Successful Assembly Research Articles

Microbial occurrence and symbiont detection in a global sample of lichen metagenomes.

In lichen research, metagenomes are increasingly being used for evaluating symbiont composition and metabolic potential, but the overall content and limitations of these metagenomes have not been assessed. We reassembled over 400 publicly available metagenomes, generated metagenome-assembled genomes (MAGs), constructed phylogenomic trees, and mapped MAG occurrence and frequency across the data set. Ninety-seven percent of the 1,000 recovered MAGs were bacterial or the fungal symbiont that provides most cellular mass. Our mapping of recovered MAGs provides the most detailed survey to date of bacteria in lichens and shows that 4 family-level lineages from 2 phyla accounted for as many bacterial occurrences in lichens as all other 71 families from 16 phyla combined. Annotation of highly complete bacterial, fungal, and algal MAGs reveals functional profiles that suggest interdigitated vitamin prototrophies and auxotrophies, with most lichen fungi auxotrophic for biotin, most bacteria auxotrophic for thiamine and the few annotated algae with partial or complete pathways for both, suggesting a novel dimension of microbial cross-feeding in lichen symbioses. Contrary to longstanding hypotheses, we found no annotations consistent with nitrogen fixation in bacteria other than known cyanobacterial symbionts. Core lichen symbionts such as algae were recovered as MAGs in only a fraction of the lichen symbioses in which they are known to occur. However, the presence of these and other microbes could be detected at high frequency using small subunit rRNA analysis, including in many lichens in which they are not otherwise recognized to occur. The rate of MAG recovery correlates with sequencing depth, but is almost certainly influenced by biological attributes of organisms that affect the likelihood of DNA extraction, sequencing and successful assembly, including cellular abundance, ploidy and strain co-occurrence. Our results suggest that, though metagenomes are a powerful tool for surveying microbial occurrence, they are of limited use in assessing absence, and their interpretation should be guided by an awareness of the interacting effects of microbial community complexity and sequencing depth.

Comparative mitochondrial genomics of Thelebolaceae in Antarctica: insights into their extremophilic adaptations and evolutionary dynamics

Species of Antarctomyces and Thelebolus (Thelebolaceae), primarily found in Antarctic environments, exhibit psychrophilic adaptations, yet their mitochondrial genomes have not been extensively studied. Furthermore, few studies have compared the mitochondrial genomes of psychrophilic, psychrotrophic, and mesophilic fungi. After successful sequencing and assembly, this study annotated the mitochondrial genomes of Antarctomyces psychrotrophicus CPCC 401038 and Thelebolus microsporus CPCC 401041. We also performed a comparative analysis with the previously characterized mitochondrial genomes of psychrotrophic and mesophilic fungi. The analysis revealed that nad4L was the most conserved gene across the mitochondrial genomes, characterized by its synonymous and non-synonymous substitution rates (Ks and Ka), genetic distance, and GC content and skew within the protein-coding genes (PCGs). Additionally, the mitochondrial genomes of psychrophilic and psychrotrophic fungi showed a higher proportion of protein-coding regions and a lower GC content compared to those of mesophilic fungi, underscoring the genetic basis of cold adaptation. Phylogenetic analyses based on these mitochondrial genes also confirmed the phylogenetic relationships of Thelebolaceae in the class Leotiomycetes. These findings advance our understanding of the phylogenetic relationships and evolutionary dynamics within the family Thelebolaceae, highlighting how different environmental temperatures influence fungal mitochondrial genomic structure and adaptation.

