Synthetic Constructs Research Articles

Sustained unidirectional rotation of a self-organized DNA rotor on a nanopore

Flow-driven rotary motors such as windmills and water wheels drive functional processes in human society. Although examples of such rotary motors also feature prominently in cell biology, their synthetic construction at the nanoscale has remained challenging. Here we demonstrate flow-driven rotary motion of a self-organized DNA nanostructure that is docked onto a nanopore in a thin solid-state membrane. An elastic DNA bundle self-assembles into a chiral conformation upon phoretic docking onto the solid-state nanopore, and subsequently displays a sustained unidirectional rotary motion of up to 20 rev s−1. The rotors harness energy from a nanoscale water and ion flow that is generated by a static chemical or electrochemical potential gradient in the nanopore, which are established through a salt gradient or applied voltage, respectively. These artificial nanoengines self-organize and operate autonomously in physiological conditions, suggesting ways to constructing energy-transducing motors at nanoscale interfaces.

A Synthetic Hydrogel Composite with a Strength and Wear Resistance Greater than Cartilage

AbstractKey hurdles for replacing damaged cartilage with an equivalent synthetic construct are the development of a hydrogel with a strength that exceeds that of cartilage and fixation of this hydrogel onto the surface of an articulating joint. This article describes the first hydrogel with a tensile and compressive strength (51 and 98 MPa) that exceeds those of cartilage (40 and 59 MPa), and the first attachment of a hydrogel to a metal backing with a shear strength (2.0 MPa) that exceeds that of cartilage on bone (1.2 MPa). The hydrogel strength is achieved through reinforcement of crystallized polyvinyl alcohol with bacterial cellulose. The high attachment strength is achieved by securing freeze‐dried bacterial cellulose to a metal backing with an adhesive and a shape memory alloy clamp prior to infiltration and crystallization of the polyvinyl alcohol. The bacterial cellulose‐reinforced polyvinyl alcohol is three times more wear resistant than cartilage over one million cycles and exhibits the same coefficient of friction. These advances in hydrogel strength and attachment enable the creation of a hydrogel‐based implant for durable resurfacing of damaged articulating joints.

Robust counterselection and advanced λRed recombineering enable markerless chromosomal integration of large heterologous constructs.

Despite advances in bacterial genome engineering, delivery of large synthetic constructs remains challenging in practice. In this study, we propose a straightforward and robust approach for the markerless integration of DNA fragments encoding whole metabolic pathways into the genome. This approach relies on the replacement of a counterselection marker with cargo DNA cassettes via λRed recombineering. We employed a counterselection strategy involving a genetic circuit based on the CI repressor of λ phage. Our design ensures elimination of most spontaneous mutants, and thus provides a counterselection stringency close to the maximum possible. We improved the efficiency of integrating long PCR-generated cassettes by exploiting the Ocr antirestriction function of T7 phage, which completely prevents degradation of unmethylated DNA by restriction endonucleases in wild-type bacteria. The employment of highly restrictive counterselection and ocr-assisted λRed recombineering allowed markerless integration of operon-sized cassettes into arbitrary genomic loci of four enterobacterial species with an efficiency of 50–100%. In the case of Escherichia coli, our strategy ensures simple combination of markerless mutations in a single strain via P1 transduction. Overall, the proposed approach can serve as a general tool for synthetic biology and metabolic engineering in a range of bacterial hosts.

SRCLoc: Synthetic Radio Map Construction Method for Fingerprinting Outdoor Localization in Hybrid Networks

A precise localization system is a key enabling technology for Internet of Things (IoT) applications and location-based services. Fingerprint-based localization methods are well-known and widely used solutions. These methods, however, are time-consuming and laborious for radio map construction during an offline site survey in large-scale applications. In this article, we presented a novel semi-supervised deep convolutional generative adversarial network-based radio map construction method for real-time device localization. The proposed synthetic radio map construction method for fingerprinting outdoor localization (SRCLoc) combined the hybrid support vector machine and deep gated recurrent unit algorithms sequentially. The SRCLoc reduced the workload of site surveying required to build the fingerprint database by up to 85.7%. The results show that the average positioning error of SRCLoc is less than 39 cm, and more than 90% of the errors are less than 82 cm. That is, numerical results proved that, in comparison to traditional methods, the proposed SRCLoc method can significantly improve positioning performance and reduce radio map construction costs.

