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Results for “synthetic biology”

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At least 415 records · Page 23Linked to original sources

bioRxiv2015

Deconvolution of gene expression noise into physical dynamics of cognate promoters

When facing recalcitrant pollutants, soil bacteria exploit noise of catabolic promoters for deploying environmentally beneficial phenotypes such as metabolic bet-hedging an/or division of biochemical labor. While the origin of such noise in terms of upstream promoter-regulator interplay is hardly understood, its dynamics has to be somehow encrypted in the patterns of flow-cytometry data delivered by transcriptional reporter fusions. On this background, we have examined the behaviour of the Pm promoter of the environmental bacterium Pseudomonas putida and its cognate 3-methylbenzoate-responsive regulator XylS under different conditions by following expression of Pm-GFP fusions in single cells. Using mathematical modeling and computational simulations we elucidated the kinetic properties of the system and use them as a baseline code to interpret the observed fluorescence output in terms of upstream regulator variability. Transcriptional noise was predicted to depend on the intracellular physical distance between the regulator source (where the e.g. XylS is being produced in the cells) and the target promoter. Experiments with engineered bacteria where this distance is either minimized or enlarged proved the effects of proximity on noise patterns as predicted by the model. This approach not only allowed deconvolution of cytometry data into mechanistic information on the gene expression flow. But it also provided a mechanistic basis for selecting a given level of noise in engineered regulatory nodes e.g. in Synthetic Biology constructs.

Synthetic Biology↗

bioRxiv2020

A framework for implementing metaheuristic algorithms using intercellular communication

Metaheuristic procedures (MH) have been a trend driving Artificial Intelligence (AI) researchers for the past 50 years. A variety of tools and applications (not only in Computer Science) stem from these techniques. Also, MH frequently rely on evolution, a trademark process involved in cell colony growth. Generally, MH are used to approximate the solution to difficult problems but require a large amount of computational resources. Cell colonies harboring synthetic distributed circuits using intercell communication offer a direction for tackling this problem, as they process information in a massively parallel fashion. In this work, we propose a framework that maps MH elements to synthetic circuits in growing cell colonies. The framework relies on cell-cell communication mechanisms such as quorum sensing (QS) and bacterial conjugation. As a proof-of-concept, we also implemented the workflow associated to the framework, and tested the execution of two specific MH (Genetic Algorithms and Simulated Annealing) encoded as synthetic circuits on the gro simulator. Furthermore, we show an example of how our framework can be extended by implementing another kind of computational model: The Cellular Automaton. This work seeks to lay the foundations of mappings for implementing AI algorithms in a general manner using Synthetic Biology constructs in cell colonies.

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bioRxiv2021

The Functional Nanopore (FuN) Screen: A Versatile Genetic Assay to Study and Engineer Protein Nanopores in Escherichia coli

Nanopores comprise a versatile class of membrane proteins that carry out a range of key physiological functions and are increasingly developed for different biotechnological applications. Yet, a capacity to study and engineer protein nanopores by combinatorial means has so far been hampered by a lack of suitable assays that combine sufficient experimental resolution with throughput. Addressing this technological gap, the Functional Nanopore (FuN) screen now provides a quantitative and dynamic read-out of nanopore assembly and function in E. coli. The assay is based on genetically-encoded fluorescent protein sensors that resolve the nanopore-dependent influx of Ca2+ across the inner membrane of E. coli. Illustrating its versatile capacity, the FuN screen is first applied to dissect the molecular features that underlie the assembly and stability of nanopores formed by the S2168 holin. In a subsequent step, nanopores are engineered by recombining the transmembrane module of S2168 with different ring-shaped oligomeric protein structures that feature defined hexa-, hepta- and octameric geometries. Library screening highlights substantial plasticity in the ability of the S2168 transmembrane module to oligomerize in alternative geometries while the functional properties of the resultant nanopores can be fine-tuned through the identity of the connecting linkers. Overall, the FuN screen is anticipated to facilitate both fundamental studies and complex nanopore engineering endeavors with many potential applications in biomedicine, biotechnology and synthetic biology.

