SEARCH · bioRxiv Science

Results for “Synthetic Biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 973 records · Page 54Linked to original sources

bioRxiv2026

Handwritten Digit classification with neural cultures is influenced by neural architecture, network dynamics, and decoding methods

As silicon-based computing approaches fundamental physical limits, neurocomputing offers an energy-efficient alternative by leveraging the intrinsic non-linear dynamics of biological systems. To harness these dynamics, it is vital to understand the structure-function relationship governing how neural cultures process complex spatio-temporal information and how to appropriately decode the resulting neural electrophysiological activity. We investigated this utilizing a closed-loop electrophysiology platform, the CL1, to implement reservoir computing in human iPSC-derived neuronal networks. To systematically evaluate the variables driving neurocomputational capacity, we explored how cellular composition (cortical vs. hippocampal lineages), and the physical architecture (unstructured monolayers, 3D neural organoids, and modular networks confined by microfluidic devices) influenced electrophysiological properties and interacted with different decoding methodologies. Using a spatio-temporal version of a handwritten digit pattern recognition task (MNIST), we analyzed how these biological and analytical factors influenced classification accuracy. To ensure robust interpretation this required us to first demonstrated that reservoir computing decoding methods require strict artifact control and trial-based cross-validation to distinguish network computation from artifactual signal separability or temporal data leakage. Applying this validated frequency-domain pipeline, we suggest a clear functional hierarchy where structural modularity acts as a vital functional regularizer. Modular cortical cultures significantly outperformed unconstrained monolayers and organoids on MNIST. Furthermore, decoding frequency information from raw signals proved superior to typical time-bin decoding implementations. These findings establish that maximizing the computational potential of Synthetic Biological Intelligence, while avoiding false positives, requires a synergistic optimization of cellular identity, structural governance, and rigorous decoding logic. In doing so, this work provides a critical base establishing the criteria under which to evaluate neurocomputing implementations.

neuroscience↗

bioRxiv2025

Optimizing Bispecific Antibody Expression via Multi-Omics Analysis and Vector Redesign

Bispecific antibodies are a growing class of therapeutics that simultaneously engage two targets. However, their complex molecular structures pose challenges for production in Chinese hamster ovary cells, the current industry standard for biologics manufacturing. Here we present a case study of three IgG-scFv format bsAbs expressed in CHO cells, in which one candidate exhibited markedly lower titers despite high sequence homology to the other two. Using multi-omics analysis (RNA sequencing, splicing prediction, codon optimization assessment, and motif screening) to investigate potential causes, we identified several likely mechanisms for poor expression, including aberrant splicing motifs, ribosome pausing sites, and suboptimal codon usage. Through targeted protein and DNA sequence engineering, we generated a revised variant with an 11-fold increase in stable expression titers. This work demonstrates that integrating sequence-level bioinformatic and synthetic biology diagnostics can directly improve manufacturability, providing a generalizable framework for resolving hidden expression liabilities in complex biologics.

bioengineering↗

bioRxiv2019

Automated design of highly diverse riboswitches

Riboswitches that couple binding of ligands to recruitment of molecular machines offer sensors and control elements for RNA synthetic biology and medical biotechnology. Current approaches to riboswitch design enable significant changes in output activity in the presence vs. absence of input ligands. However, design of these riboswitches has so far required expert intuition and explicit specification of complete target secondary structures, both of which limit the structure-toggling mechanisms that have been explored. We present a fully automated method called RiboLogic for these design tasks and high-throughput experimental tests of 2,875 molecules using RNA-MaP (RNA on a massively parallel array) technology. RiboLogic designs explore an unprecedented diversity of structure-toggling mechanisms validated through experimental tests. These synthetic molecules consistently modulate their affinity to the MS2 bacteriophage coat protein upon binding of flavin mononucleotide, tryptophan, theophylline, and microRNA miR-208a, achieving activation ratios of up to 20 and significantly better performance than control designs. The data enable dissection of features of structure-toggling mechanisms that correlate with higher performance. The diversity of RiboLogic designs and their quantitative experimental characterization provides a rich resource for further improvement of riboswitch models and design methods.