The Relationship Between Benevolent Leadership and Organizational Citizenship Behavior

Purpose: The purpose of this study is to assess the relationship between benevolent leadership and employees’ organizational citizenship behavior in the context of Polish organizations. Design/methodology/approach: The data were obtained from 415 company employees. The relationship was investigated using structural equation models (SEMs). Findings: The analyses of the results showed that benevolent leadership has a positive relationship with OCB. The more benevolent the leadership, the more frequent the organizational citizenship behaviors among workers. Research limitations/implications: There are some limitations to this study. The results cannot be generalized since the sample was not selected probabilistically. It should be added that the research was conducted only in enterprises. The analyzed relationship between BL and OCB in public institutions, which are by definition established to serve the public, could also be examined. The research provided a significant contribution to the literature, particularly in the field of leadership. First, it demonstrates that benevolent leadership as a synthetic construct may be applied in a variety of cultural contexts. Secondly, the study findings support predictions derived from the social exchange theory. Benevolent leaders, and thus fair and trustworthy ones, will motivate their subordinates to emulate their positive behavior. Originality/value: The article presents a fragment of the results of research conducted for the first time in Poland on the topic of benevolent leadership.

IFLinkC-X: A Scalable Framework to Assemble Bespoke Genetically Encoded Co-polymeric Linkers of Variable Lengths and Amino Acid Composition.

Linker engineering is rapidly gaining prominence as protein engineers and synthetic biologists construct increasingly sophisticated protein assemblies capable of executing complex molecular functions in the context of biosensing, biocatalysis, or biotherapeutics. Depending on the application, the structural and functional requirements imposed on the underlying linkers can differ vastly. At the same time, there is a distinct lack of methods to effectively code linkers at the level of DNA and tailor them to the functional requirements of different fusion proteins. Addressing these limitations, a scalable framework is presented to compose co-polymeric linkers of variable lengths and amino acid composition based on a limited number of linker fragments stored in sequence-verified entry plasmids. The assembly process is exemplified for Pro-rich linkers in the context of a Zn2+-responsive dual-readout BRET/FRET sensor while examining how linker composition impacts key functional properties such as ligand affinity, dynamic range, and their ability to separate structurally distinct domains.

Dual-modality synthetic mammogram construction for breast lesion detection using U-DARTS

Multimodal image fusion is an emergent research area for cancer detection. It provides a wide variety of visual qualities for the accurate medical diagnosis. However, this process requires accurate registration of each image modality for its efficient and effective use. To address the aforementioned issue, a novel synthetic mammogram construction model is proposed. An image enhancement approach is applied to enhance the image quality. Dual-modality structural feature (DMSF) based mapping function is designed to transform a mammogram from a thermal image segment. This paper also proposes modified Differentiable ARchiTecture Search (DARTS) named as (U-DARTS) to detect and classify the breast lesion. In U-DARTS, a stochastic gradient descent optimizer is used. The proposed approach is tested over DMR and INbreast datasets. The results exhibit a significant improvement in the performance of the proposed model over the existing techniques. The validation and testing accuracies of 98% and 91%, respectively, are achieved. Overall, the proposed approach establishes supremacy in so far as the mammogram construction and further lesion detection are concerned.

Controllable assembly of synthetic constructs with programmable ternary DNA interaction.

Compared with the dual binding components in a binary interaction, the third component of a ternary interaction often serves as modulator or regulator in biochemical processes. Here, we presented a programmable ternary interaction strategy based on the natural DNA triplex structure. With the DNA triplex-based ternary interaction, we have successfully demonstrated controllable hierarchical assemblies from nanometer scale synthetic DNA nanostructure units to micrometer scale live bacteria. A selective signaling system responsive to orthogonal nucleic acid signals via ternary interaction was also demonstrated. This assembly method could further enrich the diversified design schemes of DNA nanotechnology.

Synthetic Heparan Sulfate Hydrogels Regulate Neurotrophic Factor Signaling and Neuronal Network Activity.

