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Results for “Synthetic Biology”

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bioRxiv2023

Turn-On Protein Switches for Controlling Actin Binding in Cells

Within a shared cytoplasm, filamentous actin (F-actin) plays numerous and critical roles across the cell body. Cells rely on actin-binding proteins (ABPs) to organize F-actin and to integrate its polymeric characteristics into diverse cellular processes. Yet, the multitude of ABPs that engage with and shape F-actin make studying a single ABPs influence on cellular activities a significant challenge. Moreover, without a means of manipulating actin-binding subcellularly, harnessing the F-actin cytoskeleton for synthetic biology purposes remains elusive. Here, we describe a suite of designed proteins, Controllable Actin-binding Switch Tools (CASTs), whose actin-binding behavior can be controlled with external stimuli. CASTs were developed that respond to different external inputs, providing options for turn-on kinetics and enabling orthogonality. Being genetically encoded, we show that CASTs can be inserted into native protein sequences to control F-actin association locally and engineered into new structures to control cell and tissue shape and behavior.

synthetic biology↗

bioRxiv2026

A droplet microfluidic-based platform for enhanced DNA delivery in non-model organisms

Expanding genetic engineering beyond model microorganisms is critical to unlocking novel applications in biotechnology, yet the low efficiency of DNA delivery methods like conjugation, remains a major bottleneck in non-model and environmental microbes. Here, we present an automated, high-throughput droplet microfluidic platform that enhances conjugation by encapsulating donor and recipient microbes in picoliter-scale water-in-oil microdroplets, stabilizing cell-cell contact and DNA transfer. Optimization of incubation time, donor to recipient ratio, and plasmid type yielded over a 100-fold increase in conjugation efficiency compared to conventional methods and enabled delivery of complex DNA libraries in low reaction volumes, demonstrating scalability for pooled plasmid library delivery. We further utilized a synthetic biology circuit for donor removal within microdroplets without antibiotic selection, eliminating the need for host-specific selection markers or engineered auxotrophs. When applied to a soil microbial community, this platform improved community-level conjugation, preserving microbial diversity and enabling the identification of genetically accessible chassis. Collectively, this platform establishes a scalable, generalizable solution for high throughput DNA delivery in previously inaccessible microbial hosts. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=54 SRC="FIGDIR/small/721591v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@2ce034org.highwire.dtl.DTLVardef@3399eaorg.highwire.dtl.DTLVardef@1bde851org.highwire.dtl.DTLVardef@df5727_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2017

Tools for engineering coordinated system behaviour in synthetic microbial consortia

Advancing synthetic biology to the multicellular level requires the development of multiple orthogonal cell-to-cell communication channels to propagate information with minimal signal interference. The development of quorum sensing devices, the cornerstone technology for building microbial communities with coordinated system behaviour, has largely focused on reducing signal leakage between systems of cognate AHL/transcription factor pairs. However, the use of non-cognate signals as a design feature has received limited attention so far. Here, we demonstrate the largest library of AHL-receiver devices constructed to date with all cognate and non-cognate chemical signal interactions quantified and we develop a software tool that allows automated selection of orthogonal chemical channels. We use this approach to identify up to four orthogonal channels in silico and experimentally demonstrate the simultaneous use of three channels in co-culture. The development of multiple non-interfering cell-to-cell communication channels will facilitate the design of synthetic microbial consortia for novel applications including distributed bio-computation, increased bioprocess efficiency, cell specialisation, and spatial organisation.

synthetic biology↗

bioRxiv2026

Evaluating AI-Assisted Customer Verification for Synthetic Nucleic Acid Screening

