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Results for “Synthetic Biology”

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At least 451 records · Page 25Linked to original sources

bioRxiv2018

A synthetic morphogenic perceptory cell

Reconstitution of artificial cells capable of transducing extracellular signals into cytoskeletal changes is a challenge in synthetic biology that will reveal fundamental principles of non-equilibrium phenomena of cellular morphogenesis and information processing. Here, we generated a life-like Synthetic Morphogenic Membrane System (SynMMS) by encapsulating a dynamic microtubule (MT) aster and a light-inducible signaling system driven by GTP/ATP chemical potential into cell-sized vesicles. The biomimetic design of the light-induced signaling system embodies the operational principle of morphogen induced Rho-GTPase signal transduction in cells. Activation of synthetic signaling promotes membrane-deforming growth of MT-filaments by dynamically elevating the membrane-proximal concentration of tubulin. The resulting membrane deformations enable the recursive coupling of the MT-aster with the signaling system, creating global self-organized morphologies that reorganize towards external light cues in dependence on prior sensory experience that is stored in the dynamically maintained morphology. SynMMS thereby signifies a step towards bio-inspired engineering of self-organized cellular morphogenesis.

synthetic biology↗

bioRxiv2026

Fine-tuned synthetic transcription factors for production of 3' phosphoadenosine-5'-phosphosulfate in yeast

Technologies developed over the past decade have made Saccharomyces cerevisiae a promising platform for producing various natural products. Balancing multi-enzyme expression, while maintaining robust microbial growth, remains a limiting factor for engineering long biosynthetic pathways in yeast. Here, we improved the transcriptional capacity of our previously developed isopropyl {beta}-D-1-thiogalactopyranoside (IPTG)-inducible synthetic transcription factors (synTFs) derived from the plant JUB1 DNA-binding domain. To this end, at cysteine positions within surface-exposed loop regions of a JUB1-derived DNA-binding scaffold, we introduced a short peptide to enhance loop flexibility while providing local stability and orientation. The generated synTFs, so-called JUB1-X synTFs, varying in strength, have been successfully used to improve the synthesis of 3-phosphoadenosine 5-phosphosulfate (PAPS), a universal sulfate donor necessary for the synthesis of bioactive molecules, including therapeutic glycosaminoglycans and sulfolipids. Using only this engineered yeast strain in simple batch culture, PAPS accumulation of 21.4 {+/-} 5.8 mg g-1 cdw was achieved after only 5 hours of inducing the expression of JUB1-X synTFs. Beyond PAPS production, the design principle demonstrated here provides a generalizable strategy to fine-tune other plant-derived synTFs, expanding the regulatory capabilities of existing synTF collections. Together, this work offers a modular, scalable approach to constructing high-performance gene circuits and supports the development of yeast cell factories for complex metabolic and synthetic biology applications.

synthetic biology↗

bioRxiv2026

BLAST: A blue light-assisted secretion toolkit tunable by reversible protein-protein interactions

Precise control over protein secretion is essential for programming intercellular communication and coordinating complex physiological responses. However, conventional methods relying on transcriptional regulation or chemical induction often lack the spatiotemporal precision and reversibility required to mimic endogenous signaling dynamics. Here, we present the Blue Light-Assisted Secretion Toolkit (BLAST), a genetically encoded system that orchestrates protein release from the endoplasmic reticulum via light-tunable protein-protein interactions. BLAST comprises two complementary modules utilizing both light-induced iLID/SspB association (a-BLAST) and LOV2/Zdk1 dissociation (d-BLAST). Both modules harness the highly conserved RXR motif to enforce strict ER confinement in the dark state. Most importantly, by utilizing non-destructive steric masking rather than enzymatic cleavage, BLAST achieves unprecedented temporal resolution with strict reversibility. We demonstrate that both systems can be repeatedly toggled ON and OFF, instantaneously arresting cargo release upon light withdrawal to generate highly controlled, pulsatile secretion profiles. Leveraging this dynamic control, we successfully achieved the rapid, robust, and light-triggered secretion of complex therapeutic proteins, including insulin and interleukin-12. By bypassing transcriptional delays and irreversible activation steps, BLAST provides a generalized, plug-and-play platform for the on-demand delivery of therapeutic proteins, significantly expanding the optogenetic toolbox for synthetic biology and cell-based therapies.

