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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 199 records · Page 11Linked to original sources

bioRxiv2026

UstiGate: Next generation toolkit for advanced genetic engineering of the basidiomycete chassis Ustilago maydis

The corn smut fungus Ustilago maydis is an established model for fungal biology and an emerging host for biotechnology. However, its broader application in synthetic biology has been constrained by a limited repertoire of standardized genetic parts and tools for multigene engineering. Here, we introduce UstiGate, a modular toolkit that combines MoClo-compatible DNA assembly with targeted genomic integration in U. maydis. The toolkit comprises 92 parts and vectors, including more than 20 promoter regions as well as terminators, selectable markers, fluorescent reporters and modules for different genomic integration sites. Quantitative reporter analyses at both, population and single-cell level, revealed a broad range of promoter strengths and characterized their behavior across growth phases, media, genomic loci and reporter contexts. We further identified a maltose-inducible, glucose-repressed promoter that enables autoinduction and established carotenoid-biosynthesis genes as integration sites supporting visual pre-selection of transformants. Finally, UstiGate enabled the assembly and simultaneous genomic integration of four transcriptional units in a single transformation. UstiGate thus provides a standardized and quantitatively characterized platform for standard applications as well as multigene engineering, enabling advanced synthetic biology applications such as pathway transplantation in U. maydis.

synthetic biology↗

bioRxiv2024

Quencher-free fluorescence monitoring of G-Quadruplex folding

Guanine-rich sequences exhibit a high degree of polymorphism and can form single-stranded, Watson-Crick duplex, and four-stranded G-quadruplex structures. These sequences have found a wide range of uses in synthetic biology applications, arising in part from their structural plasticity. High-throughput, low-cost tools for monitoring the folding and unfolding transitions of G-rich sequences would provide an enabling technology for accelerating prototyping of synthetic biological systems and for accelerating design-build-test cycles. Here, we show that unfolding transitions of a range of G-quadruplex-forming DNA sequences can be monitored in a FRET-like format using DNA sequences that possess only a single dye label, with no quencher. These quencher-free assays can be performed at low cost, with both cost and lead times ca. 1 order of magnitude lower than FRET-labeled strands. Thus, quencher-free secondary structure monitoring promises to be a valuable tool for testing and development of synthetic biology systems employing G-quadruplexes.

biophysics↗

bioRxiv2024

MAIA: An open-source, modular, bioreactor for cities

This work presents the design and fabrication of MAIA, an open-source, modular, low-cost, and portable bioreactor for democratizing the development of synthetic biology based projects for urban settings. The integration of open-source synthetic biology (synbio) tools in a citys infrastructure planning and design is crucial for addressing the great challenges related to urbanization. Synbio tools have great potential to help us complement our current sensing and actuating urban infrastructure. The MAIA reactor controls bacterial growth variables, making it suitable for cell-based experiments while reducing the need for expensive laboratory equipment. Its low-cost and open-source design allow for easy replication and modification, making it accessible to a broader audience. Its portability makes it suitable for use outside of traditional laboratory settings. We qualitatively and quantitatively validated the reactors capability to support cell growth, stimulate gene expression, and act as a creative tool for students and users.

synthetic biology↗

bioRxiv2025

Synthetic Control of Implanted Engineered Liver Tissue Growth

Despite the promise of engineered tissue implants for the treatment of organ failure, scaling of these constructs to sizes of therapeutic relevance remains a barrier to clinical translation. Here, we propose a strategy to circumvent this limitation: to instead implant a small-scale construct and then induce it to grow in situ after its engraftment into a host. Using engineered liver tissue as a proof-of-concept application, we integrated synthetic biology and tissue engineering tools to build liver tissues that can be expanded on-demand after implantation in vivo. To achieve this goal, we first identified the combination of YAP and growth factor signaling as sufficient to drive human hepatocyte proliferation in dense, 3D engineered tissues. We then engineered control of these signaling axes using synthetic biology tools to drive human liver tissue expansion both in vitro and in vivo. As such, this work establishes a genetic strategy for generating large organ implants through bioengineered, on-demand outgrowth using synthetic triggers (BOOST). TeaserPresenting bioengineered on-demand outgrowth via synthetic biology triggering (BOOST) for in situ solid cell therapy scale up.

bioengineering↗

bioRxiv2025

From Synthetic to Biological Nitrification Inhibition: Advancing Stabilization of Organic Fertilizers