Legitimacy screening, the process of verifying the identity and purpose of customers ordering synthetic nucleic acids, is a primary safeguard against the misuse of synthetic biology. However, the associated costs discourage the adoption of screening practices. To evaluate whether AI tools can facilitate this process, we tested five large language models on five verification tasks using customer profiles of life sciences researchers from around the world. We compared AI performance against an expert human baseline on flag accuracy, source quality, source fidelity, and cost. Flag accuracy of the best-performing model (Gemini 2.5 Pro with four bibliographic and sanctions APIs) was statistically indistinguishable from the human baseline at 90.2% and 89.0% (n = 41). Gemini 2.5 Pro performed at or above the human baseline on source quality and fidelity, at roughly one-tenth of the cost ($1.18 vs. $14.04 per customer). For information-gathering tasks, which excluded the human review step, costs averaged $0.23 per customer, around 50 times cheaper than human screening. These results support piloting AI assistance at the information-gathering step of legitimacy screening at providers of synthetic nucleic acids and other dual-use biotechnology products, with human reviewers retaining authority over follow-up communication and order fulfillment decisions.

synthetic biology↗

bioRxiv2026

Lipid-conjugated DNA enables on-demand delivery of lipids and proteins to synthetic cells

The bottom-up construction of synthetic cells based on giant unilamellar vesicles (GUVs) is a central goal in synthetic biology. Achieving targeted changes in membrane and cytoplasmic composition with temporal control remains challenging however. DNA-mediated fusion with small vesicles ([~]100 nm large unilamellar vesicles; LUVs) has been proposed as a strategy to deliver lipids and cytosolic contents in a programmable manner. However, in vitro, membrane fusion is generally found to be inefficient and poorly controllable for reasons that are poorly understood. Here, we present an approach based on lipid-conjugated DNA (LiNA) to mediate programmable fusion between LUVs and micron-sized GUVs, which we quantitatively monitor with confocal microscopy at the single-GUV level. We show that lipid and content mixing both occur with high efficiency over a wide range of LiNA concentrations, demonstrating that LiNAs indeed induce robust membrane fusion. Furthermore, we show that LiNA-mediated fusion provides a powerful tool to deliver cytosolic biomolecules, enabling control over internal activities. Our findings establish a quantitative framework for studying fusion-driven processes in synthetic cells and provide a versatile platform for the programmable delivery of lipids and cytosolic cargoes - thus advancing the development of synthetic cells that can grow and adapt through fusion-based uptake of molecular building blocks.

synthetic biology↗

bioRxiv2024

Biochemical implementation of acceleration sensing and PIDA control

Designing dependable, self-regulated biochemical systems has long posed a challenge in the field of Synthetic Biology. Here, we propose a realization of a Proportional-Integral-Derivative-Acceleration (PIDA) control scheme as a Chemical Reaction Network (CRN) governed by mass action kinetics. A constituent element of this architecture is a speed and acceleration biosensing mechanism we introduce and, subsequently, place within a feedback configuration. Our control scheme provides enhanced dynamic performance and robust steady-state tracking. In addition to our theoretical analysis, this is practically highlighted in both the deterministic and stochastic settings by regulating a specific biochemical process in-silico and drawing comparisons with a simpler PID controller.

synthetic biology↗

bioRxiv2025

Rational Development of Recombinant ELP Bolaamphiphiles for the Controlled Construction of Multifunctionalized Globular Protein Vesicles

Synthetic biology has enabled the development of new strategies for creating artificial cells that can sense and respond to external stimuli. This study introduces the bottom-up construction of globular protein vesicles (GPVs) that incorporate elastin-like peptide (ELP) bolaamphiphiles as transmembrane components. To enable this strategy, we devised a Golden Gate-based cloning strategy to streamline the design, expression, and purification of ELP bolaamphiphiles. Three ELP bolamphiphiles with varying structural complexity were developed, incorporating fluorescent proteins to facilitate visualization and characterization. The self-assembly of these bolaamphiphiles into GPVs was optimized by varying the molar ratios of recombinant building blocks. Structural characterization confirmed vesicle formation, dynamic light scattering analysis revealed size distributions dependent on modular complexity, and atomic force microscopy demonstrated that the vesicles exhibited MPa-range Youngs moduli, indicative of high mechanical robustness. Our findings demonstrate that multifunctional ELP bolaamphiphiles can be incorporated into GPVs, enabling modular vesicle engineering. This work provides a foundation for designing synthetic cells with customizable bi-functionalities and modularity, advancing compartmentalized systems.

synthetic biology↗

bioRxiv2025

A synthetic bacterium that degrades and assimilates poly(ethylene terephthalate)