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bioRxiv2025

Title: Engineering Plasmids with Synthetic Origins of Replication

Plasmids remain by far the most common medium for delivering engineered DNA to microorganisms. However, the reliance on natural plasmid replication mechanisms limits their tunability, compatibility, and modularity. Here we refactor the natural pMB1 origin and create plasmids with customizable copy numbers by tuning refactored components. We then create compatible origins that use synthetic RNA regulators to implement independent copy control. We further demonstrate that the synthetic origin of replication (SynORI) can be engineered modularly to respond to various signals, allowing for multiplexed copy-based reporting of environmental signals. Lastly, a library of 6 orthogonal SynORI plasmids is created and co-maintained in E. coli for a week. This work establishes the feasibility of creating plasmids with SynORI that can serve as a new biotechnology for synthetic biology.

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bioRxiv2025

Screening and machine-learning assisted prediction of translation-enhancing peptides reducing ribosomal stalling in Escherichia coli

We previously reported that the nascent SKIK peptide enhances translation and alleviates ribosomal stalling caused by arrest peptides (APs) such as SecM and polyproline when positioned immediately upstream of the APs in both Escherichia coli in vivo and in vitro translation systems. In this study, we performed a comprehensive screening of translation-enhancing peptides (TEPs) using a randomized artificial tetrapeptide library. The screening was based on the ability of the peptides to suppress SecM AP-induced translational stalling in E. coli cells. Various TEPs exhibiting a range of translation-enhancing activities were identified. In vitro translation analysis suggested that the fourth amino acid in the tetrapeptide plays a key role in reducing SecM AP-mediated stalling. Furthermore, we developed a machine learning model using a random forest algorithm to predict TEP activity. The predicted values showed a strong correlation with experimentally measured activities. These findings offer a compact peptide toolkit and a data-driven approach for mitigating AP-induced ribosome stalling, with potential applications in synthetic biology.

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bioRxiv2019

Noise-Reducing Negative-Feedback Optogenetic Circuits in Mammalian Cells

Gene autorepression is widely present in nature and is also employed in synthetic biology, partly to reduce gene expression noise in cells. Optogenetic systems have recently been employed for controlling gene expression levels in mammalian cells, but most have utilized activator-based proteins, neglecting negative feedback. Here, we engineer optogenetic negative-feedback gene circuits in mammalian cells to achieve noise-reduction for precise gene expression control. We build a toolset of these noise-reducing Light-Inducible Tuner (LITer) gene circuits using the TetR repressor fused with a Tet-Inhibitory peptide (TIP) or a degradation tag through the light-sensitive LOV2 protein domain. These LITers provide nearly a range of 4-fold gene expression control and up to five-fold noise reduction from existing optogenetic systems. Moreover, we use the LITer gene circuit architecture to control gene expression of the cancer oncogene KRAS(G12V) and study its downstream effects through phospho-ERK levels and cellular proliferation. Overall, these novel LITer optogenetic platforms should enable precise spatiotemporal perturbations for studying multicellular phenotypes in developmental biology, oncology, and other biomedical fields of research.

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bioRxiv2023

Information storage across a microbial community using universal RNA memory

Biological recorders can code information in DNA, but they remain challenging to apply in complex microbial communities. To program microbiome information storage, a synthetic catalytic RNA (cat-RNA) was used to write information in ribosomal RNA (rRNA) about gene transfer host range. By reading out native and modified rRNA using amplicon sequencing, we find that 140 out of 279 wastewater microbial community members from twenty taxonomic orders participate in conjugation and observe differences in information storage across amplicon sequence variants. Twenty of the variants were only observed in modified rRNA amplicons, illustrating information storage sensitivity. This autonomous and reversible RNA-addressable memory (RAM) will enable biosurveillance and microbiome engineering across diverse ecological settings and studies of environmental controls on gene transfer and cellular uptake of extracellular materials. One-Sentence SummaryRibosomal RNA sequencing detects cellular events recorded across a wastewater microbial community using synthetic biology.

