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bioRxiv2025

Systematic Investigation of Double Emulsion Dewetting Dynamics for the Droplet Microfluidic Production of Giant Unilamellar Vesicles (GUVs) under Biocompatible Conditions

Giant unilamellar vesicles (GUVs) embody biomimetic membranes with compartmentalization that serve not only as simplified models to better understand complex biochemical and biophysical processes, but also as a chassis for the bottom-up assembly of synthetic cells. Recently, double emulsion droplet microfluidics has proven to be a promising platform for their production, offering greater throughput, control, and reproducibility over traditional methods. However, the interplay of parameters--particularly under biocompatible conditions--that influence the complex multiphase fluid dynamics of the dewetting process underlying GUV production has not been thoroughly studied, limiting the democratization of the approach. In this study, we systematically investigate how lipid composition and concentration, aqueous phase conditions, droplet confinement, and fluid dynamics effects promote or impede the dewetting process. We show that the prevalent use of high concentrations of glycerol and P188 are unnecessary, and the altered dewetting dynamics with restricted surfactant usage can be tuned by adjusting chip dimensions and multi-phase compositions. Guided by these findings, we achieved robust, high throughput production of monodisperse GUVs using 0.1% P188 and no glycerol with salts. Our results improve the reliability and accessibility of droplet-microfluidics GUV platforms to catalyze advances in biophysics, synthetic biology, and drug discovery.

synthetic biology↗

bioRxiv2025

A T7 RNAP regulatory toolbox for cell-free network engineering and biosensing applications

T7 RNA polymerase is ubiquitously used in the fields of synthetic biology and biotechnology. Yet the ability to precisely and modularly regulate T7 RNAP remains surprisingly limited. Here, we developed a T7 RNAP regulatory toolbox consisting of programmable synthetic repressors, activators, and biosensors in a cell-free system. This toolbox enables the scalable design of T7 RNAP based gene regulatory networks and enables the rapid and sensitive detection of diverse biomolecules, including small-molecule drugs, antibodies, and proteins. By integrating a protein design pipeline, we generated biosensors using fully synthetic binders, demonstrating the potential for rapid development of novel protein-based sensors. We constructed a diagnostic cell-free system combining SARS-CoV-2 Spike protein sensing, gene regulatory based amplification, enzymatic amplification, and glucose based detection demonstrating the potential for point-of-care detection with high sensitivity. This work establishes a flexible and expandable framework for constructing gene circuits responsive to a wide range of biomolecules and demonstrates the potential for engineering point-of-care cell-free diagnostic assays.

synthetic biology↗

bioRxiv2022

Extending Janus lectins architecture: characterization and application to protocells

Synthetic biology is a rapidly growing field with applications in biotechnology and biomedicine. Through various approaches, remarkable achievements, such as cell and tissue engineering, have been already accomplished. In synthetic glycobiology, the engineering of glycan binding proteins is being developed for producing tools with precise topology and specificity. We developed the concept of chimeric lectins, i.e., Janus lectin, with increased valency, and additional specificity. The novel engineered lectin, assembled as a fusion protein between the {beta}-propeller domain from Ralstonia solanacearum and the {beta}-trefoil domain from fungus Marasmius oreades, is specific for fucose and -galactose and its unique protein architecture allows to bind these ligands simultaneously. The protein activity was tested with glycosylated giant unilamellar vesicles, resulting in the formation of proto-tissue-like structures through cross-linking of such protocells. The synthetic protein binds to H1299 lung epithelial cancer cells by its two domains. The biophysical properties of this new construct were compared with the two already existing Janus lectins, RSL-CBM40 and RSL-CBM77Rf. Denaturation profiles of the proteins indicate that the fold of each has a significant role in protein stability and should be considered during protein engineering.