Polyethylene terephthalate (PET) is the fourth most commonly used plastic worldwide. Like all plastics, post-consumer PET is poorly managed and accumulates in the environment, posing significant ecological threats. After 70 years of accumulation, microorganisms capable of degrading and assimilating PET have been isolated, demonstrating that PET can be broken down and converted into valuable cellular biomass or metabolic products. These natural isolates, however, are poorly characterized and challenging to genetically manipulate, which limits their further optimization and applicability. Here, we engineer a well-established synthetic biology chassis for the biodegradation and assimilation of PET. We modified the bacterium Pseudomonas putida KT2440 to heterologously express an active PET-hydrolytic enzyme extracellularly and to metabolize PET biodegradation products. The resulting strain, named PETBuster, was capable of growing on PET as the sole carbon source on solid and liquid media. We achieved 91% PET degradation after 21 days of culture, with a doubling time of 3.6 days, under mesophilic conditions. In this way, we demonstrate that PET fermentation is feasible, opening the door to the production of valuable chemicals from waste.

synthetic biology↗

bioRxiv2024

graded-CRISPRi, a novel tool for tuning the strengths of CRISPRi-mediated knockdowns in Vibrio natriegens using gRNA libraries

In recent years, the fast-growing bacterium Vibrio natriegens has gained increasing attention as it has the potential to become a next-generation chassis for Synthetic Biology. A wide range of genetic parts and genome engineering methods have already been developed. However, there is still a need for a well-characterized tool to effectively and gradually reduce the expression level of native genes. To bridge this gap, we created graded-CRISPRi, a system utilizing gRNA variants that lead to varying levels of repression strength. By incorporating multiple gRNA sequences into our design, we successfully extended this concept to simultaneously repress four distinct reporter genes. Furthermore, we demonstrated the capability of using graded-CRISPRi to target native genes, thereby examining the effect of various knockdown levels on growth.

synthetic biology↗

bioRxiv2024

High-throughput development and characterization of new functional nanobodies for gene regulation and epigenetic control in human cells

Controlling gene expression and chromatin state via the recruitment of transcriptional effector proteins to specific genetic loci has advanced the potential of mammalian synthetic biology, but is still hindered by the challenge of delivering large chromatin regulators. Here, we develop a new method for generating small nanobodies against human chromatin regulators that can repress or activate gene expression. We start with a large and diverse nanobody library and perform enrichment against chromatin regulatory complexes using yeast display, followed by high-throughput pooled selection for transcriptional control when recruited to a reporter in human cells. This workflow allows us to efficiently select tens of functional nanobodies that can act as transcriptional repressors or activators in human cells.

synthetic biology↗

bioRxiv2019

Cell-in-the-loop pattern formation with optogenetically emulated cell-to-cell signaling

1Designing and implementing synthetic biological pattern formation remains a challenge due to underlying theoretical complexity as well as the difficulty of engineering multicellular networks bio-chemically. Here, we introduce a \"cell-in-the-loop\" approach where living cells interact through in silico signaling, establishing a new testbed to interrogate theoretical principles when internal cell dynamics are incorporated rather than modeled. We present a theory that offers an easy-to-use test to predict the emergence of contrasting patterns in gene expression among laterally inhibiting cells. Guided by the theory, we experimentally demonstrated spontaneous checkerboard patterning in an optogenetic setup where cell-to-cell signaling was emulated with light inputs calculated in silico from real-time gene expression measurements. The scheme successfully produced spontaneous, persistent checkerboard patterns for systems of sixteen patches, in quantitative agreement with theoretical predictions. Our research highlights how tools from dynamical systems theory may inform our understanding of patterning, and illustrates the potential of cell-in-the-loop for engineering synthetic multicellular systems.

synthetic biology↗

bioRxiv2020

ProGen: Language Modeling for Protein Generation

Generative modeling for protein engineering is key to solving fundamental problems in synthetic biology, medicine, and material science. We pose protein engineering as an unsupervised sequence generation problem in order to leverage the exponentially growing set of proteins that lack costly, structural annotations. We train a 1.2B-parameter language model, ProGen, on [~]280M protein sequences conditioned on taxonomic and keyword tags such as molecular function and cellular component. This provides ProGen with an unprecedented range of evolutionary sequence diversity and allows it to generate with fine-grained control as demonstrated by metrics based on primary sequence similarity, secondary structure accuracy, and conformational energy.