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bioRxiv2024

Synthetic bacteria with programmed cell targeting and protein injection suppress tumor growth in vivo

Bacterial living therapeutics (BLTs) hold promise for treating cancer and other human diseases because they can be engineered and transported into the microbiota (e.g., of tumors, gastrointestinal tract) to deliver therapeutic payloads. Current approaches rely on the natural tropism of the bacterial chassis used and trigger the local release of protein cargoes, typically through active extracellular secretion or bacterial lysis. BLTs capable of targeting specific cellular subsets and delivering payloads intracellularly might provide new therapeutic opportunities and improve efficacy while reducing off-target effects. We used synthetic biology to develop BLTs that can deliver defined cargo proteins into the cytoplasm of target cells. We designed a modular synthetic bacterium with programmed adhesion to cells by targeting defined cell surface antigen and armed with an inducible type III secretion system (T3SS) for injection of a protein cargo of interest. As a proof of principle, we programmed synthetic bacteria to recognize the epidermal growth factor receptor (EGFR) and inject the catalytic fragments of the potent adenosine diphosphate-ribosyltransferase toxins ExoA and TccC3. These BLTs demonstrated the ability to trigger robust tumor cell death in vitro. Intratumoral administration of these synthetic bacteria suppressed tumor growth in vivo and prolonged the survival of treated animals when the tumor cells were recognized by the engineered bacteria. These results demonstrate the potential of programming cell targeting and controlled protein injection for the development of effective and specific BLTs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/590337v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@eb769dorg.highwire.dtl.DTLVardef@b84b39org.highwire.dtl.DTLVardef@152b8b0org.highwire.dtl.DTLVardef@1db4108_HPS_FORMAT_FIGEXP M_FIG C_FIG

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bioRxiv2019

Controlling spatiotemporal pattern formation in a concentration gradient with a synthetic toggle switch

The formation of spatiotemporal patterns of gene expression is frequently guided by gradients of diffusible signaling molecules. The toggle switch subnetwork, composed of two cross-repressing transcription factors, is a common component of gene regulatory networks in charge of patterning, converting the continuous information provided by the gradient into discrete abutting stripes of gene expression. We present a synthetic biology framework to understand and characterize the spatiotemporal patterning properties of the toggle switch. To this end, we built a synthetic toggle switch controllable by diffusible molecules in Escherichia coli. We analyzed the patterning capabilities of the circuit by combining quantitative measurements with a mathematical reconstruction of the underlying dynamical system. The toggle switch can produce robust patterns with sharp boundaries, governed by bistability and hysteresis. We further demonstrate how the hysteresis, position, timing, and precision of the boundary can be controlled, highlighting the dynamical flexibility of the circuit.

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bioRxiv2025

Generative AI-based design of hybrid transcriptional activator proteins with new DNA-binding specificity

Transcriptional control arises from the specific recognition of promoter DNA by transcription factors (TFs), forming the basis of cellular information processing and gene regulation. In synthetic biology, TF-promoter interactions are assembled into genetic circuits to program cellular behaviors. To ensure expected circuit performance, most synthetic genetic circuits rely on combinations of well-characterized and orthogonal transcriptional regulations. This reliance minimizes crosstalk but constrains circuit complexity and information integration. Creating hybrid TFs that combine or interpolate promoter specificities could therefore expand the design space of synthetic regulatory systems. However, it remains unclear whether hybrid functions can be created by mixing amino acid sequences, and how such functional integration could be achieved in a principled manner. Here we show that a variational autoencoder (VAE) trained on LuxR-family DNA-binding domains can generate transcription factors with hybrid and partially novel promoter recognition properties. By sampling intermediate regions of the VAE-learned latent space, we designed hybrid TFs that activate both the lux and las promoters. High-throughput sort-seq assays together with individual in vivo assays revealed that a subset of functional variants exhibited dual-responsive behavior while maintaining sequence-selective DNA recognition. Together, these results provide a data-driven strategy for exploring functional intermediate sequences between closely related proteins.

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bioRxiv2019

Design and assembly of DNA molecules using multi-objective optimisation

Rapid engineering of biological systems is currently hindered by limited integration of manufacturing constraints into the design process, ultimately limiting the yield of many synthetic biology workflows.\n\nHere we tackle DNA engineering as a multi-objective optimization problem aiming at finding the best tradeoff between design requirements and manufacturing constraints. We developed a new open-source algorithm for DNA engineering, called Multi-Objective Optimisation algorithm for DNA Design and Assembly (MOODA), available as a Python package and web application at http://mooda.stracquadaniolab.org.\n\nExperimental results show that our method provides near optimal constructs and scales linearly with design complexity, effectively paving the way to rational engineering of DNA molecules from genes to genomes.