Chromosome-level genome assembly and annotation of the skinnycheek lanternfish Benthosema ptertum

Lanternfish not only boast the most abundant biomass among marine fish species but also play a vital role in marine ecosystems. As one of the lanternfish species with the highest global catch, the skinnycheek lanternfish (Benthosema pterotum) is widely distributed in the Indo-Pacific region, playing a pivotal role in the marine biological pump. This study constructed the first chromosome-level genome of B. pterotum using a combination of short-read sequencing, PacBio, and Hi-C sequencing technologies. The genome size of B. pterotum is 1,272.53 Mb, with a contig N50 of 810 Kb and a scaffold N50 of 54.49 M. More than 99.65% of contigs were successfully anchored onto 24 pseudochromosomes, and 95.7% of BUSCO genes were identified within the genome, demonstrating the high level of completeness in genome assembly. A total of 24,934 protein-coding genes were predicted, of which 99.02% were functionally annotated. The successful assembly of a high-quality genome for B. pterotum provides valuable genetic resources for better understanding its biological characteristics and potentially those of all lanternfish species.

Diverse new plasmid structures and antimicrobial resistance in strains isolated from perianal abscess patients.

Plasmids, the most important and versatile bacterial extrachromosomal DNA Molecules, has have been a center central topic for bacterial genetics and biology. However, the inability of short-read high-throughput sequencing methods to reliably assemble plasmids makes it difficult to investigate the diversity of plasmid structures and functions. In this work, we used the long-read Nanopore sequencing method to address this issue, by producing high quality whole genome sequences of 33 bacterial strains from 11 perianal abscess-suffering patients. Successful high quality assemblies were generated with this method, including 20 perfect assemblies out of 33 genomes. A total of 47 plasmids were identified from the bacterial strains, including 12 unique, newly identified, high quality circular plasmids. These plasmids were further subject to structural analysis, leading to the finding of significant diversification from previously known plasmids, suggesting the diversity of plasmid structure and function. Particularly, two mcr-10.1-harboring conjugative plasmids were found from Citrobacter portucalensis and Enterobacter kobei, which were not previously reported. This works shows the feasibility of using long-read sequencing to identify plasmids, and the high diversity of plasmid structure and function that awaits further surveillance.

The conserved cysteines at position 18, 36, and 49 of Autographa californica multiple nucleopolyhedrovirus VP39 are essential for virus replication.

Autographa californica nucleopolyhedrovirus orf89 (vp39) encodes the major capsid protein VP39. Multiple alignments of protein sequences showed that VP39 has 8 conserved cysteine (Cys) residues. Cysteine residues play an important role in proper function of a protein. To determine the importance of these conserved cysteine residues for virus proliferation, a series of recombinant viruses harboring VP39-Cys mutants were constructed. Viral growth curves and transmission electron microscopy showed that mutation of Cys29, Cys132, Cys169, Cys229, or Cys232 of VP39 to alanine did not affect budded virion production; however, the mutation of Cys18, Cys36, or Cys49 to alanine resulted in interruption of capsid assembly. Co-immunoprecipitation assays showed that mutations of these 8 cysteines individually or simultaneously had no effect on self-association of VP39. Immunofluorescence analysis by confocal microscopy revealed that the subcellular localization of VP39 with mutations in Cys18, Cys36 or Cys49 was exclusively distributed in the cytoplasm of a cell regardless of virus infection or not, while the wild-type VP39 or the VP39 carrying mutations in Cys29, Cys132, Cys169, Cys229, or Cys232 was distributed throughout the cytoplasm and the nucleus. Our results demonstrated that Cys18, Cys36, and Cys49 are essential for the proper localization of VP39, which is a prerequisite for successful nucleocapsid assembly of the virus.