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bioRxiv2024

Combining positive and negative regulation for modular and robust biomolecular control architectures

Engineered biotechnologies are powered by synthetic gene regulation and control systems, known as genetic circuits, which must be modular and robust to disturbances if they are to perform reliably. An emerging family of regulatory mechanisms is mediated by clustered interspaced palindromic repeats (CRISPR) that can both interfere with (downregulate) or activate (upregulate) a given genes expression. However, all CRSIPR regulation relies on a shared resource pool of dCas9 proteins. Hence, a circuits components can indirectly affect one another via resource competition - even without any intended interactions between them - which compromises the modularity of synthetic biological designs. Using a resourceaware model of CRISPR regulation, we find that circuit modules which simultaneously subject a gene to CRISPR interference and activation are rendered robust to resource competition crosstalk. Evaluating this architectures simulated performance, we identify the scenarios where it can be advantageous over the extant resource competition mitigation strategies. We then consider different feedback architectures to demonstrate that combining opposite regulatory interactions overcomes the trade-off in robustness to perturbations of different nature. The motif of combined positive and negative regulation may therefore give rise to more robust and modular biomolecular controllers, as well as hint at the characteristics of natural systems that possess it.

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bioRxiv2025

Harnessing Coding Sequence Cleavage: Theophylline Aptazymes as Portable Gene Regulators in Bacteria

Nucleic acid-based regulatory elements capable of modulating gene expression in response to specific molecular cues have gained increasing attention in synthetic biology. These systems, which include riboswitches, allosteric DNAzymes, and aptazymes, function as Gene Expression Nucleic Allosteric actuators (GENAs) by coupling molecular recognition with genetic regulation. Their versatility can enable applications in diagnostics, therapeutics, and metabolic engineering. This study presents a novel "semi-trans" aptazyme-based system for gene regulation in bacteria, expanding the range of GENAs. The system employs theophylline-responsive hammerhead ribozyme aptazymes positioned in the 5 UnTranslated Region (UTR), designed to cleave within the coding sequence of the target gene, thereby modulating gene expression in a ligand-dependent manner. Using the tetA gene in Escherichia coli (E. coli) as a proof of concept, we demonstrate ligand-controlled regulation of tetracycline resistance and nickel sensitivity. The system's effectiveness is validated through in vitro cleavage assays and in vivo phenotypic studies in two E. coli strains, highlighting its portability across genetic backgrounds. Furthermore, the ability to design multiple aptazymes targeting different coding regions enables complex and fine-tuned regulation. This work broadens the landscape of synthetic gene regulation tools, facilitating the development of new aptazymes based on this approach. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/650125v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1fe90b3org.highwire.dtl.DTLVardef@1cdd9d1org.highwire.dtl.DTLVardef@197480aorg.highwire.dtl.DTLVardef@fa9398_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2019

Proteolytic queues at ClpXP increase antibiotic tolerance

Antibiotic tolerance is a widespread phenomenon that renders antibiotic treatments less effective and facilitates antibiotic resistance. Here we explore the role of proteases in antibiotic tolerance, short-term population survival of antibiotics, using queueing theory (i.e. the study of waiting lines), computational models, and a synthetic biology approach. Proteases are key cellular components that degrade proteins and play an important role in a multi-drug tolerant subpopulation of cells, called persisters. We found that queueing at the protease ClpXP increases antibiotic tolerance ~80 and ~60 fold in an E. coli population treated with ampicillin and ciprofloxacin, respectively. There does not appear to be an effect on antibiotic persistence, which we distinguish from tolerance based on population decay. These results demonstrate that proteolytic queueing is a practical method to probe bacterial tolerance and related genes, while limiting the unintended consequences frequently caused by gene knockout and overexpression.