biophysics↗

bioRxiv2026

Metabolic commitment and nitrogen control of diazotrophy in the diazoplast-containing diatom Epithemia adnata

Earths nitrogen cycle is central to sustaining ecosystem productivity and global biogeochemical balance. Although biological N2-fixation is well characterized in prokaryotes and plant symbioses, in other eukaryotic lineages it remains poorly understood. Diatoms of the family Rhopalodiacea harbor diazoplasts, endosymbiotic spheroid bodies specialized for N2-fixation. This makes these diatoms genuine N2-fixing eukaryotes that represent a unique model for organelle evolution, parallel but distinct from haptophyte nitroplasts. Here, we report the isolation and stable cultivation of an Epithemia adnata strain, the sequencing of its diazoplast genome and its proteomic profile when growing diazotrophically in the light or darkness, or upon exposure to ammonium. Our analyses reveal that ammonium induced broad down-regulation of diazoplast proteins, particularly those linked to N2-fixation, ATP synthesis, and central carbon metabolism underscoring a general regulatory commitment toward diazotrophic metabolism tightly coupled to host carbon and nitrogen status. The pentose phosphate pathway and ferredoxin-NADP oxidoreductase appear as likely source of reductant to nitrogenase. A striking enrichment of chaperones, peroxiredoxins, bacterioferritin-like proteins, and DpsA might stabilize nitrogenase and buffer against oxidative stress during light-driven diazotrophy. Importantly, we identified a plasmid-encoded GlpF as a putative glycerol transporter, pointing to glycerol-mediated host-symbiont metabolic integration in the extant symbiosis and possibly a crucial innovation during the early evolutionary stages of its establishment. Thus, diazoplast activity is not autonomous but requires integration with host carbon and nitrogen status, establishing glycerol transport, reductant supply, stress mitigation, and nutrient-responsive regulation as pivotal mechanisms of nitrogenase activity and host integration. These findings have broad implications for biogeochemical cycling, organellogenesis, and synthetic biology strategies aimed at engineering N2-fixation in crop plants. SignificanceN2-fixing eukaryotes are increasingly recognized as abundant algae containing bacterial-derived diazotrophic endosymbionts, representing an underappreciated component of global N cycling. Diazoplasts in rhopalodiacean diatoms represent a compelling example of such endosymbionts specialized for N2-fixation. By combining genomic sequencing and proteomic analysis, we demonstrate their metabolic specialization, host integration, and regulatory commitment to diazotrophy. These findings reinforce the emerging view that diazoplasts function as organelle-like entities dedicated to N2-fixation, dependent on host-supplied carbon while contributing fixed N in return. Beyond giving evolutionary insights into organellogenesis, this work establishes a framework for translational applications, such as engineering N2-fixation into agricultural plants. Such advances could reduce reliance on synthetic fertilizers, influence biogeochemical cycles, and promote sustainable food production.

plant biology↗

bioRxiv2021

Lipid membranes modulate the activity of RNA through sequence-specific interactions

RNA is a ubiquitous biomolecule that can serve as both catalyst and information carrier. Understanding how RNA bioactivity is controlled is crucial for elucidating its physiological roles and potential applications in synthetic biology. Here we show that lipid membranes can act as RNA organization platforms, introducing a novel mechanism for ribo-regulation. The activity of R3C ribozyme can be modified by the presence of lipid membranes, with direct RNA-lipid interactions dependent on RNA nucleotide content, base pairing and length. In particular, the presence of guanine in short RNAs is crucial for RNA-lipid interactions, and G-quadruplex formation further promotes lipid binding. Lastly, by artificially modifying the R3C substrate sequence to enhance membrane binding we generated a lipid-sensitive ribozyme reaction with riboswitch-like behavior. These findings introduce RNA-lipid interactions as a tool for developing synthetic riboswitches and novel RNA-based lipid biosensors, and bear significant implications for RNA World scenarios for the origin of life.

biochemistry↗

bioRxiv2023

Mapping the landscape of transcription factor promoter activity during vegetative development in Marchantia