synthetic biology↗

bioRxiv2022

Enzymatic assembly of small synthetic genes with repetitive elements

Gene synthesis efficiency has greatly improved in recent years but is limited when it comes to repetitive sequences and results in synthesis failure or delays by DNA synthesis vendors. This is representing a major obstacle for the development of synthetic biology, since repetitive elements are increasingly being used in design of genetic circuits and design of biomolecular nanostructures. Here, we describe a method for the assembly of small synthetic genes with repetitive elements: First, a gene of interest is split in silico into small synthons of up to 80 base pairs flanked by Golden Gate-compatible overhangs. Then, synthons are made by oligo extension and finally assembled into a synthetic gene by Golden Gate Assembly. We demonstrate the method by constructing eight challenging genes with repetitive elements e.g., multiple repeats of RNA aptamers and RNA origami scaffolds with multiple identical aptamers. The genes range in size from 133 to 456 base pairs and are assembled with fidelities of up to 87.5 %. The method was developed to facilitate our own specific research but may be of general use for constructing challenging and repetitive genes and thus a valuable addition to the molecular cloning toolbox.

synthetic biology↗

bioRxiv2025

Opto-p53: A Light-Controllable p53 Signaling for Regulating p53-Dependent Cell Fate.

p53 protein, a crucial transcription factor in cellular responses to a wide variety of stress, regulates multiple target genes involved in tumor suppression, senescence induction, and metabolic functions. However, it remains unclear how diverse cellular phenotypes are modulated by p53. In this study, we developed an optogenetic tool, Opto-p53, to control p53 signaling by light. Opto-p53 was designed to trigger p53 signaling by reconstituting p53 N-terminal and C-terminal fragments with a light-inducible dimerization (LID) system. Upon light exposure, cells expressing Opto-p53 demonstrated p53 transcriptional activation, resulting in cell death and cell cycle arrest. We further enhanced the efficacy of light-induced p53 activation by introducing specific mutations into Opto-p53 fragments. Our findings unveil the capability of Opto-p53 to serve as a powerful tool for dissecting the complex roles of p53 in cellular processes, thereby contributing to the field of synthetic biology and providing general design principles for optogenetic tools using endogenous transcription factors.

synthetic biology↗

bioRxiv2025

Engineering Artificial 5' Regulatory Sequences for Thermostable Protein Expression in the Extremophile Thermus thermophilus

The utilisation of biocatalysts in biotechnological applications often necessitates their heterologous expression in suitable host organisms. However, the range of standardised microbial hosts for recombinant protein production remains limited, with most being mesophilic and suboptimal for certain protein types. Although the thermophilic bacterium Thermus thermophilus has long been established as a valuable extremophile host, thanks to its high-temperature tolerance, robust growth, and extensively characterised proteome, its genetic toolkit has predominantly depended on a limited set of native promoters. To overcome this bottleneck, we have expanded the available regulatory repertoire in T. thermophilus by developing novel artificial 5' regulatory sequences. In this study, we applied our Gene Expression Engineering platform to engineer 53 artificial 5' regulatory sequences (ARES) in T. thermophilus. These ARES, which comprise both promoter and 5' untranslated regions (UTRs), were functionally characterised in both T. thermophilus and Escherichia coli, revealing distinct host-specific expression patterns. Furthermore, we demonstrated the utility of these ARES by demonstrating high-level expression of thermostable proteins, including {beta}-galactosidase, a superfolder citrine fluorescent protein, and phytoene synthase. A bioinformatic analysis of the novel sequences has also being carried out indicating that the ARES possess markedly lower GC content compared to native promoters. This study contributes to expanding the genetic toolkit for recombinant protein production by providing a set of functionally validated ARES, enhancing the versatility of T. thermophilus as a synthetic biology chassis for thermostable protein expression.

synthetic biology↗

bioRxiv2026

Biochemical Logic Computation Through Repurposing Natural bZIP Protein Interaction Networks