synthetic biology↗

bioRxiv2020

Frequency Spectra and the Color of Cellular Noise

The invention of the Fourier integral in the 19th century laid the foundation for modern spectral analysis methods. By decomposing a (time) signal into its essential frequency components, these methods uncovered deep insights into the signal and its generating process, precipitating tremendous inventions and discoveries in many fields of engineering, technology, and physical science. In systems and synthetic biology, however, the impact of frequency methods has been far more limited despite their huge promise. This is in large part due to the difficulties encountered in connecting the underlying stochastic reaction network in the living cell, whose dynamics is typically modelled as a continuous-time Markov chain (CTMC), to the frequency content of the observed, distinctively noisy single-cell trajectories. Here we draw on stochastic process theory to develop a spectral theory and computational methodologies tailored specifically to the computation and analysis of frequency spectra of noisy cellular networks. Specifically, we develop a generic method to obtain accurate Pade approximations of the spectrum from a handful of trajectory simulations. Furthermore, for linear networks, we present a novel decomposition result that expresses the frequency spectrum in terms of its sources. Our results provide new conceptual and practical methods for the analysis and design of noisy cellular networks based on their output frequency spectra. We illustrate this through diverse case studies in which we show that the single-cell frequency spectrum facilitates topology discrimination, synthetic oscillator optimization, cybergenetic controller design, systematic investigation of stochastic entrainment, and even parameter inference from single-cell trajectory data.

synthetic biology↗

bioRxiv2019

Comparative analysis of inducible promoters in cyanobacteria

Research in the field of synthetic biology highly depends on efficient, well-characterized promoters. While great progress has been made with other model organisms such as Escherichia coli, photosynthetic cyanobacteria still lag behind. Commonly used promoters that have been tested in cyanobacteria show weaker dynamic range or no regulation at all. Alternatives such as native metal-inducible promoters pose the problem of inducer toxicity.\n\nHere, we evaluate four different inducible promoters, both previously published and new, using the modular plasmid pSHDY, in the model cyanobacterium Synechocystis sp. PCC 6803 - namely the vanillate-inducible promoter PvanCC, the rhamnose-inducible Prha, and the aTc-inducible PL03, and the Co2+-inducible PcoaT. We estimate individual advantages and disadvantages, as well as dynamic range and strength of each promoter in comparison with well-established constitutive systems. We observed a delicate balance between transcription factor toxicity and sufficient expression to obtain a dose-dependent response to the inducer. In summary, we expand the current understanding and employability of inducible promoters in order to facilitate the construction of more complex regulatory synthetic networks, as well as more complicated biotechnological pathways for cyanobacteria.

synthetic biology↗

bioRxiv2019

Extensive Mammalian Germline Genome Engineering

Xenotransplantation, specifically the use of porcine organs for human transplantation, has long been sought after as an alternative for patients suffering from organ failure. However, clinical application of this approach has been impeded by two main hurdles: 1) risk of transmission of porcine endogenous retroviruses (PERVs) and 2) molecular incompatibilities between donor pigs and humans which culminate in rejection of the graft. We previously demonstrated that all 25 copies of the PERV elements in the pig genome could be inactivated and live pigs successfully generated. In this study, we improved the scale of porcine germline editing from targeting a single repetitive locus with CRISPR to engineering 13 different genes using multiple genome engineering methods. we engineered the pig genome at 42 alleles using CRISPR-Cas9 and transposon and produced PERVKO{middle dot}3KO{middle dot}9TG pigs which carry PERV inactivation, xeno-antigen KO and 9 effective human transgenes. The engineered pigs exhibit normal physiology, fertility, and germline transmission of the edited alleles. In vitro assays demonstrated that these pigs gain significant resistance to human humoral and cell mediated damage, and coagulation dysregulations, similar to that of allotransplantation. Successful creation of PERVKO{middle dot}3KO{middle dot}9TG pigs represents a significant step forward towards safe and effective porcine xenotransplantation, which also represents a synthetic biology accomplishment of engineering novel functions in a living organism. One Sentence SummaryExtensive genome engineering is applied to modify pigs for safe and immune compatible organs for human transplantation

synthetic biology↗