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bioRxiv2026

A modular live-cell biosensor for extracellular protease activity

Extracellular protease activity plays key roles in cell and tissue behavior, processing cell surface proteins, ligands and the extracellular matrix. Extracellular proteases can be subject to complex post-translational regulation, yet it remains challenging to quantify their activity in single cells over time. We present eNRGies (engineered neuregulin reporters as generalized indicators of extracellular shedding): modular, genetically encoded protease biosensors that translate extracellular cleavage into nuclear translocation of an intracellular (fluorescent) protein domain. We optimize the platform to report on protease activity in single cells on a timescale of minutes, and show it can be applied to soluble and cell-surface proteases including TEV protease, enterokinase, Factor Xa, MMP-9, and the sheddase ADAM17. We find ADAM17 activity can be transiently activated during mitosis and exhibit complex dynamics following EGF receptor stimulation. eNRGies biosensors enable observation of extracellular protease activity with high spatiotemporal resolution, and could be applied as synthetic biology scaffold to translate protease activity into customized cellular responses.

Synthetic Biology↗

bioRxiv2026

Directed evolution of a thermostable family-A DNA polymerase into a reverse transcriptase for 2'-modified RNA

RNA-based therapeutics increasingly rely on nuclease-resistant xeno-nucleic acids (XNAs), but engineering polymerases capable of reading and amplifying these non-canonical substrates remains a major challenge. Natural DNA polymerases cannot process C2' sugar modifications, and the complete structural determinants governing this limitation remain poorly understood, limiting rational design strategies. In this study, we apply directed evolution methods to Thermus thermophilus DNA polymerase using a microfluidic implementation of the Compartmentalized Self-Replication (CSR). We identify an engineered variant, termed E5, capable of reverse transcribing C2'-modified RNA, a previously inaccessible activity for this enzyme. Next-generation sequencing allow us to track evolutionary trajectories across nine rounds of evolution. Through an integrated analysis combining statistical methods, protein language models, and crystallographic data, we reveal unknown epistatic networks controlling polymerase function. This large-scale experimental dataset (>196,000 thermostable enzyme variants) provides a robust basis for predicting sequence-to-function relationships, accelerating development of enzymes for aptamer discovery and synthetic biology applications.

synthetic biology↗

bioRxiv2015

Layering genetic circuits to build a single cell, bacterial half adder

Gene regulation in biological systems is impacted by the cellular and genetic context-dependent effects of the biological parts which comprise the circuit. Here, we have sought to elucidate the limitations of engineering biology from an architectural point of view, with the aim of compiling a set of engineering solutions for overcoming failure modes during the development of complex, synthetic genetic circuits. Using a synthetic biology approach that is supported by computational modelling and rigorous characterisation, AND, OR and NOT biological logic gates were layered in both parallel and serial arrangements to generate a repertoire of Boolean operations that include NIMPLY, XOR, half adder and half subtractor logics in single cell. Subsequent evaluation of these near-digital biological systems revealed critical design pitfalls that triggered genetic context dependent effects, including 5 UTR interference and uncontrolled switch-on behaviour of {sigma}54 promoter. Importantly, this work provides a representative case study to the debugging of genetic context dependent effects through principles elucidated herein, thereby providing a rational design framework to program single prokaryotic cell with diversified digital operations.

Synthetic Biology↗

bioRxiv2023

A synthetic signalling network imitating the action of immune cells in response to bacterial metabolism

State-of-the-art bottom-up synthetic biology allows us to replicate many basic biological functions in artificial cell-like devices. To mimic more complex behaviours, however, artificial cells would need to perform many of these functions in a synergistic and coordinated fashion, which remains elusive. Here we considered a sophisticated biological response, namely the capture and deactivation of pathogens by neutrophil immune cells, through the process of netosis. We designed a consortium consisting of two synthetic agents - responsive DNA-based particles and antibiotic-loaded lipid vesicles - whose coordinated action mimics the sought immune-like response when triggered by bacterial metabolism. The artificial netosis-like response emerges from a series of interlinked sensing and communication pathways between the live and synthetic agents, and translates into both physical and chemical antimicrobial actions, namely bacteria immobilisation and exposure to antibiotics. Our results demonstrate how advanced life-like responses can be prescribed with a relatively small number of synthetic molecular components, and outlines a new strategy for artificial-cell-based antimicrobial solutions.