Competitive protein dimerization networks offer an alternative to transcriptional genetic circuits by enabling fast molecular decision-making through direct protein-protein interactions. Implementing these networks currently requires challenging de novo protein design. In this work, we circumvent this limitation by repurposing natural, experimentally pre-characterized basic leucine zipper (bZIP) transcription factor networks. By keeping natural binding affinities and modulating only component monomer concentrations via a customized genetic algorithm, we comprehensively evaluate the computational versatility and robustness of these natural substrates. We identified 135 individual networks capable of implementing Boolean logic, with the most versatile natural clusters computing up to 15 of the 16 possible two-input logic gates, including the non-linearly separable XOR and XNOR. Computational versatility increased with network size and connectivity, and robustness analysis revealed that many optimized networks preserved reliable logical behavior despite substantial stochastic expression noise. These results demonstrate that natural bZIP networks possess substantial latent computational capacity and can perform reliable biochemical computation through concentration tuning alone, providing a realistic foundation for developing scalable protein-based biocomputing platforms for future synthetic biology applications.

synthetic biology↗

bioRxiv2026

PRIME: A modular plasmid-based platform for continuous in vivo evolution across bacteria

Directed evolution enables the engineering of proteins with novel or improved functions, yet existing approaches often require extensive manual intervention and are difficult to sustain over long evolutionary trajectories. Here we introduce PRIME (Protein-primed Replication for In vivo Mutagenesis and Evolution), a plasmid-based orthogonal DNA replication system for continuous directed evolution in Escherichia coli. PRIME harnesses protein-primed DNA replication to establish an autonomous replicon that operates independently of the host genome. Coupling this system to an error-prone DNA polymerase enables targeted diversification of constructs encoded on the orthogonal plasmid while preserving genomic integrity. Using only standard laboratory equipment, we demonstrate the evolution of a construct expressing msfGFP with an 11.8-fold increase in fluorescence. The platform is fully compatible with standard molecular biology workflows and requires no specialised instrumentation. We further demonstrate its portability across bacterial hosts by establishing PRIME in Pseudomonas putida. PRIME thus provides a broadly accessible framework for continuous in vivo evolution, with wide-ranging applications in protein engineering and synthetic biology.

synthetic biology↗

bioRxiv2023

SYNTHETIC CIRCUITS BASED ON SPLIT CAS9 TO DETECT CELLULAR EVENTS

Synthetic biology involves the generation of logic circuits to create or control biological functions and behaviors by engineering interconnected genetic elements such as promoters, repressors, and transcriptional activators. CRISPR discovery, and its adaptations to mammalian cells, has made it the tool of choice in molecular biology and revolutionized genome engineering in biomedical sciences. Here, we describe an adaptation of a split Cas9 to generate synthetic logic gates to sense biological events. As proof-of-concept, the complementing halves of split Cas9 were placed under different promoters, one unique to cancer cells of epithelial origin (phCEA) and one universal promoter (pCMV). We used self-assembling inteins to reunite the halves when co-expressed. Only cancer cells with epithelial origin expressed both halves and activated a reporter becoming green fluorescent. We then investigated whether we could apply this system to the detection of biological processes such as epithelial to mesenchymal transition (EMT). We designed another logic gate where one halve is expressed only by cancer cells of epithelial origin, while the other is activated during EMT - under the control of TWIST1. Indeed, cells undergoing EMT were detected by the activation of the reporter. Finally, the split-Cas9 logic gate was applied as a sensor to detect cell-cell fusion events in multiple cell lines. Each cell type expressed only one halve of split Cas9, and only induction of fusion resulted in the appearance of multinucleated syncytia and the expression of the reporter system. The simplicity and flexibility of the split Cas9 system reported here can be integrated to many other cellular processes, not only as a sensor but as an actuator. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/533022v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1f63cf0org.highwire.dtl.DTLVardef@11ab298org.highwire.dtl.DTLVardef@1c3270eorg.highwire.dtl.DTLVardef@1394488_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2017

Low cost and open source multi-fluorescence imaging system for teaching and research in biology and bioengineering