A facile method for patterned assembly of nanoparticles using SAM-guided solvent dewetting

The directed assembly of functional nanoparticles (NPs) into desired geometries is crucial for various NP-based devices and applications. Over the past two decades, numerous bottom-up and top-down approaches have been reported for achieving this goal. In this study, we employed a bottom-up approach using a capillary-driven self-assembled monolayer (SAM)-defined template-assisted self-assembly of silver nanocubes (SNCs) and nanodiamonds (NDs). First, different SAM patterns were created on a gold-coated Si substrate using microcontact printing. Upon immersion of the SAM-patterned substrates in the NP solution, the SAM patterns guide the solvent to specific regions during drying. The directed solvent carries the NPs (SNCs and NDs) into these regions during drying owing to their lower energy state in the solvent compared with the SAM regions. Subsequently, the solvent deposits the NPs in these regions because of their interactions with the substrate. Micron-sized lines and rectangular patterns are explored to observe successful NP assembly. The density of the assembled SNCs depends on their concentration in the solvent, with higher concentrations resulting in densely packed assemblies. This effect is not observed for the NDs because of the differing regular and irregular shapes of the SNCs and NDs. This process can be extended to other NPs with suitable solvents and SAMs.

Assembling Zn2+ on surface of kevlar NanoFibers as functionalized separator for dendrite-free Zn anode and high-performance Zn metal batteries

The proliferation of wearable electronic products has made the development of flexible energy storage devices increasingly important. Among various options, aqueous Zn metal batteries are promising due to their low cost, safety, non-toxicity, environmental friendliness, and abundant element storage. However, the growth of dendrites on the anode surface of Zn metal batteries is a significant challenge to efficient transport of Zn2+, which leads to reduced battery capacity or even failure. This limitation hinders practical applications for these batteries. This study develops a functionalized separator with high ion transport performance and excellent mechanical performances by electrostatic assembling deprotonated PPTA− molecular chains with Zn2+ using Kevlar as a raw material. The successful assembly of Zn2+ is confirmed through chemical composition and functional group analysis. The prepared separator significantly shows the increased the transfer number of Zn2+ and decreased activation energy of Zn2+ deposition. The separator is used in Zn||Zn symmetric batteries, achieving a reversible deposition/stripping process of more than 180 h at a current density of 1 mA cm−2 and a deposition capacity of 1 mAh·cm−2. When applied to Zn||MnO2 batteries, an initial discharging specific capacity of up to 220 mAh·g−1 is obtained at a high current density of 2 A g−1, and the battery could be cycled stably for more than 1, 200 cycles, with a smooth and flat Zn anode surface and no obvious dendrite growth. The separator has good potential for flexible energy storage applications due to its excellent mechanical strength, which enables it to maintain high electrochemical activity even after bending 500 times at 180°. Overall, the functionalized separator effectively inhibits dendrite growth, improves the electrochemical stability of Zn metal batteries, and demonstrates significant application potential for other flexible energy storage applications.

EA-CTFVS: An Environment-Agnostic Coarse-to-Fine Visual Servoing Method for Sub-Millimeter-Accurate Assembly

Peg-in-hole assembly, a crucial component of robotic automation in manufacturing, continues to pose challenges due to its strict tolerance requirements. To date, most conventional peg-in-hole assembly algorithms have been validated only within simulated environments or under limited observational scenarios. In this paper, an environment-agnostic coarse-to-fine visual servoing (EA-CTFVS) assembly algorithm is proposed. Firstly, to solve the frequent issue of visual blindness during visual servoing, a bottleneck pose is proposed to be used as the desired pose for the visual servoing. Secondly, to achieve accurate assembly, a coarse-to-fine framework is constructed, in which a rough pose is given by the coarse controller to remove large initial alignment errors. For the fine controller, a twin network-based fine controller is provided to improve assembly accuracy. Furthermore, EA-CTFVS utilizes the Oriented Bounding Box (OBB) of objects as the input for visual servoing, which guarantees the system’s ability to operate effectively in diverse and complex scenes. The proposed EA-CTFVS achieves a successful assembly rate of 0.92/0.89 for initial alignment errors of 15/30 cm and 0.6 mm tolerance in real-world D-sub plug assembly tasks under complex scenarios.

Immunizing Mice with Influenza Virus-like Particles Expressing the Leishmania amazonensis Promastigote Surface Antigen Alleviates Inflammation in Footpad.