Transcription factors (TFs) are essential for the regulation of gene expression and cell fate determination. Characterising the transcriptional activity of TF genes in space and time is a critical step towards understanding complex biological systems. The vegetative gametophyte meristems of bryophytes share some characteristics with the shoot-apical meristems of flowering plants. However, the identity and expression profiles of TFs associated with gametophyte organization are largely unknown. With only [~]450 TF genes, Marchantia polymorpha is an outstanding model system for plant systems biology. We have generated a near-complete collection of promoter elements derived from Marchantia TF genes. We experimentally tested in planta reporter fusions for all the TF promoters in the collection and systematically analysed expression patterns in Marchantia gemmae. This allowed us to build a map of precise expression domains and identify a unique set of TFs expressed in the stem-cell zone, providing new insight into the dynamic regulation of the gametophytic meristem and its evolution. In addition, we provide an online database of expression patterns for all promoters in the collection. We expect that the promoter elements characterised here will be useful for cell-type specific expression, synthetic biology applications, and functional genomics.

plant biology↗

bioRxiv2024

Tumor-specific antibodies elicited by engineered bacteria promote bladder cancer immunotherapy

The intratumoral microbiome has recently emerged as a new hallmark of cancer, with implications for response or resistance to therapy. While bacteria can either promote or inhibit cancer growth, intratumoral bacteria can also be engineered using synthetic biology to remodel the tumor microenvironment. Here, we engineered the probiotic bacterium E. coli Nissle 1917 (EcN) to express the human chemokine CXCL13, a critical component of germinal center (GC) formation. The GC reaction is a fundamental aspect of adaptive immunity by which antibody affinity develops in secondary lymphoid organs for defense against pathogens. Using orthotopic models of bladder cancer, engineered CXCL13-expressing EcN colonized bladder tumors and elicited GC responses in bladder tumor-draining lymph nodes after intravesical delivery. Furthermore, when combined with PD-1 blockade, engineered EcN amplified the antitumor antibody response and promoted long-term survival and protective immunity upon tumor rechallenge. Thus, we demonstrate that synthetically engineered CXCL13-expressing EcN can enhance the efficacy of PD-1 checkpoint blockade immunotherapy by amplifying tumor-specific humoral immunity.

immunology↗

bioRxiv2026

Metabolic reprogramming and stress mitigation of Chlamydomonas reinhardtii using protective metal-phenolic networks

Single-cell encapsulation is widely used in biotechnology to protect highly sensitive living cells from environmental stress. However, the impact of encapsulation on biological function and metabolism remains rarely explored despite its importance for understanding stress response and modulating the production of valuable metabolites. Herein, we show that coating individual cells with protective metal-phenolic networks not only improves survival against stress, but also modifies cellular metabolism. Importantly, this encapsulation technique induces a reversible state of quiescence, which enables accumulation of high-energy-density compounds through selective nutrient diffusion and the resulting carbon flux redistribution. Specifically, light exposure favors the accumulation of nearly eightfold higher starches, while incubation in darkness leads to twofold higher lipid accumulation compared to native cells. This metabolic engineering approach via individual cell encapsulation expands the cell editing toolbox and will facilitate applications in synthetic biology, bioengineering, and cell-based therapeutics. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=184 SRC="FIGDIR/small/711231v2_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@d27478org.highwire.dtl.DTLVardef@dc73baorg.highwire.dtl.DTLVardef@131dcaeorg.highwire.dtl.DTLVardef@8f3dc4_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Rewiring native post-transcriptional carbon regulators to build multi-layered genetic circuits and optimize engineered microbes for bioproduction