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bioRxiv2020

Genetic Circuits Combined with Machine Learning Provides Fast Responding Living Sensors

Whole cell biosensors (WCBs) have become prominent in many fields from environmental analysis to biomedical diagnostics thanks to advanced genetic circuit design principles. Despite increasing demand on cost effective and easy-to-use assessment methods, a considerable amount of WCBs retains certain drawbacks such as long response time, low precision and accuracy. Furthermore, the output signal level does not correspond to a specific analyte concentration value but shows comparative quantification. Here, we utilized a neural network-based architecture to improve the aforementioned features of WCBs and engineered a gold sensing WCB which has a long response time (18 h). Two Long-Short Term-Memory (LSTM)-based networks were integrated to assess both ON/OFF and concentration dependent states of the sensor output, respectively. We demonstrated that binary (ON/OFF) network was able to distinguish between ON/OFF states as early as 30 min with 78% accuracy and over 98% in 3 h. Furthermore, when analyzed in analog manner, we demonstrated that network can classify the raw fluorescence data into pre-defined analyte concentration groups with high precision (82%) in 3 h. This approach can be applied to a wide range of WCBs and improve rapidness, simplicity and accuracy which are the main challenges in synthetic biology enabled biosensing.

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bioRxiv2022

A split ribozyme that links detection of a native RNA to orthogonal protein outputs

Individual RNA remains a challenging signal to synthetically transduce into different types of cellular information. Here, we describe Ribozyme-ENabled Detection of RNA (RENDR), a plug-and-play strategy that uses cellular transcripts to template the assembly of split ribozymes, triggering splicing reactions that generate orthogonal protein outputs. To identify split ribozymes that require templating for splicing, we used laboratory evolution to evaluate the activities of different split variants of the Tetrahymena thermophila ribozyme. The best design delivered a 93-fold dynamic range of splicing with RENDR controlling fluorescent protein production in response to an RNA input. We resolved a thermodynamic model to guide RENDR design, showed how input signals can be transduced into diverse visual, chemical, and regulatory outputs, and used RENDR to detect an antibiotic resistance phenotype in bacteria. This work shows how transcriptional signals can be monitored in situ using RNA synthetic biology and converted into different types of biochemical information.

synthetic biology↗

bioRxiv2022

Engineering Escherichia coli to utilize erythritol as sole carbon source

Erythritol, one of the natural sugar alcohols, is widely used as a sugar substitute sweetener in food industries. Human themselves are not able to catabolize erythritol and their gut microbes lack related catabolic pathways either to metabolize erythritol. Here, we engineer Escherichia coli to utilize erythritol as sole carbon source aiming for defined applications. First, we isolate the erythritol metabolic gene cluster and experimentally characterize the erythritol-binding transcriptional repressor and its DNA-binding site. Transcriptome analysis suggests that carbohydrate metabolism-related genes in the engineered E. coli are overall upregulated, which then guides the selection of four genes for overexpression that notably enhances cell growth. Finally, engineered E. coli strains can be used as a living detector to distinguish erythritol-containing soda soft drinks and can grow in the simulated intestinal fluid supplemented with erythritol. We expect our work will inspire the engineering of more hosts to respond and utilize erythritol for broad applications in metabolic engineering, synthetic biology, and biomedical engineering.

synthetic biology↗

bioRxiv2023

Cell surface sculpting using logic-gated protein actuators

Cell differentiation and tissue specialization lead to unique cellular surface landscapes and exacerbated or loss of expression patterns can result in further heterogenicity distinctive of pathological phenotypes1-3. Immunotherapies and emerging protein therapeutics seek to exploit such differences by engaging cell populations selectively based on their surface markers. Since a single surface antigen rarely defines a specific cell type4,5, the development of programmable molecular systems that integrate multiple cell surface features to convert on-target inputs to user-defined outputs is highly desirable. Here, we describe an autonomous decision-making protein device driven by proximity-gated protein trans-splicing that allows local generation of an active protein from two otherwise inactive fragments. We show that this protein actuator platform can perform various Boolean logic operations on cell surfaces, allowing highly selective recruitment of enzymatic and cytotoxic activities to specific cells within mixed populations. Due to its intrinsic modularity and tunability, this technology is expected to be compatible with different types of inputs, targeting modalities and functional outputs, and as such will have broad application in the synthetic biology and biotechnology areas.

synthetic biology↗

bioRxiv2024

Orthogonalized human protease control of secreted signals

Synthetic circuits that regulate protein secretion in human cells could support cell-based therapies by enabling control over local environments. While protein-level circuits enable such potential clinical applications, featuring orthogonality and compactness, their non-human origin poses a potential immunogenic risk. Here, we developed Humanized Drug Induced Regulation of Engineered CyTokines (hDIRECT) as a platform to control cytokine activity exclusively using human-derived proteins. We sourced a specific human protease and its FDA-approved inhibitor. We engineered cytokines (IL-2, IL-6, and IL-10) whose activities can be activated and abrogated by proteolytic cleavage. We utilized species specificity and re-localization strategies to orthogonalize the cytokines and protease from the human context that they would be deployed in. hDIRECT should enable local cytokine activation to support a variety of cell-based therapies such as muscle regeneration and cancer immunotherapy. Our work offers a proof of concept for the emerging appreciation of humanization in synthetic biology for human health.