Three-dimensional (3D) synthetic heparan sulfate (HS) constructs possess promising attributes for neural tissue engineering applications. However, their sulfation-dependent ability to facilitate molecular recognition and cell signaling has not yet been investigated. We hypothesized that fully sulfated synthetic HS constructs (bearing compound 1) that are functionalized with neural adhesion peptides will enhance fibroblast growth factor-2 (FGF2) binding and complexation with FGF receptor-1 (FGFR1) to promote the proliferation and neuronal differentiation of human neural stem cells (hNSCs) when compared to constructs with unsulfated controls (bearing compound 2). We tested this hypothesis in vitro using 2D and 3D substrates consisting of different combinations of HS tetrasaccharides (compounds 3 and 4) and an engineered integrin-binding chimeric peptide (CP), which were assembled using strain-promoted alkyne-azide cycloaddition (SPAAC) chemistry. Results indicated that the adhesion of hNSCs increased significantly when cultured on 2D glass substrates functionalized with chimeric peptide. hNSCs encapsulated in 1-CP hydrogels and cultured in media containing the mitogen FGF2 exhibited significantly higher neuronal differentiation when compared to hNSCs in 2-CP hydrogels. These observations were corroborated by Western blot analysis, which indicated the enhanced binding and retention of both FGF2 and FGFR1 by 1 as well as downstream phosphorylation of extracellular signal-regulated kinases (ERK1/2) and enhanced proliferation of hNSCs. Lastly, calcium activity imaging revealed that both 1 and 2 hydrogels supported the neuronal growth and activity of pre-differentiated human prefrontal cortex neurons. Collectively, these results demonstrate that synthetic HS hydrogels can be tailored to regulate growth factor signaling and neuronal fate and activity.

Platforms for Optogenetic Stimulation and Feedback Control

Harnessing the potential of optogenetics in biology requires methodologies from different disciplines ranging from biology, to mechatronics engineering, to control engineering. Light stimulation of a synthetic optogenetic construct in a given biological species can only be achieved via a suitable light stimulation platform. Emerging optogenetic applications entail a consistent, reproducible, and regulated delivery of light adapted to the application requirement. In this review, we explore the evolution of light-induction hardware-software platforms from simple illumination set-ups to sophisticated microscopy, microtiter plate and bioreactor designs, and discuss their respective advantages and disadvantages. Here, we examine design approaches followed in performing optogenetic experiments spanning different cell types and culture volumes, with induction capabilities ranging from single cell stimulation to entire cell culture illumination. The development of automated measurement and stimulation schemes on these platforms has enabled researchers to implement various in silico feedback control strategies to achieve computer-controlled living systems—a theme we briefly discuss in the last part of this review.

Sugar-Assisted Cryopreservation of Stem Cell-Laden Gellan Gum-Collagen Interpenetrating Network Hydrogels.

Tissue engineering involves the transplantation of stem cell-laden hydrogels as synthetic constructs to replace damaged tissues. However, their time-consuming fabrication procedures are hurdles to widespread application in clinics. Fortunately, similar to cell banking, synthetic tissues could be cryopreserved for subsequent central distribution. Here, we report the use of trehalose and gellan gum as biomacromolecules to form a cryopreservable yet directly implantable hydrogel system for adipose-derived stem cell (ADSC) delivery. Through a modified cell encapsulation method and a preincubation step, adequate cryoprotection was afforded at 0.75 M trehalose to the encapsulated ADSCs. At this concentration, trehalose demonstrated lower propensity to induce apoptosis than 10% DMSO, the current gold standard cryoprotectant. Moreover, when cultured along with trehalose after thawing, the encapsulated ADSCs retained their stem cell-like phenotype and osteogenic differentiation capacity. Taken together, this study demonstrates the feasibility of an “off-the-shelf” biomacromolecule-based synthetic tissue to be applied in widespread tissue engineering applications.

Easy efficient HDR-based targeted knock-in in Saccharomyces cerevisiae genome using CRISPR-Cas9 system

ABSTRACT During the last two decades, yeast has been used as a biological tool to produce various small molecules, biofuels, etc., using an inexpensive bioprocess. The application of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein (Cas) techniques in yeast genetic and metabolic engineering has made a paradigm shift, particularly with a significant improvement in targeted chromosomal integration using synthetic donor constructs, which was previously a challenge. This study reports the CRISPR-Cas9-based highly efficient strategy for targeted chromosomal integration and in-frame expression of a foreign gene in the genome of Saccharomyces cerevisiae (S. cerevisiae) by homology-dependent recombination (HDR); our optimized methods show that CRISPR-Cas9-based chromosomal targeted integration of small constructs at multiple target sites of the yeast genome can be achieved with an efficiency of 74%. Our study also suggests that 15 bp microhomology flanked arms are sufficient for 50% targeted knock-in at minimal knock-in construct concentration. Whole-genome sequencing confirmed that there is no off-target effect. This study provides a comprehensive and streamlined protocol that will support the targeted integration of essential genes into the yeast genome for synthetic biology and other industrial purposes. Highlights • CRISPR-Cas9 based in-frame expression of foreign protein in Saccharomyces cerevisiae using Homology arm without a promoter. • As low as 15 base pairs of microhomology (HDR) are sufficient for targeted integration in Saccharomyces cerevisiae. • The methodology is highly efficient and very specific as no off-targeted effects were shown by the whole-genome sequence.