As the bioprocessing and synthetic biology spaces rapidly expand, post-transcriptional regulation is emerging as driver for maximizing signal response rate and for minimizing cost per protein within cells. Designing robust post-transcriptional control systems that have precise tunability and can achieve diverse regulatory outcomes are paramount to advance the field. Herein, we develop a new approach for engineered post-transcriptional control in bacteria by rewiring a native regulatory system in Escherichia coli, the Carbon Storage Regulatory (Csr) Network, to create tunable, complex genetic circuits. First, by co-opting native components of the Csr Network to regulate translation of a target mRNA transcript, we establish a Csr-regulated Buffer Gate. Next, by rationally engineering the interactions between our synthetic construct and the native components of the Csr Network, we expand our original design into a genetic toolbox of 12 Buffer Gates that achieve precise tunability across a 10-fold range of target gene expression. Subsequently, to further regulatory capabilities using this approach, we develop a Csr-regulated NOT Gate through engineering a Csr-activated sequence into our synthetic constructs. We then build upon the Csr Buffer and Not Gates to create post-transcriptional dual input Boolean OR, NOR, AND and NAND Logic Gates, as well as a genetic pulse circuit. As a third step, we demonstrate portability of our Csr-regulated Buffer Gates into three industrially relevant bacteria by recapitulating Buffer Gate activity simply by leveraging the conserved homologous Csr Network in each species. Lastly, as a demonstration of downstream application, we apply our system to a proof-of-concept synthetic mevalonate pathway. Using our engineered constructs, we optimize mevalonate production in E. coli resulting in a three-fold increase in production relative to a transcriptionally controlled mevalonate pathway. As a whole, we establish a novel approach to rewire post-transcriptional regulatory networks for complex bacterial computation that can be utilized for efficient bioproduction in engineered microbes.

bioengineering↗

bioRxiv2023

Structure-function analysis of the cyclic beta-1,2-glucan synthase

The synthesis of complex sugars is a key aspect of microbial biology. Cyclic {beta}-1,2-glucan (C{beta}G) is a circular polysaccharide critical for host interactions of many bacteria, including major pathogens of humans (Brucella) and plants (Agrobacterium). C{beta}G is produced by the cyclic glucan synthase (Cgs), a massive multi-domain membrane protein. So far, its structure as well as the mechanisms underlining the synthesis have not been clarified. Here we use cryo-electron microscopy (cryo-EM) and functional approaches to study Cgs from A. tumefaciens. We were able to determine the structure of this complex protein machinery and clarify key aspects of C{beta}G synthesis. Our research opens new possibilities for combating pathogens that rely on polysaccharide virulence factors and can lead to new synthetic biology approaches for producing complex cyclic sugars.

molecular biology↗

bioRxiv2020

Capsid Proteins are Necessary for Replication of a Densovirus

AO_SCPLOWBSTRACTC_SCPLOWDespite tight genetic compression, viral genomes are often organized in functional gene clusters, a modular structure that might favor their evolvability. This has greatly facilitated biotechnological developments, such as the recombinant Adeno-Associated Virus (AAV) systems for gene therapy. Following this lead, we endeavored to engineer the related insect parvovirus Junonia coenia densovirus (JcDV) to create addressable vectors for insect pest biocontrol. To enable safer manipulation of capsid mutants, we translocated the non-structural (ns) gene cluster outside the viral genome. To our dismay, this yielded a virtually non-replicable clone. We linked the replication defect to an unexpected modularity breach, as ns translocation truncated the overlapping 3 UTR of the capsid transcript (vp). We found that native vp 3UTR is necessary to high VP production, but that decreased expression do not adversely impact the expression of NS proteins, which are known replication effectors. As nonsense vp mutations recapitulate the replication defect, VP proteins appear directly implicated in the replication process. Our findings suggest intricate replication-encapsidation couplings that favor maintenance of genetic integrity. We discuss possible connections with an intriguing cis-packaging phenomenon previously observed in parvoviruses, whereby capsids preferentially package the genome from which they were expressed. IO_SCPLOWMPORTANCEC_SCPLOWDensoviruses could be used as biological control agents to manage insect pests. Such applications require in depth biological understanding and associated molecular tools. However, the genomes of these viruses remain hard to manipulate due too poorly tractable secondary structures at their extremities. We devised a construction strategy that enable precise and efficient molecular modifications. Using this approach, we endeavored to create a split clone of the Junonia coenia densovirus (JcDV) that can be used to safely study the impact of capsid mutations on host specificity. Our original construct proved to be non-functional. Fixing this defect led us to uncover that capsid proteins and their correct expression are essential for continued rolling-hairpin replication. This points to an intriguing link between replication and packaging, which might be shared with related viruses. This serendipitous discovery illustrates the power of synthetic biology approaches to advance our knowledge of biological systems.