Cutaneous leishmaniasis (CL) is a tropical disease endemic in many parts of the world. Characteristic clinical manifestations of CL include the formation of ulcerative skin lesions that can inflict life-long disability if left untreated. Although drugs are available, they are unaffordable and out of reach for individuals who need them the most. Developing a highly cost-efficient CL vaccine could address this problem but such a vaccine remains unavailable. Here, we developed a chimeric influenza virus-like particle expressing the Leishmania amazonensis promastigote surface antigen (LaPSA-VLP). LaPSA-VLPs were self-assembled in Spodoptera frugiperda insect cell lines using the baculovirus expression system. After characterizing the vaccines and confirming successful VLP assembly, BALB/c mice were immunized with these vaccines for efficacy assessment. Sera acquired from mice upon subcutaneous immunization with the LaPSA-VLP specifically interacted with the L. amazonensis soluble total antigens. LaPSA-VLP-immunized mice elicited significantly greater quantities of parasite-specific IgG from the spleens, popliteal lymph nodes, and footpads than unimmunized mice. LaPSA-VLP immunization also enhanced the proliferation of B cell populations in the spleens of mice and significantly lessened the CL symptoms, notably the footpad swelling and IFN-γ-mediated inflammatory response. Overall, immunizing mice with the LaPSA-VLPs prevented mice from developing severe CL symptoms, signifying their developmental potential.

Tailored Polymersomes for Enhanced Oral Drug Delivery: pH-Sensitive Systems for Intestinal Delivery of Immunosuppressants.

Ensuring precise drug release at target sites is crucial for effective treatment. Here, pH-responsive nanoparticles for oral administration of mycophenolate mofetil, an alternative therapy for patients with inflammatory bowel disease unresponsive to conventional treatments is developed. However, its oral administration presents challenges due to its low solubility in the small intestine and high solubility and absorption in the stomach. Therefore, this aim is to design a drug delivery system capable of maintaining drug solubility compared to the free drug while delaying absorption from the stomach to the intestine. Successful synthesis and assembly of a block copolymer incorporating a pH-responsive functional group is achieved. Dynamic light scattering indicated a significant change in hydrodynamic size when the pH exceeded 6.5, confirming successful incorporation of the pH-responsive group. Encapsulation and controlled release of mycophenolate mofetil are efficiently demonstrated, with 90% release observed at intestinal pH. In vitro cell culture studies confirmed biocompatibility, showing no toxicity or adverse effects on Caco-2 cells. In vivo oral rat studies indicated reduced drug absorption in the stomach and enhanced absorption in the small intestine with the developed formulation. This research presents a promising drug delivery system with potential applications in the treatment of inflammatory bowel disease.

Chitosan/PCL fibrous membranes loaded potassium cinnamate/β-CD clathrate compounds developed by ELS for fruit preservation

In this study, Potassium cinnamate (PC)/β-cyclodextrin (β-CD) clathrate compounds were self-assembled by ultrasound-guided, and Chitosan (CS)/Polycaprolactone (PCL) fibrous membranes were fabricated by electrospinning (ELS) to load the clathrate compounds. FTIR and XRD patterns confirmed the successful assembly of the clathrate compounds and the formation of hydrogen bonds between the clathrate compounds and the CS/PCL fibrous membrane. With the addition of the clathrate compounds, the membrane showed decreased crystallinity, increased thermal stability, hydrophilicity, and brittleness, and exhibited a sustained release of PC for 240 h. More importantly, the series of fibrous membranes showed good antibacterial activity in vitro and on cherries, which provided an important reference value for the application of fibrous composites in prolonging the shelf life of fruits.