The advent of easy-to-use open source microcontrollers, off-the-shelf electronics and customizable manufacturing technologies has facilitated the development of inexpensive scientific devices and laboratory equipment. In this study, we describe an imaging system that integrates low-cost and open-source hardware, software and genetic resources. The multi-fluorescence imaging system consists of readily available 470 nm LEDs, a Raspberry Pi camera and a set of filters made with low cost acrylics. This device allows imaging in scales ranging from single colonies to entire plates. We developed a set of genetic components (e.g. promoters, coding sequences, terminators) and vectors following the standard framework of Golden Gate, which allowed the fabrication of genetic constructs in a combinatorial, low cost and robust manner. In order to provide simultaneous imaging of multiple wavelength signals, we screened a series of long stokes shift fluorescent proteins that could be combined with cyan/green fluorescent proteins. We found CyOFP1, mBeRFP and sfGFP to be the most compatible set for 3-channel fluorescent imaging. We developed open source Python code to operate the hardware to run time-lapse experiments with automated control of illumination and camera and a Python module to analyze data and extract meaningful biological information. To demonstrate the potential application of this integral system, we tested its performance on a diverse range of imaging assays often used in disciplines such as microbial ecology, microbiology and synthetic biology. We also assessed its potential for STEM teaching in a high school environment, using it to teach biology, hardware design, optics, and programming. Together, these results demonstrate the successful integration of open source hardware, software, genetic resources and customizable manufacturing to obtain a powerful, low cost and robust system for STEM education, scientific research and bioengineering. All the resources developed here are available under open source licenses.

synthetic biology↗

bioRxiv2017

Data-Driven Prediction of CRISPR-Based Transcription Regulation for Programmable Control of Metabolic Flux

Multiplex and multi-directional control of metabolic pathways is crucial for metabolic engineering to improve product yield of fuels, chemicals, and pharmaceuticals. To achieve this goal, artificial transcriptional regulators such as CRISPR-based transcription regulators have been developed to specifically activate or repress genes of interest. Here, we found that by deploying guide RNAs to target on DNA sites at different locations of genetic cassettes, we could use just one synthetic CRISPR-based transcriptional regulator to simultaneously activate and repress gene expressions. By using the pairwise datasets of guide RNAs and gene expressions, we developed a data-driven predictive model to rationally design this system for fine-tuning expression of target genes. We demonstrated that this system could achieve programmable control of metabolic fluxes when using yeast to produce versatile chemicals. We anticipate that this master CRISPR-based transcription regulator will be a valuable addition to the synthetic biology toolkit for metabolic engineering, speeding up the \"design-build-test\" cycle in industrial biomanufacturing as well as generating new biological insights on the fates of eukaryotic cells.

synthetic biology↗

bioRxiv2022

Inteins in the Loop: A Framework for Engineering Advanced Biomolecular Controllers for Robust Perfect Adaptation

Homeostasis is one of the cornerstones of life shaped by billions of years of evolution. A notion that is similar to homeostasis, but yet more stringent, is Robust Perfect Adaptation (RPA). A system is endowed with RPA if it is capable of driving a variable of interest to a prescribed level despite the presence of disturbances and uncertainties in the environment. Designing and building biomolecular controllers capable of achieving RPA have been identified as an important task which has immediate implications for various disciplines. Here, we develop systematic theoretical and experimental frameworks for custom-built proteins that exploit split inteins -- short amino acid sequences capable of performing protein-splicing reactions -- to design, genetically build and analyze a wide class of RPA-achieving integral feedback controllers. We first lay down a theoretical foundation that facilitates the screening of intein-based controller networks for RPA, and then usher an easy-to-use recipe to simplify their, otherwise complex, underlying mathematical models. Furthermore, we genetically engineer and test various controller circuits based on commonly used transcription factors in mammalian cells. We experimentally and theoretically demonstrate their ability of robustly rejecting external disturbances (that is achieving RPA) over an exquisitely broad dynamic range. Due to their small size, flexibility, modularity, lack of side effects and applicability across various forms of life, inteins serve as promising genetic parts to implement RPA-achieving controllers. To this end, we believe "inteins in the control loop" will leave a significant impact on various disciplines spanning synthetic biology, biofuel production, metabolic engineering and cell therapy among others.