synthetic biology↗

bioRxiv2018

CRISPR-interference based modulation of mobile genetic elements in bacteria

Spontaneous mutagenesis of synthetic genetic constructs by mobile genetic elements frequently results in the rapid loss of advantageous functions. Previous efforts to minimize such mutations required the exceedingly time-consuming manipulation of bacterial chromosomes and the complete removal of insertional sequences (ISes). To this aim, we developed a single plasmid-based system (pCRIS) that applies CRISPR-interference to inhibit the transposition of bacterial ISes. pCRIS expresses multiple guide RNAs to direct inactivated Cas9 (dCas9) to simultaneously silence IS1, IS3, IS5, and IS150 at up to 38 chromosomal loci in Escherichia coli, in vivo. As a result, the transposition rate of all four targeted ISes dropped to negligible levels at both chromosomal and episomal targets, increasing the half-life of exogenous protein expression. Most notably, pCRIS, while requiring only a single plasmid delivery performed within a single day, provided a reduction of IS-mobility comparable to that seen in genome-scale chromosome engineering projects. Global transcriptomics analysis revealed nevertheless only minute alterations in the expression of untargeted genes. Finally, the transposition-silencing effect of pCRIS was easily transferable across multiple E. coli strains. The plasticity and robustness of our IS-silencing system make it a promising tool to stabilize bacterial genomes for synthetic biology and industrial biotechnology applications.

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bioRxiv2025

Engineering a Covalent Linkage into a Dimeric De Novo Enzyme Reveals a Novel Life-Sustaining Mechanism

Designing novel proteins that share no homology with natural sequences, but which nonetheless provide life sustaining functions, is an important goal for synthetic biology. Towards this goal, we previously reported Syn-F4, the first de novo enzyme capable of catalyzing a life-sustaining reaction, both in vitro and in vivo. Syn-F4 catalyzes hydrolysis of the siderophore, ferric enterobactin, thereby releasing iron and enabling growth in iron-limited media of an otherwise inviable {Delta}fes strain of Escherichia coli. Although Syn-F4 provides a direct enzymatic replacement of the natural ferric enterobactin esterase encoded by Fes, it has a dramatically different structure and enzymatic mechanism than the natural Fes enzyme. The novel Syn-F4 enzyme forms a 4-helix bundle, comprising a homodimer of two -helical hairpins. Here we describe the engineering of a covalent peptide linkage into the homodimer to generate a single chain 4-helix bundle. As expected, the resulting linked protein (Syn-F4-Link) also rescued {Delta}fes cells in iron-limited media. Moreover, X-ray crystallography revealed a 3D structure similar to the parental homodimer. Surprisingly, however, the linked protein was not enzymatically active. Instead, Syn-F4-Link rescues {Delta}fes cells by upregulating biosynthesis of the enterobactin siderophore thereby enabling assimilation of sufficient iron to sustain cell growth. These findings demonstrate that two very similar de novo proteins can sustain cell growth using dramatically different biological mechanisms.

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bioRxiv2020

Systematic tools for reprogramming plant gene expression in a simple model, Marchantia polymorpha.

We present the OpenPlant toolkit, a set of interlinked resources and techniques to develop Marchantia as testbed for bioengineering in plants. Marchantia is a liverwort, a simple plant with an open form of development that allows direct visualization of gene expression and dynamics of cellular growth in living tissues. We describe new techniques for simple and efficient axenic propagation and maintenance of Marchantia lines with no requirement for glasshouse facilities. Marchantia plants spontaneously produce clonal propagules within a few weeks of regeneration, and lines can be amplified million-fold in a single generation by induction of the sexual phase of growth, crossing and harvesting of progeny spores. The plant has a simple morphology and genome with reduced gene redundancy, and the dominant phase of its life cycle is haploid, making genetic analysis easier. We have built robust Loop assembly vector systems for nuclear and chloroplast transformation and genome editing. These have provided the basis for building and testing a modular library of standardized DNA elements with highly desirable properties. We have screened transcriptomic data to identify a range of candidate genes, extracted putative promoter sequences, and tested them in vivo to identify new constitutive promoter elements. The resources have been combined into a toolkit for plant bioengineering that is accessible for laboratories without access to traditional facilities for plant biology research. The toolkit is being made available under the terms of the OpenMTA and will facilitate the establishment of common standards and the use of this simple plant as testbed for synthetic biology.

