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Results for “Synthetic Biology”

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bioRxiv2026

Chemically tunable permeability of engineered alpha-Hemolysin in synthetic cells

Controlling molecular transport across membranes is a defining feature of living cells, yet replicating this functionality in synthetic systems remains a major challenge. Self-inserting protein nanopores, such as -hemolysin (HL), offer a promising route towards programmable membrane permeability due to their robust assembly, compatibility with diverse membrane systems, and intrinsic permeability for diverse biomolecules. Here, we explored the use of chemically functionalized HL nanopores as tunable transport modules. To quantify translocation of peptide substrates across HL-containing membranes, we developed a high-throughput luminescence-based breakage-controlled assay using large unilamellar vesicles. With this assay we introduce a one-pot nanopore modification and strategy, compatible with scalable workflows. Electrophysiology and molecular simulations demonstrate that the introduction of cysteine residues at defined pore locations, combined with targeted chemical modification, enables controlled tuning of HL-nanopore selectivity based on peptide structure and charge. Together, these findings position engineered protein nanopores as versatile and responsive components for controlling membrane transport in synthetic biology.

synthetic biology↗

bioRxiv2022

ACtivE: Assembly and CRISPR-targeted in vivo Editing for Yeast Genome Engineering Using Minimum Reagents and Time

Among the numerous genetic tools developed for yeast, CRISPR/Cas system has been a widely used genome editing method thanks to its sophistication. However, CRISPR methods for yeast generally rely on pre-assembled DNAs and extra cloning steps to deliver gRNA, Cas protein, and donor DNA. These laborious steps might hinder its usefulness. Here, we propose a convenient, rapid, standardizable CRISPR method, named Assembly and CRISPR-targeted in vivo Editing (ACtivE), which only relies on in vivo assembly of linear DNA fragments for both plasmid and donor DNA construction. Thus, depending on the users need, these parts can be easily selected and combined from a repository, serving as a toolkit for rapid genome editing without any expensive reagent. The toolkit contains verified linear DNA fragments, which are easy to store, share and transport at room temperature, drastically reducing expensive shipping costs and assembly time. After optimizing this technique, eight ARS-close loci in the yeast genome were also characterized in terms of integration and gene expression efficiencies and the impacts of the disruptions of these regions on cell fitness. The flexibility and multiplexing capacity of the ACtivE were shown by constructing {beta}-carotene pathway. In only a few days, > 80% integration efficiency for single gene integration and > 50% integration efficiency for triplex integration were achieved from scratch without using in vitro DNA assembly methods, restriction enzymes, or extra cloning steps. This study presents a standardizable method to be readily employed to accelerate yeast genome engineering and provides well-defined genomic location alternatives for yeast synthetic biology and metabolic engineering purposes.

synthetic biology↗

bioRxiv2024

PreciCE: Precision engineering of cell fates via data-driven multi-gene control of transcriptional networks

The directed differentiation of stem cells into specific cell types is critical for regenerative medicine and cell-based applications. However, current methods for cell fate control are inefficient, imprecise, and rely on laborious trial-and-error. To address these limitations, we present a method for data-driven multi-gene modulation of transcriptional networks. We develop bidirectional CRISPR-based tools based on dCas12a, Cas13d, and dCas9 for simultaneously activating and repressing many genes. Due to the vast combinatorial complexity of multi-gene regulation, we introduce a machine learning-based computational algorithm that uses single-cell RNA sequencing data to predict multi-gene perturbation sets for converting a starting cell type into a desired target cell type. By combining these technologies, we establish a unified workflow for data-driven cell fate engineering and demonstrate its efficacy in controlling early stem cell differentiation while suppressing alternative lineages through logic-based cell fate operations. This approach represents a significant advancement in the use of synthetic biology to engineer cell identity.

synthetic biology↗

bioRxiv2025

Super Recombinator (SuRe): An in vivo recombination system for scalable and efficient transgene assembly at a single genomic locus

The capacity to engineer organisms with multiple transgenic components is crucial to synthetic biology and basic biology research. For the former field, transgenic organisms allow the creation of novel biological functions; for the latter, such organisms provide potent means of dissecting complex biological pathways. However, the size limitations of a single transgenesis event and challenges associated with the assembly of multiple DNA fragments hinder the efficient integration of multiple transgenes. To overcome these hurdles, here we introduce a building block for synthetic design termed an integrated genetic array (IGA), which incorporates all genetic components into a single locus to prevent their separation during genetic manipulations. Since the natural recombination rate for genes located in the same locus is near zero, to construct IGAs we developed the Super Recombinator (SuRe) system, which uses CRISPR/Cas9, alone or in combination with site-specific serine recombinases, for in vivo transgene recombination at a single genomic locus. SuRe effectively doubles the number of elements assembled in each recombination round, exponentially accelerating IGA construction. By preventing the separation of transgenic elements, SuRe greatly reduces screening burdens, as validated here through studies of Drosophila melanogaster and Caenorhabditis elegans. To optimize SuRe, we compared CRISPR/Cas9-induced homology-directed recombination to site-specific recombination using various serine recombinases. Optimized versions of SuRe achieved efficiency and fidelity values near their theoretical maxima and allowed the generation of recombinant products up to 4.2 Mbp in size in Drosophila. Using SuRe, we created fruit flies with 12 transgenic elements for fluorescence voltage imaging of neural activity in precisely defined cell-types. Mathematical modeling of the scalability of SuRe to large transgene assemblies showed that integration times and gene assembly workloads respectively scale logarithmically and linearly with the number of transgenes, both major improvements over conventional approaches. Overall, SuRe enables the efficient integration of multiple genes at individual loci, up to the chromosomal scale.