(Un)Easily Possible Synthetic Biology

AbstractSynthetic biology has a strong modal dimension that is part and parcel of its engineering agenda. In turning hypothetical biological designs into actual synthetic constructs, synthetic biologists reach toward potential biology instead of concentrating on naturally evolved organisms. We analyze synthetic biology’s goal of making biology easier to engineer through the combinatorial theory of possibility, which reduces possibility to (re)combinations of individuals and their attributes in the actual world. While the last decades of synthetic biology explorations have shown biology to be much more difficult to engineer than originally conceived, synthetic biology has not given up its combinatorial approach.

Achieving High-Performance Molecular Rectification through Fast Screening Alkanethiol Carboxylate-Metal Complexes Electroactive Units

Achieving High-Performance Molecular Rectification through Fast Screening Alkanethiol Carboxylate-Metal Complexes Electroactive Units

Intronic gRNAs for the Construction of Minimal Gene Drive Systems.

Gene drives are promising tools for the genetic control of insect vector or pest populations. CRISPR-based gene drives are generally highly complex synthetic constructs consisting of multiple transgenes and their respective regulatory elements. This complicates the generation of new gene drives and the testing of the behavior of their constituent functional modules. Here, we explored the minimal genetic components needed to constitute autonomous gene drives in Drosophila melanogaster. We first designed intronic gRNAs that can be located directly within coding transgene sequences and tested their functions in cell lines. We then integrated a Cas9 open reading frame hosting such an intronic gRNA within the Drosophila rcd-1r locus that drives the expression in the male and female germlines. We showed that upon removal of the fluorescent transformation marker, the rcd-1r d allele supports efficient gene drive. We assessed the propensity of this driver, designed to be neutral with regards to fitness and host gene function, to propagate in caged fly populations. Because of their simplicity, such integral gene drives could enable the modularization of drive and effector functions. We also discussed the possible biosafety implications of minimal and possibly recoded gene drives.

A systematic dissection of determinants and consequences of snoRNA-guided pseudouridylation of human mRNA.

RNA can be extensively modified post-transcriptionally with >170 covalent modifications, expanding its functional and structural repertoire. Pseudouridine (Ψ), the most abundant modified nucleoside in rRNA and tRNA, has recently been found within mRNA molecules. It remains unclear whether pseudouridylation of mRNA can be snoRNA-guided, bearing important implications for understanding the physiological target spectrum of snoRNAs and for their potential therapeutic exploitation in genetic diseases. Here, using a massively parallel reporter based strategy we simultaneously interrogate Ψ levels across hundreds of synthetic constructs with predesigned complementarity against endogenous snoRNAs. Our results demonstrate that snoRNA-mediated pseudouridylation can occur on mRNA targets. However, this is typically achieved at relatively low efficiencies, and is constrained by mRNA localization, snoRNA expression levels and the length of the snoRNA:mRNA complementarity stretches. We exploited these insights for the design of snoRNAs targeting pseudouridylation at premature termination codons, which was previously shown to suppress translational termination. However, in this and follow-up experiments in human cells we observe no evidence for significant levels of readthrough of pseudouridylated stop codons. Our study enhances our understanding of the scope, ‘design rules’, constraints and consequences of snoRNA-mediated pseudouridylation.

Content Swapping: A New Image Synthesis for Construction Sign Detection in Autonomous Vehicles.