Experimental Behavior of a Prototype 3m-Span Modular Glass Pedestrian Bridge

This research is related to the structural performance of a shell-type system made of hollow glass units (HGU) that utilizes glass as the primary structural material. The efficient structural function of the proposed shell-type system is designed using Polyhedral Graphic Statics to achieve a system geometry that maximizes in-plane compression and limits the presence of tension. The large-scale shell structure is constructed using a modular assembly of individual hollow glass units. To date, the research team has completed studies on individual HGU strength and stiffness, and the performance of the interface material necessary to transmit the high in-plane compression forces between neighboring HGUs. As well, the feasibility of the proposed modular system has also been demonstrated through the successful assembly and disassembly of a 3 m span prototype pedestrian bridge known as Tortuca. Tortuca is comprised of 13 individual interlocking HGUs assembled into a compression-dominant form supported on steel abutments. In the current research study, the physical performance of Tortuca is experimentally investigated under controlled laboratory testing and using an extensive assortment of displacement and strain sensors. Significant findings related to experimental testing of Tortuca will be reported.

Flight verification of cooling self-sustaining high-temperature superconducting motor

The global shift towards sustainable development and technological advancements has propelled the energy transition trend. Recognizing the substantial environmental impact of conventional commercial airplanes, there is a growing urgency to develop a sophisticated superconducting motor system for commercial aviation. The advent of high-temperature superconducting motors presents a transformative leap, offering significant advantages in power density and efficiency when compared to traditional motors. To validate the issues that future liquid-hydrogen superconducting electric airplanes may encounter, a kilowatt-class aerospace high-temperature superconducting motor is designed. Based on the requirements of airborne applications, critical parameters such as electromagnetic characteristics, operating characteristics, and AC losses have been analyzed. Furthermore, extensive research and testing have been conducted on the superconducting motor magnet, leading to the successful assembly of a prototype. The superconducting motor has a rated output power of 2.7 kW and a rated speed of 5000 rpm. Rigorous ground operation performance tests have also been conducted to ensure the feasibility and reliability of the motor in practical applications. Benefiting from the topological structure design, the superconducting motor has an excellent sealing performance at low temperatures. The superconducting motor can maintain low temperature and high vacuum for a long time, when the vacuum pump is removed and the liquid nitrogen inlet is closed after the motor is completely cooled. The culmination of these endeavors is the realization of a successful flight validation of an unmanned aerial vehicle equipped with a high-temperature superconducting motor, demonstrating a sustained flight of nearly one hour.

Investigation on the Torsional–Flexural Instability Phenomena during the Bending Process of Hairpin Windings: Experimental Tests and FE Model Validation

Modern electric motors developed for the automotive industry have an ever higher power density with a relatively compact size. Among the various existing solutions to improve torque and power density, a reduction in the dimensions of the end-windings has been explored, aiming to decrease volume, weight, and losses. However, more compact end-windings often lead to complex shapes of the conductors, especially when preformed hairpin windings are considered. The rectangular cross-section of hairpin conductors makes them prone to deviating out of the bending plane during the forming process. This phenomenon, known as torsional–flexural instability, is influenced by the specific aspect ratio of the cross-section dimensions and the bending direction. This study focuses on understanding this instability phenomenon, aiming to identify a potential threshold of the cross-section aspect ratio. The instability makes it difficult to predict the final geometry, potentially compromising the compliance with the geometric tolerances. A finite element model is developed to analyse a single planar bend in a hairpin conductor. Various cross-section dimensions with different aspect ratios are simulated identifying those that experience instability. Moreover, an experimental campaign is conducted to confirm the occurrence of instability by testing the same single planar bending. The experimental data obtained are used to validate the finite element model for the tested dimensions. The aim is to provide designers with a useful tool to select hairpin geometries that are more suitable for the folding process, contributing to successful assembly and improving the overall design process of preformed hairpin conductors.