Fertilizer type plays a critical role in nitrogen (N) cycling, influencing nitrous oxide (N2O) emissions, soil mineral N dynamics, and microbial communities. Understanding these interactions is essential for developing sustainable fertilization strategies that balance agricultural productivity with environmental protection. This study examined the effects of mineral and organic fertilizers (OFs) on N transformations and evaluated the efficiency of the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) in mitigating N2O losses. Results showed that OFs exhibited variable impacts on N2O emissions depending on their composition and C/N ratio. DMPP effectively reduced nitrification-driven N2O emissions, particularly in treatments with high ammoniacal N content. However, its efficiency was limited with animal-based OFs, suggesting a complex interaction between fertilizer properties and inhibitor effectiveness. DMPP had not direct impact on soil microbial diversity but specifically targeted the Nitrosomonaceae family and Nitrospira class. Beyond synthetic inhibitors, biological nitrification inhibition (BNI) emerged as a promising alternative, which we explored using rhizospheric soils from wheat landrace Persia 44 and white mustard (cv. Pole Position, Verdi). These soils significantly reduced N2O emissions, particularly when combined with OFs. The integration of BNI with organic fertilizers, especially liquid digestate, represents a promising strategy for reducing N losses while maintaining soil fertility. This study underscores the need for tailored fertilization strategies that combine chemical and biological tools to optimize N use efficiency and support environmentally sustainable agriculture.

plant biology↗

bioRxiv2024

Uncovering the class II-bacteriocin predatiome in salivarius streptococci

Facing the surge of antibiotic resistance, the medical field has a critical need for alternatives to treat bacterial infections. Among these, the use of bacteriocins, ribosomally-synthesized antimicrobial peptides produced by bacteria, is considered to be a promising route. In the human commensal Streptococcus salivarius, the production of unmodified class II bacteriocins is directly controlled at the transcriptional level by the quorum-sensing ComRS system. Here, we used an integrated approach combining bioinformatics and synthetic biology to identify novel bacteriocins from salivarius streptococci active against human pathogens. We developed a bioinformatic pipeline that combines conservation of DNA motifs for genetic regulation and features of bacteriocin primary sequences to uncover cryptic class II bacteriocins. Notably, we discovered more than 50 novel bacteriocin candidates clustered into 21 groups from 100 genomes of S. salivarius. Strain-based analysis of bacteriocin cocktails revealed an important diversity restricted to seven distinct loci, probably resulting from bacteriocin intra- and inter- species exchanges. Using in vitro or in vivo production and synthetic biology tools, we showed that most of them are active against a panel of Gram-positive bacteria, including clinically- relevant pathogens. Overall, this work provides a new search-to-test generic pipeline for the discovery of novel bacteriocins in Gram-positive bacteria that could be used in cocktails for broad applications in the food and biomedical fields. IMPORTANCETo survive in highly challenging environments, streptococci have evolved a competence- predation coupling mechanism for genome plasticity. This developmental process is highly regulated at the transcriptional level, masking the predation killing effects in usual laboratory conditions. Here, we present a general strategy that combines bioinformatics and synthetic biology to unveil class II bacteriocins in streptococci. Its implementation to the beneficial species Streptococcus salivarius revealed around 40 class II salivaricin cocktails explained by the plasticity of seven independent loci. Notably, the salivaricin predatiome includes a subtle blend of fratricins, sobrinicins, and broad-spectrum bacteriocins with overlapping activities against a wide spectrum of low-GC Gram-positive bacteria, including notorious pathogens. Furthermore, most of those bacteriocins are predicted to be variants of a common -hairpin structure, indicating that their mode of action evolved convergently. Finally, the discovery of ca. 50 novel bacteriocins offers perspectives for the rational assembly of potent cocktails active against pathogenic staphylococci, streptococci or enterococci.

microbiology↗

bioRxiv2022

Optogenetic Maxwell Demon to Exploit Intrinsic Noise and Control Cell Differentiation Despite Time Delays and Extrinsic Variability

The field of synthetic biology focuses on creating modular components which can be used to generate complex and controllable synthetic biological systems. Unfortunately, the intrinsic noise of gene regulation can be large enough to break these systems. Noise is largely treated as a nuisance and much past effort has been spent to create robust components that are less influenced by noise. However, extensive analysis of noise combined with smart microscopy tools and optognenetic actuators can create control opportunities that would be difficult or impossible to achieve in the deterministic setting. In previous work, we proposed an Optogenetic Maxwells Demons (OMD) control problem and found that deep understanding and manipulation of noise could create controllers that break symmetry between cells, even when those cells share the same optogenetic input and identical gene regulation circuitry. In this paper, we extend those results to analyze (in silico) the robustness of the OMD control under changes in system volume, with time observation/actuation delays, and subject to parametric model uncertainties.