synthetic biology↗

bioRxiv2024

Spheroplasted cells: a game changer for DNA delivery to diatoms

Diatoms produce 20% of the worlds fixed organic carbon annually, making them vital to global carbon fixation and climate change mitigation. Their potential as cell factories for biofuels, proteins, and other high value chemicals remains underutilized due to a lack of genetic engineering tools, with DNA delivery being one of the biggest challenges. Here, we present optimized electroporation and polyethylene glycol transformation methods for delivering DNA and ribonucleoprotein complexes to Phaeodactylum tricornutum, a model diatom species and emerging chassis for algal biotechnology. It was possible to recover transformants with as little as 1 ng of DNA, and to transform linear or circular episomes as large as 55.6 kb. With the optimized electroporation protocol, episomes can be assembled in the algal cell de novo through diatom in vivo assembly (DIVA), forgoing the need for time-consuming traditional cloning steps in Escherichia coli and Saccharomyces cerevisiae. It was also possible to electroporate a Cas9 ribonucleoprotein complex in P. tricornutum, providing an alternative to biolistics for DNA free genome engineering. We have demonstrated that the PEG approach can be adapted to successfully transform Thalassiosira pseudonana, demonstrating the applicability of our methods for engineering other diatom species. These tools can be used to accelerate diatom synthetic biology projects and, therefore, the development of sustainable technologies.

synthetic biology↗

bioRxiv2025

Design of post-translational, ligand-controlled inverters and switches

Rational control over diverse, ligand-responsive output networks is a foundational challenge in synthetic biology, particularly for systems based on post-translational signaling. Here, we present the design and engineering of minimal, modular protein architectures for chemically responsive molecular inverters and digital switches. Our inverter design modifies the plant-derived PYR1-HAB1 chemically inducible dimerization module by incorporating a constitutive activator, which is then competitively displaced by the ligand-bound PYR1 receptor, converting the native dimerization-on mechanism into a signal-off inverter. We establish key design features and demonstrate predictable tuning of the inverters transfer function, including its maximum output, half-maximal inhibitory concentration, and minimum output, solely by adjusting protein stoichiometry. The architecture is modular, enabling plug-and-play response to diverse, user-defined drug-like small molecules. We show that an inverter biosensor for an environmental contaminant functions in engineered living cells with a low nanomolar sensitivity. Additionally, we convert the PYR1-HAB1 sensor into a digital switch by adding an engineered molecular titrant to the system. Overall, this work provides a generalizable, minimal, and tunable protein scaffold for programming complex, post-translational signaling logic, significantly expanding the toolkit for sophisticated biological circuit design.

synthetic biology↗

bioRxiv2023

Assembling membraneless organelles from de novo designed proteins

Recent advances in de novo protein design have delivered a diversity of discrete de novo protein structures and complexes. A new challenge for the field is to use these designs directly in cells to intervene in biological process and augment natural systems. The bottom-up design of self-assembled objects like microcompartments and membraneless organelles is one such challenge, which also presents opportunities for chemical and synthetic biology. Here, we describe the design of genetically encoded polypeptides that form membraneless organelles in Escherichia coli (E. coli). To do this, we combine de novo -helical sequences, intrinsically disordered linkers, and client proteins in single-polypeptide constructs. We tailor the properties of the helical regions to shift protein assembly from diffusion-limited assemblies to dynamic condensates. The designs are characterised in cells and in vitro using biophysical and soft-matter physics methods. Finally, we use the designed polypeptide to co-compartmentalise a functional enzyme pair in E. coli.

