403,627 × g. Analysis shows that the small size of prokaryotic cells is essential for their proliferation under conditions of hyperacceleration. Our results indicate that microorganisms cannot only survive during hyperacceleration but can display such robust proliferative behavior that the habitability of extraterrestrial environments must not be limited by gravity.
Asunto(s)
Bacterias/crecimiento & desarrollo , Medio Ambiente Extraterrestre , Hongos/crecimiento & desarrollo , Hipergravedad , Aceleración , Bacterias/citología , Escherichia coli/crecimiento & desarrollo , Exobiología , Hongos/citología , Bacterias Gramnegativas/crecimiento & desarrollo , Bacterias Grampositivas/crecimiento & desarrollo , Presión Hidrostática , Paracoccus denitrificans/crecimiento & desarrollo , Saccharomyces cerevisiae/crecimiento & desarrollo , Estrés MecánicoRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0244464.].
RESUMEN
ß-N-Acetylgalactosamine-containing glycans play essential roles in several biological processes, including cell adhesion, signal transduction, and immune responses. ß-N-Acetylgalactosaminidases hydrolyze ß-N-acetylgalactosamine linkages of various glycoconjugates. However, their biological significance remains ambiguous, primarily because only one type of enzyme, exo-ß-N-acetylgalactosaminidases that specifically act on ß-N-acetylgalactosamine residues, has been documented to date. In this study, we identify four groups distributed among all three domains of life and characterize eight ß-N-acetylgalactosaminidases and ß-N-acetylhexosaminidase through sequence-based screening of deep-sea metagenomes and subsequent searching of public protein databases. Despite low sequence similarity, the crystal structures of these enzymes demonstrate that all enzymes share a prototype structure and have diversified their substrate specificities (oligosaccharide-releasing, oligosaccharide/monosaccharide-releasing, and monosaccharide-releasing) through the accumulation of mutations and insertional amino acid sequences. The diverse ß-N-acetylgalactosaminidases reported in this study could facilitate the comprehension of their structures and functions and present evolutionary pathways for expanding their substrate specificity.
Asunto(s)
Acetilgalactosamina , Glicósido Hidrolasas , Metagenoma , Metagenoma/genética , Especificidad por Sustrato , Acetilgalactosamina/metabolismo , Acetilgalactosamina/química , Glicósido Hidrolasas/metabolismo , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/química , beta-N-Acetilhexosaminidasas/metabolismo , beta-N-Acetilhexosaminidasas/genética , beta-N-Acetilhexosaminidasas/química , Filogenia , Cristalografía por Rayos X , Secuencia de Aminoácidos , AnimalesRESUMEN
Assaying enzymatic degradation of water-insoluble substrates like cellulose is challenging because only the substrate surface is accessible to the enzymes resulting in low reaction rates. Here, we describe a protocol for surface pitting observation technology (SPOT), an ultra-sensitive quantitative assay for analyzing enzymatic hydrolysis of cellulose. We describe the use of a porous substrate to accelerate the hydrolysis rate of cellulose. We also detail the steps for combining inkjet patterning and optical profilometry to analyze volume loss upon hydrolysis. For complete details on the use and execution of this protocol, please refer to Tsudome et al. (2022).1.
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Celulosa , Tecnología , Celulosa/metabolismo , HidrólisisRESUMEN
An inulinase-producing Microbulbifer sp. strain, JAM-3301, was isolated from a deep-sea sediment. An inulin operon that contained three open reading frames was cloned and sequenced. Two of the three genes were expressed. One product was an endo-inulinase, and the other was a ß-fructofuranosidase. Both enzymes worked together to effectively degrade inulin.