synthetic biology↗

bioRxiv2022

Improved cell-free transcription-translation reactions in microfluidic chemostats augmented with hydrogel membranes for continuous small molecule dialysis

Increasing protein production capacity of the PURE cell-free transcription-translation (TX-TL) system will be key to implementing complex synthetic biological circuits, and to establish a fully self-regenerating system as a basis for the development of a synthetic cell. Under steady-state conditions, the protein synthesis capacity of the PURE system is likely at least one order of magnitude too low to express sufficient quantities of all PURE protein components. This is in part due to the fact that protein synthesis cant be sustained during the entire dilution cycle, especially at low dilution rates. We developed a microfluidic chemostat augmented with semi-permeable membranes that combines steady-state reactions and continuous dialysis as a possible solution to enhance protein synthesis at steady-state. In batch operation, the continuous dialysis of low molecular weight components via the membranes extended protein synthesis by over an order of magnitude from 2 hours to over 30 hours, leading to a seven-fold increase in protein yield. In chemostat operation, continuous dialysis enabled sustained protein synthesis during the entire dilution cycle even for low dilution rates, leading to six-fold higher protein levels at steady state. The possibility to combine and independently manipulate continuous dialysis and chemostat operation renders our dialysis chemostat a promising technological basis for complex cell-free synthetic biology applications that require enhanced protein synthesis capacity.

synthetic biology↗

bioRxiv2022

Excel-SBOL Converter: Creating SBOL from Excel Templates and Vice Versa

Standards support synthetic biology research by enabling the exchange of component information. However, using formal representations, such as the Synthetic Biology Open Language (SBOL), typically requires either a thorough understanding of these standards or a suite of tools developed in concurrence with the ontologies. Since these tools may be a barrier for use by many practitioners, the Excel-SBOL Converter was developed to allow easier use of SBOL and integration into existing workflows. The converter consists of two Python libraries: one that converts Excel templates to SBOL, and another that converts SBOL to an Excel workbook. Both libraries can be used either directly or via a SynBioHub plugin. We illustrate the operation of the Excel-SBOL Converter with two case studies: uploading experimental data with the studys metadata linked to the measurements and downloading the Cello part repository. Graphical TOC Entry O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/505873v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@c06d7aorg.highwire.dtl.DTLVardef@153724eorg.highwire.dtl.DTLVardef@1758791org.highwire.dtl.DTLVardef@11779c2_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2022

Engineering functional membrane-membrane interface by InterSpy

Engineering synthetic interfaces between membranes has potential applications in designing non-native cellular communication pathways and creating synthetic tissues. Here, InterSpy is introduced as a synthetic biology tool consisting of a heterodimeric protein engineered to form and maintain membrane-membrane interfaces between apposing synthetic as well as cell membranes through SpyTag/SpyCatcher interaction. Inclusion of split fluorescent protein fragments in the designed InterSpy toolkit allows tracking the formation of membrane-membrane interface and reconstitution of functional fluorescent protein in the space between apposing membranes. We first demonstrate InterSpy by testing split protein designs using a mammalian cell-free expression system. By utilizing co-translational helix insertion, cell-free synthesized InterSpy fragments are incorporated into the membrane of liposomes and supported lipid bilayers with a desired topology. Functional reconstitution of split fluorescent protein between the membranes is strictly dependent on SpyTag/SpyCatcher. Finally, since InterSpy is fully genetically encoded, the engineered system is adapted to cells and showcased. InterSpy demonstrates the power of cell-free expression systems in functional reconstitution of synthetic membrane interfaces via proximity-inducing proteins. This technology may also prove useful for synthetic biology where cell-cell contacts and communication are recreated in a controlled manner using minimal components.

synthetic biology↗

bioRxiv2018

Design Guidelines For Sequestration Feedback Networks

Integral control is commonly used in mechanical and electrical systems to ensure perfect adaptation. A proposed design of integral control for synthetic biological systems employs the sequestration of two biochemical controller species. The unbound amount of controller species captures the integral of the error between the current and the desired state of the system. However, implementing integral control inside bacterial cells using sequestration feedback has been challenging due to the controller molecules being degraded and diluted. Furthermore, integral control can only be achieved under stability conditions that not all sequestration feedback networks fulfill. In this work, we give guidelines for ensuring stability and good performance (small steady-state error) in sequestration feedback networks. Our guidelines provide simple tuning options to obtain a flexible and practical biological implementation of sequestration feedback control. Using tools and metrics from control theory, we pave the path for the systematic design of synthetic biological circuits.