synthetic biology↗

bioRxiv2024

Translational activation by a synthetic PPR protein elucidates control of psbA translation in Arabidopsis chloroplasts

Translation initiation on psbA mRNA in plant chloroplasts scales with light intensity, providing its gene product, D1, to replace photodamaged D1 in Photosystem II. The psbA translational activator HCF173 has been hypothesized to mediate this regulation. HCF173 belongs to the short-chain dehydrogenase/reductase superfamily, associates with the psbA 5-untranslated region (5-UTR), and has been hypothesized to enhance translation by binding an RNA segment that would otherwise pair with and mask the ribosome binding region. To test these hypotheses, we examined whether a synthetic pentatricopeptide repeat (sPPR) protein can substitute for HCF173 when bound to the HCF173 binding site. We show that an sPPR designed to bind HCF173s footprint in the psbA 5-UTR bound the intended site in vivo and partially substituted for HCF173 to activate psbA translation. However, sPPR-activated translation did not respond to light. These results imply that HCF173 activates translation, at least in part, by sequestering the RNA it binds to maintain an accessible ribosome binding region, and that HCF173 is also required to regulate psbA translation in response to light. Translational activation can be added to the functions that can be programmed with sPPR proteins for synthetic biology applications in chloroplasts. One sentence summaryA synthetic PPR protein substitutes for HCF173, a non-PPR translational activator in chloroplasts, elucidating HCF173 functions and demonstrating the ability of synthetic PPRs to activate translation.

plant biology↗

bioRxiv2024

A spring-loaded and leakage-tolerant synthetic gene switch for in-vitro detection of DNA and RNA

Nucleic acid tests (NATs) are essential for biomedical diagnostics. Traditional NATs, often complex and expensive, have prompted the exploration of Toehold-Mediated Strand Displacement (TMSD) circuits as an economical alternative. However, the wide application of TMSD-based reactions is limited by leakage--the spurious activation of the reaction leading to high background signals and false positives. Here we introduce a new TMSD cascade that recognizes a custom nucleic acid input and generates an amplified output. The system is based on a pair of thermodynamically spring-loaded DNA modules. The binding of a predefined nucleic acid target triggers an intermolecular reaction that activates a T7 promoter, leading to the perpetual transcription of a fluorescent aptamer that can be detected by a smartphone camera. The system is designed to permit the selective depletion of leakage byproducts to achieve high sensitivity and zero-background signal in the absence of the correct trigger. Using Zika virus (ZIKV)- and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-derived nucleic acid sequences, we show that the assay generates a reliable target-specific readout. Native RNA can be directly detected under isothermal conditions, without requiring reverse transcription, with a sensitivity as low as 200 attomole. The modularity of the assay allows easy re-programming for the detection of other targets by exchanging a single sequence domain. This work provides a low-complexity and high-fidelity synthetic biology tool for point-of-care diagnostics and for the construction of more complex biomolecular computations.

synthetic biology↗

bioRxiv2024

Self-Authenticating Genomic Materials with Advanced Genome Signatures

The authenticity and integrity of synthetic genomic materials containing valuable intellectual property are essential for advancing scientific knowledge and enhancing biosafety. Existing DNA tags and watermarks, however, have limited efficacy due to low mutation tolerance and inadequate digital encoding capacity. Here, we present "Genome Signature", a biochemically stable and tamper-resistant DNA labeling system that enables the creation of self-authenticating genomes. Central to this system is a novel Golomb-ruler-derived Genome-Comb, which efficiently maps extensive nucleotide sequences onto limited codons within endogenous genes, significantly improving error correction and data encoding across millions of nucleotides. Our method successfully recorded a 4.5 million-nucleotide genome in living E. coli. The Genome Signature autonomously identifies and corrects mutations and effectively encodes data within codon orders, ensuring genome integrity and authenticity. Furthermore, it allows precise tracking of sequences across different cells, potentially revolutionizing the development of reliable genomic materials in synthetic biology.

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