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bioRxiv2019

Engineering combinatorial and dynamic decoders using synthetic immediate-early genes

For tissues to grow and function properly, cells must coordinate actions such as proliferation, differentiation and apoptosis. This coordination is achieved in part by the activation of intracellular signaling pathways that trigger the expression of context-specific target genes. While the function of these natural circuits has been actively studied, synthetic biology provides additional powerful tools for deconstructing, repurposing, and designing novel signal-decoding circuits. Here we report the construction of synthetic immediate-early genes (synIEGs), target genes of the Erk signaling pathway that implement complex, user-defined regulation and can be monitored through the use of live-cell biosensors to track transcription and translation. We demonstrate the power and flexibility of this approach by confirming Erk duration-sensing by the FOS immediate-early gene, elucidating how the BTG2 gene is regulated by transcriptional activation and translational repression after growth-factor stimulation, and by designing a synthetic immediate-early gene that responds with AND-gate logic to the combined presence of growth factor and DNA damage stimuli. Our work paves the way to defining the molecular circuits that link signaling pathways to specific target genes, highlighting an important role for post-transcriptional regulation in signal decoding that may be masked by analyses of RNA abundance alone.

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bioRxiv2020

Systematic Engineering of Artificial Metalloenzymes for New-to-Nature Reactions

Artificial metalloenzymes (ArMs) catalyzing new-to-nature reactions under mild conditions could play an important role in the transition to a sustainable, circular economy. While ArMs have been created for a variety of bioorthogonal transformations, attempts at optimizing their performance by enzyme engineering have been case-specific and resulted only in modest improvements. To realize the full potential of ArMs, methods that enable the rapid discovery of highly active ArM variants for any reaction of interest are required. Here, we introduce a broadly applicable, automation-compatible ArM engineering platform, which relies on periplasmic compartmentalization in Escherichia coli to rapidly and reliably identify improved ArM variants based on the biotin-streptavidin technology. We systematically assess 400 ArM mutants for five bioorthogonal transformations involving different metal cofactors, reaction mechanisms and substrate-product pairs, including novel ArMs for gold-catalyzed hydroamination and hydroarylation. The achieved activity enhancements of up to fifteen-fold over wild type highlight the potential of the systematic approach to ArM engineering. We further capitalize on the sequence-activity data to suggest and validate smart strategies for future screening campaigns. This systematic, multi-reaction study has important implications for the development of highly active ArMs for novel applications in biocatalysis and synthetic biology.

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bioRxiv2022

A gene expression control technology for cell-free systems and synthetic cells via targeted gene silencing and transfection

Cell-free transcription-translation (TXTL) is an in vitro protein expression platform. In synthetic biology, TXTL is utilized for a variety of technologies, such as genetic circuit construction, metabolic pathway optimization, and building prototypes of synthetic cells. For all these purposes, the ability to precisely control gene expression is essential. Various strategies to control gene expression in TXTL have been developed; however, further advancements on gene-specific and straightforward regulation methods are still demanded. Here, we designed a novel method to control gene expression in TXTL, called a "silencing oligo." The silencing oligo is a short oligonucleotide that binds to the target mRNA. We demonstrated that addition of the silencing oligo inhibits eGFP expression in TXTL in a sequence-dependent manner. We investigated one of the silencing oligos inhibitory mechanisms and confirmed that silencing is associated with RNase H activity in bacterial TXTL reactions. We also engineered a transfection system that can be used in synthetic cells. We screened two dozen different commercially available transfection reagents to identify the one that works most robustly in our system. Finally, we combined the silencing oligo with the transfection technology, demonstrating that we can control the gene expression by transfecting silencing oligo-containing liposomes into the synthetic cells.

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bioRxiv2025

Development of a Toolkit for High-Efficiency, Markerless, and Iterative Genome Editing in Shouchella clausii