synthetic biology↗

bioRxiv2024

Engineering prolyl hydroxylase-dependent proteolysis enables the orthogonal control of hypoxia responses in plants

Vascular plants and metazoans use selective proteolysis of transcription factors to control the adaptive responses to hypoxia, although through distinct biochemical mechanisms. The reason for this divergence is puzzling, especially when considering that the molecular components necessary to establish both strategies are conserved across the two kingdoms. To explore an alternative evolutionary scenario where plants sense hypoxia as animals do, we engineered a three-components system aimed to target proteins for degradation in an oxygen dependent manner in Arabidopsis thaliana. Applying the synthetic biology framework, we produced a hypoxia-responsive switch independent of endogenous pathways. When applied to control transcription, the synthetic system partially restored hypoxia responsiveness in oxygen-insensitive mutants. Additionally, we demonstrated its potential to regulate growth under flood-induced hypoxia. Our work highlights the use of synthetic biology to reprogram signalling pathways in plants, providing insights into the evolution of oxygen sensing and ofering tools for crop improvement under stress conditions.

synthetic biology↗

bioRxiv2021

Implementation of a novel optogenetic tool in mammalian cells based on a split T7 RNA polymerase

Optogenetic tools are widely used to control gene expression dynamics both in prokaryotic and eukaryotic cells. These tools are used in a variety of biological applications from stem cell differentiation to metabolic engineering. Despite some tools already available in bacteria, no light-inducible system currently exists to orthogonally control gene expression in mammalian cells. Such a tool would be particularly important in synthetic biology, where orthogonality is advantageous to achieve robust activation of synthetic networks. Here we implement, characterize and optimize a new orthogonal optogenetic tool in mammalian cells based on a previously published system in bacteria called Opto-T7RNAPs. The tool consists of a split T7 RNA polymerase coupled with the blue light-inducible magnets system (mammalian OptoT7 - mOptoT7). In our study we exploited the T7 polymerases viral origins to tune our systems expression level, reaching up to 20-fold change activation over the dark control. mOptoT7 is used here to generate mRNA for protein expression, shRNA for protein inhibition and Pepper aptamer for RNA visualization. Moreover, we show that mOptoT7 can mitigate gene expression burden when compared to other optogenetic constructs. These properties make mOptoT7 a new powerful tool to use when orthogonality and viral-like RNA species are desired in both synthetic biology and basic science applications.

synthetic biology↗

bioRxiv2019

Bacterial Controller Aided Wound Healing: A Case Study in Dynamical Population Controller Design

Wound healing is a complicated biological process consisting of many types of cellular dynamics and functions regulated by chemical and molecular signals. Recent advances in synthetic biology have made it possible to predictably design and build closed-loop controllers that can function appropriately alongside biological species. In this paper we develop a simple dynamical population model mimicking the sequential relay-like dynamics of cellular populations involved in the wound healing process. Our model consists of four nodes and five signals whose parameters we can tune to simulate various chronic healing conditions. We also develop a set of regulator functions based on type-1 incoherent feed forward loops (IFFL) that can sense the change from acute healing to incomplete chronic wounds, improving the system in a timely manner. Both the wound healing and type-1 IFFL controller architectures are compatible with available synthetic biology experimental tools for potential applications.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗

bioRxiv2023

A versatile in situ cofactor enhancing system for meeting cellular demands for engineered metabolic pathways

Cofactor imbalance obstructs the productivities of metabolically engineered cells. Herein, we employed a minimally perturbing system, xylose reductase and lactose (XR/lactose), to increase levels of a pool of sugar-phosphates which are connected to the biosynthesis of NAD(P)H, FAD, FMN and ATP in Escherichia coli. The XR/lactose system could increase the amounts of the precursors of these cofactors and was tested with three different metabolically engineered cell systems (fatty alcohol biosynthesis, bioluminescence light generation and alkane biosynthesis) with different cofactor demands. Productivities of these cells were increased 2-4-fold by the XR/lactose system. Untargeted metabolomic analysis revealed different metabolite patterns among these cells; demonstrating that only metabolites involved in relevant cofactor biosynthesis were altered. The results were also confirmed by transcriptomic analysis. Another sugar reducing system (glucose dehydrogenase, GDH) could also be used to increase fatty alcohol production but resulted in less yield enhancement than XR. This work demonstrates that the approach of increasing cellular sugar phosphates can be a generic tool to increase in vivo cofactor generation upon cellular demand for synthetic biology. TeaserUse of sugar and sugar reductase to increase sugar phosphates for enhancing in situ synthesis of cofactors upon cellular demand for synthetic biology.