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Alteromonadaceae/enzimología , Clonación Molecular , Glicósido Hidrolasas/genética , Proteínas Recombinantes/metabolismo , Agua de Mar/microbiología , beta-Fructofuranosidasa/genética , Alteromonadaceae/clasificación , Alteromonadaceae/genética , Alteromonadaceae/aislamiento & purificación , Glicósido Hidrolasas/química , Glicósido Hidrolasas/aislamiento & purificación , Glicósido Hidrolasas/metabolismo , Inulina/metabolismo , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Operón , ARN Ribosómico 16S/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Análisis de Secuencia de ADN , Especificidad por Sustrato , beta-Fructofuranosidasa/química , beta-Fructofuranosidasa/aislamiento & purificación , beta-Fructofuranosidasa/metabolismoRESUMEN
A novel, facultatively anaerobic bacterium (strain JAM-BA0501(T)) was isolated from a deep subseafloor sediment sample at a depth of 247 m below seafloor off the Shimokita Peninsula of Japan in the north-western Pacific Ocean (Site C9001, 1180 m water depth). Cells of strain JAM-BA0501(T) were gram-negative, filamentous, non-spore-forming and motile on solid medium by gliding. Phylogenetic analysis based on the 16S rRNA gene sequence of strain JAM-BA0501(T) indicated a distant relationship to strains representing genera within the order Bacteroidales, such as Alkaliflexus imshenetskii Z-7010(T) (91.1â% similarity), Marinilabilia salmonicolor ATCC 19041(T) (86.2â%) and Anaerophaga thermohalophila Fru22(T) (89.3â%). The new isolate produced isoprenoid quinones with menaquinone MK-7 as the major component, and the predominant fatty acids were iso-C(15â:â0) and anteiso-C(15â:â0). The DNA G+C content of the isolate was 42.9 mol%. Based on its taxonomic distinctiveness, strain JAM-BA0501(T) is considered to represent a novel species of a new genus within the family Marinilabiliaceae, for which the name Geofilum rubicundum gen. nov., sp. nov. is proposed. The type strain of Geofilum rubicundum is JAM-BA0501(T) (â=âJCM 15548(T) â=âNCIMB 14482(T)).
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Bacteroidetes/clasificación , Bacteroidetes/aislamiento & purificación , Sedimentos Geológicos/microbiología , Anaerobiosis , Técnicas de Tipificación Bacteriana , Bacteroidetes/genética , Bacteroidetes/fisiología , Composición de Base , Análisis por Conglomerados , Citosol/química , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Ácidos Grasos/análisis , Japón , Locomoción , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Océano Pacífico , Filogenia , Quinonas/análisis , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
An alkaline κ-carrageenase, Cgk-K142, was found in the culture broth of a deep-sea bacterium, Pseudoalteromonas tetraodonis JAM-K142. A gene for the enzyme was cloned and expressed. Purified recombinant Cgk-K142 (rCgk-K142) showed an optimal pH of about 8.8 in glycine-NaOH buffer at 30 °C and of about 8.0 in MOPS buffer at 50 °C. The optimal temperature for the enzyme was 55 °C at pH 8.0. rCgk-K142 was unstable, but λ- and ι-carrageenans, non-degradative substrate homologs, extensively enhanced its stability. The nucleotide sequence of the gene for Cgk-K142 comprised 1,194 bp, and the deduced amino acid sequence (397 amino acids) showed a high level of similarity to the κ-carrageenase of P. carrageenovora, with 94% identity. Another gene for a κ-carrageenase-like protein was found downstream of the gene for Cgk-K142. The nucleotide sequence of that gene consisted of 966 bp (321 amino acids), and it showed the highest similarity, at 64% identity, to protein CgkB of P. carrageenovora, which has been reported as an incomplete 57-amino acid sequence.
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Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Pseudoalteromonas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Glicósido Hidrolasas/química , Glicósido Hidrolasas/aislamiento & purificación , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Pseudoalteromonas/enzimología , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Análisis de Secuencia de ARN , Especificidad por Sustrato , TemperaturaRESUMEN
Substrates for enzymatic reactions, such as cellulose and chitin, are often insoluble in water. The enzymatic degradation of these abundant organic polymers plays a dominant role in the global carbon cycle and has tremendous technological importance in the production of bio-based chemicals. In addition, biodegradation of plastics is gaining wide attention. However, despite the significance, assaying these degradation reactions remains technically challenging owing to the low reaction rate, because only the surface of the substrate is accessible to the enzymes. We developed a nanofiber-based assay for the enzymatic hydrolysis of cellulose. This assay facilitated the quantification of the enzymatic hydrolysis of <1 ng crystalline cellulose. Utilization of the assay for the functional screening of cellulolytic microorganisms revealed an unprecedented genetic diversity underlying the production of deep-sea cellulase. This study reiterates that interdisciplinary efforts, such as from nanotechnology to microbiology, are critical for solving sustainability challenges.