Developing stable, simplified, functional consortia from Brachypodium rhizosphere for microbial application in sustainable agriculture

The rhizosphere microbiome plays a crucial role in supporting plant productivity and ecosystem functioning by regulating nutrient cycling, soil integrity, and carbon storage. However, deciphering the intricate interplay between microbial relationships within the rhizosphere is challenging due to the overwhelming taxonomic and functional diversity. Here we present our systematic design framework built on microbial colocalization and microbial interaction, toward successful assembly of multiple rhizosphere-derived Reduced Complexity Consortia (RCC). We enriched co-localized microbes from Brachypodium roots grown in field soil with carbon substrates mimicking Brachypodium root exudates, generating 768 enrichments. By transferring the enrichments every 3 or 7 days for 10 generations, we developed both fast and slow-growing reduced complexity microbial communities. Most carbon substrates led to highly stable RCC just after a few transfers. 16S rRNA gene amplicon analysis revealed distinct community compositions based on inoculum and carbon source, with complex carbon enriching slow growing yet functionally important soil taxa like Acidobacteria and Verrucomicrobia. Network analysis showed that microbial consortia, whether differentiated by growth rate (fast vs. slow) or by succession (across generations), had significantly different network centralities. Besides, the keystone taxa identified within these networks belong to genera with plant growth-promoting traits, underscoring their critical function in shaping rhizospheric microbiome networks. Furthermore, tested consortia demonstrated high stability and reproducibility, assuring successful revival from glycerol stocks for long-term viability and use. Our study represents a significant step toward developing a framework for assembling rhizosphere consortia based on microbial colocalization and interaction, with future implications for sustainable agriculture and environmental management.

Towards Complex Tissues Replication: Multilayer Scaffold Integrating Biomimetic Nanohydroxyapatite/Chitosan Composites.

This study explores an approach to design and prepare a multilayer scaffold mimicking interstratified natural tissue. This multilayer construct, composed of chitosan matrices with graded nanohydroxyapatite concentrations, was achieved through an in situ biomineralization process applied to individual layers. Three distinct precursor concentrations were considered, resulting in 10, 20, and 30 wt% nanohydroxyapatite content in each layer. The resulting chitosan/nanohydroxyapatite (Cs/n-HAp) scaffolds, created via freeze-drying, exhibited nanohydroxyapatite nucleation, homogeneous distribution, improved mechanical properties, and good cytocompatibility. The cytocompatibility analysis revealed that the Cs/n-HAp layers presented cell proliferation similar to the control in pure Cs for the samples with 10% n-HAp, indicating good cytocompatibility at this concentration, while no induction of apoptotic death pathways was demonstrated up to a 20 wt% n-Hap concentration. Successful multilayer assembly of Cs and Cs/n-HAp layers highlighted that the proposed approach represents a promising strategy for mimicking multifaceted tissues, such as osteochondral ones.

Evaluation of the Impact of Concentration and Extraction Methods on the Targeted Sequencing of Human Viruses from Wastewater.

Sequencing human viruses in wastewater is challenging due to their low abundance compared to the total microbial background. This study compared the impact of four virus concentration/extraction methods (Innovaprep, Nanotrap, Promega, and Solids extraction) on probe-capture enrichment for human viruses followed by sequencing. Different concentration/extraction methods yielded distinct virus profiles. Innovaprep ultrafiltration (following solids removal) had the highest sequencing sensitivity and richness, resulting in the successful assembly of several near-complete human virus genomes. However, it was less sensitive in detecting SARS-CoV-2 by digital polymerase chain reaction (dPCR) compared to Promega and Nanotrap. Across all preparation methods, astroviruses and polyomaviruses were the most highly abundant human viruses, and SARS-CoV-2 was rare. These findings suggest that sequencing success can be increased using methods that reduce nontarget nucleic acids in the extract, though the absolute concentration of total extracted nucleic acid, as indicated by Qubit, and targeted viruses, as indicated by dPCR, may not be directly related to targeted sequencing performance. Further, using broadly targeted sequencing panels may capture viral diversity but risks losing signals for specific low-abundance viruses. Overall, this study highlights the importance of aligning wet lab and bioinformatic methods with specific goals when employing probe-capture enrichment for human virus sequencing from wastewater.