synthetic biology↗

bioRxiv2018

CyanoGate: A Golden Gate modular cloning suite for engineering cyanobacteria based on the plant MoClo syntax

Recent advances in synthetic biology research have been underpinned by an exponential increase in available genomic information and a proliferation of advanced DNA assembly tools. The adoption of plasmid vector assembly standards and parts libraries has greatly enhanced the reproducibility of research and exchange of parts between different labs and biological systems. However, a standardised Modular Cloning (MoClo) system is not yet available for cyanobacteria, which lag behind other prokaryotes in synthetic biology despite their huge potential in biotechnological applications. By building on the assembly library and syntax of the Plant Golden Gate MoClo kit, we have developed a versatile system called CyanoGate that unites cyanobacteria with plant and algal systems. We have generated a suite of parts and acceptor vectors for making i) marked/unmarked knock-outs or integrations using an integrative acceptor vector, and ii) transient multigene expression and repression systems using known and novel replicative vectors. We have tested and compared the CyanoGate system in the established model cyanobacterium Synechocystis sp. PCC 6803 and the more recently described fast-growing strain Synechococcus elongatus UTEX 2973. The system is publicly available and can be readily expanded to accommodate other standardised MoClo parts.

synthetic biology↗

bioRxiv2026

Towards interoperable modeling of toehold-mediated strand exchange circuits across DNA nanotechnology and engineering biology

Originally developed for DNA nanotechnology, toehold-mediated strand exchange (TMSE) circuits are gaining traction in synthetic biology due to their high programmability, seamless integration with biological components, and robust operation across diverse environments and cell types. However, while forward-engineering in synthetic biology has benefited from automated genetic circuit modeling pipelines, there is currently a lack of accessible, automated tools for the mechanistic modeling of TMSE circuits integrated with these systems, hindering the development of new biotechnologies. The TMSE-BioCRNpyler Library allows TMSE molecules to be transcribed RNAs or fixed-concentration nucleic acids while leveraging existing BioCRNpyler features, such as upstream transcription regulation and downstream gene regulation. We demonstrate this librarys applicability by modeling published applications of TMSE circuits spanning a wide range of applications, including simple in vitro reactions, cell-free biosensors, and in vivo microbial and mammalian systems. Additionally, we validated that models compiled using the TMSE-BioCRNpyler Library produced results with < 0.2 % relative error compared to multiple models of TMSE previously developed in the literature. Finally, to streamline interoperability with existing models, we developed txt2biocrnpyler. This accompanying tool converts chemical reaction networks from the literature into a BioCRNpyler-ready source script and a Systems Biology Markup Language XML file -- a standard data format for sharing and simulating biological models. The TMSE-BioCRNpyler Library serves as a powerful new resource for the rational, automated design of molecular information processing systems.

bioengineering↗

bioRxiv2017

Expressing Biologically Active Membrane Proteins in a Cell-Free Transcription-Translation Platform

Cell-free transcription-translation platforms have been widely utilized to express soluble proteins in basic synthetic biological circuit prototyping. From a synthetic biology point of view, it is critical to express membrane proteins in cell-free transcription-translation systems, and use them directly in biocircuits, considering the fact that histidine kinases, G-protein coupled receptors (GPCRs) and other important biosensors are all membrane proteins. Previous studies have expressed membrane proteins in cell-free systems with the help of detergents, liposomes or nanodiscs, but have not demonstrated the ability to prototype circuit behavior for the purpose of testing more complex circuit functions involving membrane-bound proteins. Built on previous efforts, in this work we demonstrated that we could co-translationally express solubilized and active membrane proteins in our cell-free TX-TL platform with membrane-like materials. We first tested the expression of several constructs with {beta}1 and {beta}2 adrenergic receptors in TX-TL and observed significant insoluble membrane protein production. The addition of nanodiscs to the cell free expression system enabled solubilization of membrane proteins. Nanodisc is lipoprotein-based membrane-like material. The activity of {beta}2 adrenergic receptor was tested with both fluorescence and Surface Plasmon Resonance (SPR) binding assays by monitoring the specific binding response of small-molecule binders, carazolol and norepinephrine. Our results suggest that it is promising to use cell-free expression systems to prototype synthetic biocircuits involving single chain membrane proteins without extra procedures. This data made us one step closer to testing complex membrane protein circuits in cell-free environment.

bioengineering↗