microbiology↗

bioRxiv2026

Coacervate Droplets as a Liquefier for the Solid-to-Liquid Transition of RNA Aggregates

Biomolecular condensates are functionally gated by their material properties. While RNA is a primary structural scaffold, its sequence-specific interactions can aberrantly drive condensates into dysfunctional solid aggregates. Yet, actively reversing this solidification to restore function remains a fundamental challenge, impeding progress in synthetic biology and therapeutics interventions. Here, we establish that complex coacervates can be engineered as liquefiers to actively remodel solid RNA-peptide aggregates into functional liquid droplets. Integrating systematic experiments with all-atom simulations, we decode a multiscale mechanism: coacervate infiltration at the micrometer scale mediates molecular buffering at the nanometer scale, which fluidizes the crosslinked network to drive macroscopic liquefaction. This capability is governed by a design rule, where coacervates formed by small-molecular anions exhibit optimal efficacy. We deploy this principle to functionally rescue silenced RNA within a model of pathologically solidified ribonucleoprotein assemblies. Our work provides a general framework for the active, compositional control of biomolecular phase behavior, with direct implications for managing pathological aggregation and engineering functional condensates in synthetic and living systems.

biophysics↗

bioRxiv2022

Optimizing Oscillators for Specific Tasks Predicts Preferred Biochemical Implementations

Oscillatory processes are used throughout cell biology to control time-varying physiology including the cell cycle, circadian rhythms, and developmental patterning. It has long been understood that free-running oscillations require feedback loops where the activity of one component depends on the concentration of another. Oscillator motifs have been classified by the positive or negative net logic of these loops. However, each feedback loop can be implemented by regulation of either the production step or the removal step. These possibilities are not equivalent because of the underlying structure of biochemical kinetics. By computationally searching over these possibilities, we find that certain molecular implementations are much more likely to produce stable oscillations. These preferred molecular implementations are found in many natural systems, but not typically in artificial oscillators, suggesting a design principle for future synthetic biology. Finally, we develop an approach to oscillator function across different reaction networks by evaluating the biosynthetic cost needed to achieve a given phase coherence. This analysis predicts that phase drift is most efficiently suppressed by delayed negative feedback loop architectures that operate without positive feedback. PACS numbers47.15.-x

systems biology↗

bioRxiv2021

Telomere-to-telomere genome assembly of Phaeodactylum tricornutum

Phaeodactylum tricornutum is a marine diatom with a growing genetic toolbox available and is being used in many synthetic biology applications. While most of the genome has been assembled, the currently available genome assembly is not a completed telomere-to-telomere assembly. Here, we used Oxford Nanopore long reads to build a telomere-to-telomere genome for Phaeodactylum tricornutum. We developed a graph-based approach to extract all unique telomeres, and used this information to manually correct assembly errors. In total, we found 25 nuclear chromosomes that comprise all previously assembled fragments, in addition to the chloroplast and mitochondrial genomes. We found that chromosome 19 has filtered long-read coverage and a quality estimate that suggests significantly less haplotype sequence variation than the other chromosomes. This work improves upon the previous genome assembly and provides new opportunities for genetic engineering of this species, including creating designer synthetic chromosomes.

genomics↗

bioRxiv2025

CasGen: A Regularized Generative Model for CRISPR Cas Protein Design with Classification and Margin-Based Optimization

