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• High-efficiency methods for DNA assembly have enabled the routine assembly of synthetic DNAs of increased size and complexity. However, these techniques require customization, elaborate vector sets or serial manipulations for the different stages of assembly. • We have developed Loop assembly based on a recursive approach to DNA fabrication. The system makes use of two Type IIS restriction endonucleases and corresponding vector sets for efficient and parallel assembly of large DNA circuits. Standardized level 0 parts can be assembled into circuits containing 1, 4, 16 or more genes by looping between the two vector sets. • The vectors also contain modular sites for hybrid assembly using sequence overlap methods. - Loop assembly enables efficient and versatile DNA fabrication for plant transformation. We show the construction of plasmids up to 16 genes and 38 kb with high efficiency (> 80%). We have characterized Loop assembly on over 200 different DNA constructs and validated the fidelity of the method by high-throughput Illumina plasmid sequencing. • Our method provides a simple generalized solution for DNA construction with standardized parts. The cloning system is provided under an OpenMTA license for unrestricted sharing and open access.

The ability to clone large DNA fragments from genomes is valuable for both basic and applied research, such as the construction of synthetic genomes, and the expression of biosynthetic gene clusters (BGCs) for natural product discovery. Here, we report a fast and efficient platform for the direct capture of genome DNAs, by combining CRISPR and Gibson assembly. We demonstrate this method with the ability of cloning large DNA fragments ranging from 30 to 77 kb from various host genomes, achieving a near 100% cloning fidelity for DNA fragments below 50 kb. We next demonstrate this method by the cloning of a 40 kb fragment from Streptomyces ceruleus A3(2), which is rich in BGCs for natural products; and used this method cloning the 40 kb fengycin synthetic gene cluster from B. subtilis 168, encoding for a class of peptides with bioactivity. This method provides efficient and simple opportunities for assembling large DNA constructs from distant sources.

Industrial production of lignocellulosic ethanol requires a microorganism utilizing both hexose and pentose, and tolerating inhibitors. In this study, a hydrolysate-cofermenting Saccharomyces cerevisiae strain was obtained through one step in vivo DNA assembly of pentose-metabolizing pathway genes, followed by consecutive adaptive evolution in pentose media containing acetic acid, and direct screening in biomass hydrolysate media. The strain was able to coferment glucose and xylose in synthetic media with the respective maximal specific rates of glucose and xylose consumption, and ethanol production of 3.47, 0.38 and 1.62 g/g DW/h, with an ethanol titre of 41.07 g/L and yield of 0.42 g/g. Industrial wheat straw hydrolysate fermentation resulted in maximal specific rates of glucose and xylose consumption, and ethanol production of 2.61, 0.54 and 1.38 g/g DW/h, respectively, with an ethanol titre of 54.11 g/L and yield of 0.44 g/g. These are among the best for wheat straw hydrolysate fermentation through separate hydrolysis and cofermentation.

Yeast integrating plasmids (YIPs) are a versatile tool for stable integration in Saccharomyces cerevisiae. However, current YIP systems necessitate time‐ and labor‐intensive methods for cloning and selection marker rescue. Here, we describe the design, construction, and validation of a new YIP system capable of accelerating the stable integration of multiple expression constructs into different loci in the yeast S. cerevisiae. These ‘directed pop‐out’ plasmids enable a simple, two‐step integration protocol that results in a scarless integration alongside a complete rescue of the selection marker. These plasmids combine three key features: a dedicated ‘YIPout’ fragment directs a recombination event that rescues the selection marker while avoiding undesired excision of the target DNA sequence, a multifragment modular DNA assembly system simplifies cloning, and a new set of counterselectable markers enables serial integration followed by a transformation‐free marker rescue event. We constructed and tested directed pop‐out YIPs for integration of fluorescent reporter genes into four yeast loci. We validated our new YIP design by integrating three reporter genes into three different loci with transformation‐free rescue of selection markers. These new YIP designs will facilitate the construction of yeast strains that express complex heterologous metabolic pathways.