Shouchella clausii is a spore-forming, Gram-positive bacterium with intrinsic antibiotic resistance and promising potential in biotherapeutics, industrial biotechnology, and environmental applications. Its genetic intractability--due to a rigid cell wall and lack of natural competence--has limited its development as a microbial chassis. To facilitate its genetic transformation, a hyperosmotic electroporation protocol was optimized using cell wall weakening agents, achieving efficiencies comparable to other recalcitrant bacilli. A comprehensive and reusable genetic toolkit was developed centred on a temperature-sensitive E. coli-S. clausii shuttle vector (pM4B522), specifically engineered for compatibility with Golden Gate assembly. The plasmid backbone includes a spectinomycin resistance marker and an integrated red fluorescent protein reporter for transformants selection. A removable AmilCP chromoprotein cassette streamlines the assembly process by enabling blue/white screening in E. coli. This platform has demonstrated its versatility in genome editing for both S. clausii and Bacillus subtilis, as evidenced by its use in several applications: (i) sequential, markerless deletions of the non-essential catabolic genes xylA and lacA in S. clausii DSM 8716, with a success rate exceeding 60%; (ii) replacement of the lacA coding sequence with a GFP coding sequence, resulting in fluorescence induction in lactose-supplemented medium; (iii) introduction of a single-base substitution generating a premature stop codon in lacA, showcasing scar-free point mutagenesis; and (iv) transfer of the system to B. subtilis 168, highlighting its broader applicability across Gram-positive bacteria. Given the precision and scarless nature of these genetic modifications, this toolkit holds strong potential for the development of next-generation probiotics and synthetic biology applications.

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bioRxiv2019

Construction of an inducible amyloid expression circuit in Bacillus megaterium: A case study with CsgA and TasA

Environmental applications of synthetic biology such as water remediation require engineered strains to function robustly in a fluctuating and potentially hostile environment. The construction of synthetic biofilm formation circuits could potentially alleviate this issue by promoting cell survival. Towards this end, we construct a xylose-inducible system for the expression of the functional amyloids CsgA and TasA in the soil bacterium Bacillus megaterium. We find that although both amyloids are expressed, only TasA is successfully exported from the cells. Furthermore, expression of CsgA results in a significant growth penalty for the cells while expression of TasA does not. Finally, we show that TasA expression conveys a small but detectable increase in cells adhesion to nickel beads. These results suggest that TasA is a promising candidate for future work on synthetic biofilm formation in B. megaterium.

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bioRxiv2026

Split-Indigoidine synthetase as optical reporter for benchmarking protein-protein interactions

Indigoidine is a blue pigment biosynthesized by a single-module Non-Ribosomal Peptide Synthetase (NRPS) using L-glutamine as substrate. Despite its potential as a colorimetric reporter, no such system has been established from it to date. We used a recently characterized interdomain fusion site located between its adenylation (A) and thiolation (T) domains to develop the Indi2GO system, which provides a naked-eye detectable and quantitative optical readout of transient and covalent protein-protein-interaction (PPI) in living cells. Indi2GO enables high-throughput benchmarking and optimization of PPI tools in a standard 96-well plate reader format, without requiring exogenous substrates, specialized equipment or complex analytical workflows. We demonstrate its broad applicability with three widely used protein-protein interaction tools: SYNZIPS, inteins, and the SpyTag:SpyCatcher system. We used Indi2GO to validate novel SYNZIP pairs, which we used in NRPS engineering, highlighting its applicability for the development of novel PPI-mediating tools in the context of NRPS engineering and synthetic biology.

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bioRxiv2026

MozClo: An Expanded MoClo Toolset for Large Multigene Assembly and Plant Transformations

The Modular Cloning (MoClo) and PhytoBrick standards have revolutionized plant synthetic biology by establishing a standardized, hierarchical assembly grammar. However, as the engineering of complex metabolic pathways, multi-trait stacks, and synthetic gene circuits expands, existing toolkits hit practical boundaries in assembly capacity and fixed grammars. To overcome these bottlenecks, we present MozClo, an expansion of the MoClo/PhytoBrick architecture. MozClo expands the standard Level 1 assembly framework to 10 positions using new L1 acceptors, end-linkers and dummy parts. We also identify and resolve a critical, sticky-end collision at L1 position 7 that has caused assembly failures during L2 cloning of large plasmids. To address commercial DNA synthesis length constraints and to lower cloning costs, we designed a universal 5-in-1 gene fragment multiplexing system. This architecture embeds up to five distinct parts flanked by orthogonal pairs of BpiI restriction sites into a single synthesized fragment, allowing them to sort independently into their respective L0 acceptor plasmids while maintaining complete modular flexibility of part types. Finally, we provide Level 2 cloning backbones with built in selection genes for common soybean transformation methods to facilitate downstream plant selection. Together, these advancements reduce DNA synthesis overhead and accelerate the construction of complex multigene payloads for plant biotechnology.

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