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated proteins (Cas) systems have revolutionized genome editing by providing high precision and versatility. However, most genome editing applications rely on a limited number of well-characterized Cas9 and Cas12 variants, constraining the potential for broader genome engineering applications. In this study, we extensively explored Cas9 and Cas12 proteins and developed CasGen, a novel transformer-based deep generative model with margin-based latent space regularization to enhance the quality of newly generative Cas9 and Cas12 proteins. Specifically, CasGen employs a strategies that combine classification to filter out non-Cas sequences, Bayesian optimization of the latent space to guide functionally relevant designs, and thorough structural validation using AlphaFold-based analyses to ensure robust protein generation. We collected a comprehensive dataset with 3,021 Cas9, 597 Cas12, and 597 Non-Cas protein sequences from reputable biological databases such as InterPro and PDB. To validate the generated proteins, we performed sequence alignment using the BLAST tool to ensure novelty and filter out highly similar sequences to existing Cas proteins. Structural prediction using AlphaFold2 and AlphaFold3 confirmed that the generated proteins exhibit high structural similarity to known Cas9 and Cas12 variants, with TM-scores between 0.70 and 0.85 and root-mean-square deviation (RMSD) values below 2.00 [A]. Sequence identity analysis further demonstrated that the generated Cas9 orthologs exhibited 28% to 55% identity with known variants, while Cas12a variants show up to 48% identity. Our results demonstrate that the proposed Cas generative model has significant potential to expand the genome editing toolkit by designing diverse Cas proteins that retain functional integrity. The developed deep generative approach offers a promising avenue for synthetic biology and therapeutic applications, enableling the development of more precise and versatile Cas-based genome editing tools.

genomics↗

bioRxiv2021

Bioengineering of genetically encoded gene promoter repressed by flavonoids for constructing intracellular sensor for molecular events

In recent years, Synthetic Biology has emerged as a new discipline where functions that were traditionally performed by electronic devices are replaced by "cellular devices". Those are genetically encoded circuits, constructed of DNA that are built from biological parts (aka bio-parts). The cellular devices can be used for sensing and responding to natural and artificial signals. However, a major challenge in the field is that the crosstalk between many cellular signaling pathways use the same signaling endogenous molecules that can result in undesired activation. To overcome this problem, we utilized a specific promoter that can activate genes with a natural, non-toxic ligand at a highly-induced transcription level with low background or undesirable off-target expression. Here we used the orphan aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor that upon activation binds to specific AHR response elements (AHRE) of the Cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) promoter. Flavonoids have been identified as AHR ligands. Data presented here shows successful creation of a synthetic gene "off" switch that can be monitored directly using an optical reporter gene. This is the first step towards bioengineering of a synthetic, nanoscale bio-part for constructing a sensor for molecular events.

bioengineering↗

bioRxiv2025

NanoporeDB: A Structural Resource Of Multimeric Protein Nanopores In Single-Molecule Sensing

Protein nanopores are essential molecular gateways in biology and have inspired transformative technologies in biosensing and single-molecule sequencing. While this technology has transformed genomics and biosensing, the discovery of novel nanopore scaffolds remains limited due to the scarcity of experimentally resolved pore structures. Here, we present NanoporeDB, an open-access structural resource comprising over 6,600 high-confidence multimeric models across four representative pore types. Candidate proteins were systematically mined from large-scale datasets, including the AlphaFold Protein Structure Database, UniRef90, and MGnify90, and assembled using AlphaFold-Multimer and AlphaFold3. We performed membrane embedding, pore axis annotation, and constriction profiling to enable functional interpretation. NanoporeDB features an interactive web interface with 3D visualization and quantitative metrics such as insertion depth, tilt angle, and pore geometry. This resource provides a structural foundation for advancing nanopore-based molecular sensing, precision diagnostics, and synthetic biology.

bioinformatics↗

bioRxiv2026

Ecological constraints to mirror life

Our biosphere exhibits remarkable diversity yet is constrained by universal organizational principles, including molecular homochirality. Advances in synthetic biology have raised the possibility of engineering alternative life forms based on mirror-image biomolecules, prompting both technological interest and biosecurity concerns. While current discussions of mirror life largely emphasize molecular feasibility and cellular function, its potential establishment in natural environments remains poorly understood. Here, we develop a theoretical framework to assess the invasion potential of mirror organisms within existing ecosystems. Using population-level models that incorporate resource competition, metabolic constraints, and ecological network interactions, we show that mirror life might face severe limitations arising from both nutrient incompatibility and competitive exclusion by established biota. In particular, the reliance on rare or achiral substrates and the asymmetry of interactions with natural organisms constrain growth and persistence across a broad range of ecological conditions. These results highlight the importance of ecological constraints in evaluating the risks and feasibility of synthetic life.

ecology↗