RESUMEN
A chitosanase of deep-biosphere Bacillus thuringiensis strain JAM-GG01 was purified. The optimal pH and temperature for the purified enzyme (Cho-GG) were about pH 6 and 60 °C, but Cho-GG was unexpectedly unstable under incubation at over 40 °C. This discrepancy between higher activity and lower stability in the same range of temperature was abolished by the addition of reaction products, chitotriose and chitotetraose. The Cho-GG gene was amplified by PCR and sequenced. The deduced amino acid sequence of Cho-GG showed more than 98% identity to those of other Bacillus enzymes belonging to GH family 8. Although Cho-GG did not show the definite characteristics of a sub-seafloor ectoenzyme, the thermal stability of many chitosanases of B. turingienesis and other related strains can be improved by adding chitotriose or chitotetraose.
Asunto(s)
Bacillus thuringiensis/enzimología , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Secuencia de Aminoácidos , Bacillus thuringiensis/genética , Secuencia de Bases , Estabilidad de Enzimas , Glicósido Hidrolasas/química , Concentración de Iones de Hidrógeno , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Análisis de Secuencia de ARN , Especificidad por Sustrato , TemperaturaRESUMEN
HYPOTHESIS: Monodisperse nanodroplet generation in quenched hydrothermal solution (MAGIQ) is a newly developed bottom-up process for preparing nanoemulsions. In this process, homogeneous solutions of oil in supercritical water are quenched by adding cold water containing a surfactant to induce rapid phase-separation, during which oil molecules self-assemble to form nano-sized oil droplets. The droplet size in MAGIQ is known to be influenced by the interplay of the phase-separation dynamics, coalescence kinetics of the droplets, and adsorption kinetics of the surfactant on the droplet surface; however, the primary stages of the droplet formation are still elusive. EXPERIMENTS: Octaneinwater nanoemulsions containing 0.5, 1, and 3 vol% octane were prepared by the MAGIQ method. Their ripening was studied by dynamic light scattering, and the phase diagram was established. FINDINGS: The nanoemulsions containing 0.5 and 1 vol% octane transformed to thermodynamically stable microemulsions containing swollen micelles, whereas the nanoemulsion containing 3 vol% octane underwent Ostwald ripening. The initial formation of the nano-sized droplets in the former was ascribed to a unique mechanism of droplet formation in MAGIQ-the droplets are first formed by the phase separation of homogeneous binary solutions of oil in supercritical water and then stabilized upon surfactant adsorption.
RESUMEN
Restriction endonucleases play a central role in the microbial immune system against viruses and are widely used in DNA specific cleavage, which is called restriction digestion, for genetic engineering. Herein, we applied digital cell-free protein synthesis as an easy-to-use orthogonal readout means to assess the restriction digest efficiency, a new application of digital bioassays. The digital counting principle enabled an unprecedentedly sensitive trace analysis of undigested DNA at the single-molecule level in a PCR-free manner. Our approach can quantify the template DNA of much lower concentrations that cannot be detected by ensemble-based methods such as gold-standard DNA electrophoresis techniques. The sensitive and quantitative measurements revealed a considerable variation in the digest efficiency among restriction endonucleases, from less than 70% to more than 99%. Intriguingly, none of them showed truly complete digestion within reasonably long periods of reaction time. The same rationale was extended to a multiplexed assay and applicable to any DNA-degrading or genome-editing enzymes. The enzyme kinetic parameters and the flanking sequence-dependent digest efficiency can also be interrogated with the proposed digital counting method. The absolute number of residual intact DNA molecules per microliter was concluded to be at least 107, drawing attention to the residual issue of genetic materials associated with the interpretation of nucleases' behaviors and functions in daily genetic engineering experiments.