Nucleic acid‐based cryptographic approaches are an innovative emerging field for information process. However, the poor reproducibility and interference from bioenvironment of the existing decryption led to different binary translation according to the fixed threshold defined by “Sender”, which seriously affects the authenticity during message communication. Here, a programmed DNA constitutional dynamic network (CDN)‐derived adaptive threshold is shown, which is defined by the difference value of the two groups of the output patterns from CDN. Under external stimuli, the threshold is adaptive to the generated dynamic output patterns, which avoids the contrary binary translation from a slight difference on the output under fixed threshold. Importantly, there are two self‐calibrating patterns in each output group and the total concentration of constituents from the CDN system are constant, which greatly eliminates the data error. The CDN system is accompanied by computational simulation, which can predict the output patterns of the system at different states. The CDN is used to control the orthogonal and cascaded nanoparticle‐based molecular amplifiers to expand the volume of the transmitting message, as well as allow the accurate and specific sensing of DNA. Various state‐of‐the‐art representation is demonstrated by coding and decoding different types of messages. This study presents a DNA‐based constitutional dynamic network (CDN) with adaptive thresholds for secure information decryption. The system self‐calibrates output patterns, ensuring reproducibility and accuracy, and integrates molecular amplifiers for expanded message capacity and precise DNA sensing.

Liquid–liquid phase separation (LLPS) phenomena are ubiquitous in biological systems, as various cellular LLPS structures control important biological processes. Due to their ease of in vitro assembly into membraneless compartments and their presence within modern cells, LLPS systems have been postulated to be one potential form that the first cells on Earth took on. Recently, liquid crystal (LC)-coacervate droplets assembled from aqueous solutions of short double-stranded DNA (s-dsDNA) and poly-L-lysine (PLL) have been reported. Such LC-coacervates conjugate the advantages of an associative LLPS with the relevant long-range ordering and fluidity properties typical of LC, which reflect and propagate the physico-chemical properties of their molecular constituents. Here, we investigate the structure, assembly, and function of DNA LC-coacervates in the context of prebiotic molecular evolution and the emergence of functional protocells on early Earth. We observe through polarization microscopy that LC-coacervate systems can be dynamically assembled and disassembled based on prebiotically available environmental factors including temperature, salinity, and dehydration/rehydration cycles. Based on these observations, we discuss how LC-coacervates can in principle provide selective pressures effecting and sustaining chemical evolution within partially ordered compartments. Finally, we speculate about the potential for LC-coacervates to perform various biologically relevant properties, such as segregation and concentration of biomolecules, catalysis, and scaffolding, potentially providing additional structural complexity, such as linearization of nucleic acids and peptides within the LC ordered matrix, that could have promoted more efficient polymerization. While there are still a number of remaining open questions regarding coacervates, as protocell models, including how modern biologies acquired such membraneless organelles, further elucidation of the structure and function of different LLPS systems in the context of origins of life and prebiotic chemistry could provide new insights for understanding new pathways of molecular evolution possibly leading to the emergence of the first cells on Earth.

Plant genetic engineering vectors, such as RNA interference (RNAi) and CRISPR/Cas9 vectors, are important tools for plant functional genomics. Efficient construction of these functional vectors can facilitate the study of gene function. Although some methods for vector construction have been reported, their operations are still complicated and costly. Here, we describe a simpler and low-cost vector construction method by nicking endonucleases-mediated DNA assembly (NEMDA), which uses nicking endonucleases to generate single-strand overhanging complementary ends for rapid assembly of DNA fragments into plasmids. Using this approach, we rapidly completed the construction of four RNAi vectors and a CRISPR/Cas9 knockout vector with five single-guide RNA (sgRNA)-expression cassettes for multiplex genome editing, and successfully achieved the goal of decreasing the expression of the target genes and knocking out the target genes at the same time in rice. These results indicate the great potential of NEMDA in assembling DNA fragments and constructing plasmids for molecular biology and functional genomics.