synthetic biology↗

bioRxiv2021

A chemogenetic platform for controlling plasma membrane signaling and synthetic signal oscillation

Chemogenetic methods that enable the rapid translocation of specific signaling proteins in living cells using small molecules are powerful tools for manipulating and interrogating intracellular signaling networks. However, existing techniques rely on chemically induced dimerization of two protein components and have certain limitations, such as a lack of reversibility, bioorthogonality, and usability. Here, by expanding our self-localizing ligand-induced protein translocation (SLIPT) approach, we have developed a versatile chemogenetic system for plasma membrane (PM)-targeted protein translocation. In this system, a novel engineered Escherichia coli dihydrofolate reductase in which a hexalysine (K6) sequence is inserted in a loop region (iK6DHFR) is used as a universal protein tag for PM-targeted SLIPT. Proteins of interest that are fused to the iK6DHFR tag can be specifically recruited from the cytoplasm to the PM within minutes by addition of a myristoyl-O_SCPLOWDC_SCPLOW-Cys-tethered trimethoprim ligand (mDcTMP). We demonstrated the broad applicability and robustness of this engineered protein-synthetic ligand pair as a tool for the conditional activation of various types of signaling molecules, including protein and lipid kinases, small GTPases, heterotrimeric G proteins, and second messengers. In combination with a competitor ligand and a culture-medium flow chamber, we further demonstrated the application of the system for chemically manipulating protein localization in a reversible and repeatable manner to generate synthetic signal oscillations in living cells. The present bioorthogonal iK6DHFR/mDcTMP-based SLIPT system affords rapid, reversible, and repeatable control of the PM recruitment of target proteins, offering a versatile and easy-to-use chemogenetic platform for chemical and synthetic biology applications.

synthetic biology↗

bioRxiv2022

Macroscopic chemical to electrical signal converter in microbial communities

Excitable cells can convert electrical signals into chemical outputs to facilitate the active transport of information across larger distances. This electrical-to-chemical conversion requires a tightly regulated expression of ion channels. Alterations of ion channel expression provide landmarks of numerous pathological diseases, such as cardiac arrhythmia, epilepsy, or cancer. Although the activity of ion channels can be locally regulated by external light or chemical stimulus, it remains challenging to coordinate the expression of ion channels on extended spatial-temporal scales in a non-invasive manner. Here, we have engineered yeast S. cerevisiae to read and convert local chemical concentrations into a dynamic electrical field distributed across cell populations. The core mechanism encodes a chemically-excitable dual-feedback gene circuit that precisely tunes the expression domain of potassium channels, globally coordinating cyclic firing of the plasma membrane potential (PMP). We demonstrate that this mechanism leverages an engineered constitutively open bacterial potassium channel KcsA to directly couple chemical stimuli with ion flux through gene expression and it can interface with the host ion channels through the pulsatile production of toxins. Our study provides a robust synthetic transcriptional toolbox underlying the conversion of local chemical environments into spatiotemporally organized electrical impulses for various cellular engineering, synthetic biology, and potential therapeutic applications.

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bioRxiv2024

Size Selection of Giant Unilamellar Vesicles (GUVs) via Modified cDICE Method

The production of giant unilamellar vesicles (GUVs) plays a pivotal role in various scientific disciplines, particularly in the development of synthetic cells. While numerous methods exist for GUV preparation, the modified continuous droplet interface crossing encapsulation (cDICE) method offers the advantages of simplicity and high encapsulation efficiency. However, a significant limitation of this technique is the generation of vesicles with a broad size distribution and the inability to control the desired size range. This raises a key question: Can the modified cDICE method be optimized to produce GUVs with controlled size distribution? In this study, we examined the effects of two experimental parameters--rotation time (tROT) and the angular frequency ({omega}) of the cDICE chamber--on the size distribution of GUVs. Our results show that reducing either the angular frequency or rotation time shifts the size distribution toward larger vesicles, enabling effective size selection. These findings are further supported by a physical model, which provides insights into the mechanisms underlying size selection. This work demonstrates that control over GUV size distribution can be achieved through straightforward adjustments of system parameters. The ability to fine-tune vesicle size offers researchers a powerful tool for developing customizable experimental systems for synthetic biology and related fields.

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