Determination of the cervical cancer biomarker SCCA using CMK-3@UiO-66@AuNPs composite-modified glassy carbon electrodes

In order to detect the cervical cancer biomarker squamous cell carcinoma antigen (SCCA) in human serum, an electrochemical immunosensor was constructed. Zirconium 1,4-dicarboxybenzene MOF (UiO-66), with a high specific surface area, was coupled with gold nanoparticles (AuNPs), creating the UiO-66@AuNPs nanomaterial. This coupling increased the sensitivity of the electrochemical immunosensor by providing many biological binding sites for antibodies to bind to Au-S bonds. Then, mesoporous carbon (CMK-3) was added to the UiO-66@AuNPs nanomaterial to obtain a CMK-3@UiO-66@AuNPs nanocomposite. This further increased the electron transfer proficiency of the material and enhanced the electrochemical signal. Finally, the sensitive detection of squamous cell carcinoma antigen (SCCA) was made possible by unique immunological responses between antigens and antibodies. Transmission electron microscopy (TEM) analysis of CMK-3@UiO-66@AuNPs revealed embedded surfaces and voids; cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) confirmed the successful assembly of the electrochemical immunosensor. The developed electrochemical immunosensor demonstrated good linearity (R12 = 0.999) from 0.01 pg/mL to 10,000 pg/mL, which is the concentration range of SCCA in human serum. The limit of detection was 0.0034 pg/mL (at an S/N ratio of 3). The immunosensor exhibited excellent precision, stability and selectivity, with a high rate of recovery (98.26–101.8 %). The experimental validation studies demonstrated that the electrochemical immunosensor was effective for the detection of SCCA in human serum, offering a novel method for the early diagnosis and dynamic surveillance of cervical cancer.

Cell-free Protein Synthesis System for Building Synthetic Cells.

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for the core machinery of the Central Dogma, standing as an optimal chassis for the integration and assembly of diverse artificial cellular mimicry systems. Despite its frequent use in the fabrication of synthetic cells, establishing a tailored and robust CFPS system for a specific application remains a nontrivial challenge. In this methods paper, we present a comprehensive protocol for the CFPS system, routinely employed in constructing synthetic cells. This protocol encompasses key stages in the preparation of the CFPS system, including the cell extract, template preparation, and routine expression optimization utilizing a fluorescent reporter protein. Additionally, we show representative results by encapsulating the CFPS system within various micro-compartments, such as monolayer droplets, double-emulsion vesicles, and chambers situated atop supported lipid bilayers. Finally, we elucidate the critical steps and conditions necessary for the successful assembly of these CFPS systems in distinct environments. We expect that our approach will facilitate the establishment of good working practices among various laboratories within the continuously expanding synthetic cell community, thereby accelerating progress in the field of synthetic cell development.

Enantioselective [2π + 2σ] Cycloadditions of Bicyclo[1.1.0]butanes with Vinylazaarenes through Asymmetric Photoredox Catalysis.

Here we present a highly enantioselective [2π + 2σ] photocycloaddition of bicyclo[1.1.0]butanes (BCBs). The reaction uses a variety of vinylazaarenes as partners and is catalyzed by a polycyclic aromatic hydrocarbon (PAH)-containing chiral phosphoric acid as a bifunctional chiral photosensitizer. A wide array of pharmaceutically important bicyclo[2.1.1]hexane (BCH) derivatives have been synthesized with high yields, enantioselectivity, and diastereoselectivity. In addition to the diverse 1-ketocarbonyl-3-substituted BCBs, α/β-substituted vinylazaarenes are compatible with such an unprecedented photoredox catalytic pathway, resulting in the successful assembly of an all-carbon quaternary stereocenter or two adjacent tertiary stereocenters on the product.