Construction signs alert drivers to the dangers of abnormally blocked roads. In the case of autonomous vehicles, construction signs should be detected automatically to prevent accidents. One might think that we can accomplish the goal easily using the popular deep-learning-based detectors, but it is not the case. To train the deep learning detectors to detect construction signs, we need a large amount of training images which contain construction signs. However, collecting training images including construction signs is very difficult in the real world because construction events do not occur frequently. To make matters worse, the construction signs might have dozens of different construction signs (i.e., contents). To address this problem, we propose a new method named content swapping. Our content swapping divides a construction sign into two parts: the board and the frame. Content swapping generates numerous synthetic construction signs by combining the board images (i.e., contents) taken from the in-domain images and the frames (i.e., geometric shapes) taken from the out-domain images. The generated synthetic construction signs are then added to the background road images via the cut-and-paste mechanism, increasing the number of training images. Furthermore, three fine-tuning methods regarding the region, size, and color of the construction signs are developed to make the generated training images look more realistic. To validate our approach, we applied our method to real-world images captured in South Korea. Finally, we achieve an average precision (AP50) score of 84.98%, which surpasses that of the off-the-shelf method by 9.15%. Full experimental results are available online as a supplemental video. The images used in the experiments are also released as a new dataset CSS138 for the benefit of the autonomous driving community.

Precision killing of T cells via targeting of T cell receptors using two novel chimeric antigen receptor designs

Abstract Pathologic T cells drive T cell leukemia, solid organ transplant rejection, and many autoimmune diseases. Currently approved treatments have limited ability to target pathogenic vs non-pathogenic T cells. While pan-T cell treatments can be effective, they have significant risks such as infection and malignancy. Chimeric antigen receptors (CAR) are synthetic constructs that retarget T cells to a specific antigen of interest; we hypothesized that we could use this technology for targeted killing of T cells. We developed two novel CAR designs which target T cells via their T cell receptor (TCR). We opted to target OT1 T cells, a model system with a known antigenic epitope. The anti-Vβ5 CAR is a second-generation CAR with a traditional extracellular scFv domain that targets Vβ5 of the OT1 TCR. The H2Kb bait CAR instead has an extracellular H2Kb complex loaded with the SIINFEKL cognate epitope. Both anti-Vβ5 and H2Kb bait CAR T cells demonstrate degranulation, activation, and proliferation upon binding OT1 T cells. In vitro co-culture with OT1 T cells results in internalization of the OT1 TCR and specific killing of OT1 T cells. Interestingly, the changes induced by the H2Kb bait CAR were delayed relative to the anti-Vβ5 CAR. In vivo experiments show both CAR designs kill adoptively transferred OT1 T cells without significant effect on the other T cell populations. In conclusion, we report the development of two novel CAR designs which allow for the targeted killing of T cells via their T cell receptor. While the anti-Vβ5 CAR T cells generated a more rapid response, the H2Kb bait CAR offers the promise of more precisely targeting T cells. This work has the potential to transform the treatment of conditions where removal of pathogenic T cells is critical for cure. Supported by NIH HL152756

Wolffia, a minimalist plant and synthetic biology chassis.

A highly simplified species for genome engineering would facilitate rational design of a synthetic plant. A candidate species is the aquatic, non-grass monocot wolffia (Wolffia australiana) in the Lemnaceae family. Commonly known as watermeal, wolffia is a rootless ball of several thousand cells the size of a pinhead and the fastest growing plant known on Earth. Its extreme morphological reduction is coupled to transposon-mediated streamlining of its transcriptome, which represents a core set of nonredundant protein coding genes. Despite its body plan and transcriptome being highly specialized for continuous growth, wolffia retains cell types relevant to higher plants. Systems level studies with this species could enable the creation of a defined biological chassis for synthetic plant construction.

Functional genome-wide short hairpin RNA library screening identifies key molecules for extracellular vesicle secretion from microglia

SUMMARYActivated microglia release extracellular vesicles (EVs) as modulators of brain homeostasis and innate immunity. However, the molecules critical for regulating EV production from microglia are poorly understood. Here we establish a murine microglial cell model to monitor EV secretion by measuring the fluorescence signal of tdTomato, which is linked to tetraspanin CD63. Stimulation of tdTomato+ cells with ATP induces rapid secretion of EVs and a reduction in cellular tdTomato intensity, reflecting EV secretion. We generate a GFP+ tdTomato+ cell library expressing TurboGFP and barcoded short hairpin RNAs for genome-wide screening using next-generation sequencing. We identify Mcfd2, Sepp1, and Sdc1 as critical regulators of ATP-induced EV secretion from murine microglia. Small interfering RNA (siRNA-based) silencing of each of these genes suppresses lipopolysaccharide- and ATP-induced inflammasome activation, as determined by interleukin-1β release from primary cultured murine microglia. These molecules are critical for microglial EV secretion and are potential therapeutic targets for neuroinflammatory disorders.