Bacterial DNA methylases are a diverse group of enzymes which have been pivotal in the development of technologies with applications including genetic engineering, bacteriology, biotechnology and agriculture. This review describes bacterial DNA methylase types, the main technologies for targeted methylation or demethylation and the recent roles of these enzymes in molecular and synthetic biology. Bacterial methylases can be exocyclic or endocyclic and can exist as orphan enzymes or as a part of the restriction-modifications (R-M) systems. As a group, they display a rich diversity of sequence-specificity. Additional technologies for targeting methylation involve using fusion proteins combining a methylase and a DNA-binding protein (DNBP) such as a zinc-finger (ZF), transcription activator-like effector (TALE) or CRISPR/dCas9. Bacterial methylases have contributed significantly to the creation of novel DNA assembly techniques, to the improvement of bacterial transformation and to crop plant engineering. Future studies to define the characteristics of more bacterial methylases have potential to identify new tools of value in synthetic and molecular biology and with widespread applications. Key points • Bacterial methylases can be used to direct methylation to specific sequences in target DNA • DNA methylation using bacterial methylases has been applied to improve DNA assembly and to increase the efficiency of bacterial transformation • Site-selective methylation using bacterial methylases can alter plant gene expression and phenotype

Circulating tumor cells (CTCs) carry intact tumor molecular information, making them invaluable for personalized cancer monitoring. However, conventional capture methods, relying on passive diffusion, suffer from low efficiency due to insufficient collision frequency, severely limiting clinical utility. Herein, a magnetic micromotor‐functionalized DNA‐array hunter (MMDA hunter) is developed by integrating enzyme‐propelled micromotors, magnetic nanoparticles, and nucleic acid aptamers into distinct functional partitions of a DNA tile self‐assembly structure. This design ensured independent and compatible running of autonomous propulsion, targeted recognition, and magnetic enrichment, enabling efficient capture and subsequent identification of CTCs in clinical blood samples. The autonomous motion of the MMDA hunter is powered by O2 bubbles generated through the dual enzymatic cascade reactions of glucose oxidase and catalase under physiological glucose conditions. Compared to static Fe3O4 arrays (without micromotors), the MMDA hunter shows more than 2‐fold improvement in capture efficiency. Meanwhile, it achieved superb precision, simple operation, rapid response, high biocompatibility, excellent stability, and superior specificity for CTC enrichment. This method provides a reliable tool for tumor diagnosis in multiple clinical application scenarios, even in primary medical care, simultaneously offering a clever solution for the bottleneck of functional‐module interference in multifunctional nanomaterials. The magnetic micromotor‐functionalized DNA‐array hunter (MMDA hunter), integrating autonomous propulsion and precision‐partitioned functionalities, utilizes DNA array programmability and 5'‐3' directional control to achieve spatially partitioned module arrangement. The autonomous motion of the MMDA hunter is powered by O2 bubbles generated through the dual enzymatic cascade reactions of glucose oxidase and catalase under physiological glucose conditions. This system ensures independent yet compatible running of autonomous propulsion, magnetic enrichment, and targeted recognition, enabling efficient capture and precise identification of circulating tumor cells (CTCs) in clinical blood samples.

The error-free repair of double-stranded DNA breaks by homologous recombination requires processing of broken ends. These processed ends are substrates for assembly of DNA strand exchange proteins that mediate DNA strand invasion. Here, we establish that human BLM helicase, a member of the RecQ family, stimulates the nucleolytic activity of human exonuclease 1 (hExo1), a 5'[rightward arrow]3' double-stranded DNA exonuclease. The stimulation is specific because other RecQ homologs fail to stimulate hExo1. Stimulation of DNA resection by hExo1 is independent of BLM helicase activity and is, instead, mediated by an interaction between the 2 proteins. Finally, we show that DNA ends resected by hExo1 and BLM are used by human Rad51, but not its yeast or bacterial counterparts, to promote homologous DNA pairing. This in vitro system recapitulates initial steps of homologous recombination and provides biochemical evidence for a role of BLM and Exo1 in the initiation of recombinational DNA repair.