synthetic biology↗

bioRxiv2026

Quantitative Engineering and Investigation of Synthetic Sponge RNAs in E. coli

Sponge RNAs (spRNAs) play an important regulatory role in bacterial small RNA (sRNA) networks, but their engineering and quantitative systems-level properties are unexplored. Here, we design, build, and quantitatively characterise synthetic spRNA-based gene circuits in E. coli. We establish multiple design strategies for synthetic spRNAs, engineering the first synthetic spRNAs. We show that these synthetic spRNAs can reversibly de-repress sRNA-regulated gene expression, demonstrate tuneable control of gene expression, and extend these designs to multi-target regulation. Through the use of time-resolved continuous-culture characterisation in Chi.Bio together with absolute fluorescent protein quantification, we generated a quantitative dynamical dataset for model fitting and mechanistic analysis. Sequential model development showed that recapitulating the observed circuit dynamics required incorporation of Hfq-mediated resource competition, often overlooked in models of sRNA-based synthetic gene circuits. The extended model captured promoter, sRNA, and sponge circuit behaviour and was used to investigate quantitative properties of spRNA-mediated regulation, the first such quantitative investigation of spRNA-based regulation. Model-based quantitative investigations further suggest that spRNAs can tune response functions, modulate thresholds and leakiness, alter response times, improve disturbance rejection in some regimes, increase effective specificity, and buffer regulatory output against sRNA mutation. Together, these results establish synthetic spRNAs as a new post-transcriptional tool for bacterial synthetic biology and provide a quantitative framework for understanding natural and engineered spRNA-mediated regulation.

synthetic biology↗

bioRxiv2024

Directed evolution of an orthogonal transcription engine for programmable gene expression in eukaryotes

T7 RNA polymerase has enabled orthogonal control of gene expression and recombinant protein production across diverse prokaryotic host chassis organisms for decades. However, the absence of 5 methyl guanosine caps on T7 RNAP derived transcripts has severely limited its utility and widespread adoption in eukaryotic systems. To address this shortcoming, we evolved a fusion enzyme combining T7 RNAP with the single subunit capping enzyme from African swine fever virus using Saccharomyces cerevisiae. We isolated highly active variants of this fusion enzyme, which exhibited roughly two orders of magnitude higher protein expression compared to the wild-type enzyme. We demonstrate the programmable control of gene expression using T7 RNAP-based genetic circuits in yeast and validate enhanced performance of these engineered variants in mammalian cells. This study presents a robust, orthogonal gene regulatory system applicable across diverse eukaryotic hosts, enhancing the versatility and efficiency of synthetic biology applications.

synthetic biology↗

bioRxiv2025

Design-driven optimization of low-cost reagent formulations for reproducible and high-yielding cell-free gene expression

Access to recombinant proteins is vital in basic science and biotechnology research. Cell-free gene expression systems provide one approach to address this need, but widespread utilization remains limited by the cost, complexity, and inconsistency of current platforms. To address these limitations, we carry out a multi-dimensional definitive screening design to reduce the number of reagent components and remove costly secondary energy substrates. From more than 1,200 reagent formulations, we discover a simple and reproducible system based on 12 components. The optimized reagent formulation can produce 2.4 {+/-} 0.3 g/L of protein product at the 15-L scale ([~]$55/gprotein) and 3.7 {+/-} 0.2 g/L ([~]$36/gprotein) at the 4-mL scale with oxygen supplementation. This provides an 84 to 99% reduction in cost over previous cell-free reagent formulations. We further show that the optimized reagent formulation can produce nucleoside triphosphates from nucleotides and ribose and that it is robust to failure across batches of cell lysates, users/locations, and in the synthesis of different proteins. Specifically, we demonstrate the production of fifteen therapeutically relevant products, including full-length aglycosylated monoclonal antibodies. We anticipate that our optimized reagent formulation will further democratize the use of cell-free systems for protein manufacturing and synthetic biology applications.

synthetic biology↗

bioRxiv2020

Turning the screw: engineering extreme pH resistance in Escherichia coli through combinatorial synthetic operons

Adoption of microorganisms as platforms for sustainable biobased production requires host cells to be able to withstand harsh industrial conditions, which are usually far from the ones where these organisms are naturally adapted to thrive. However, novel survival mechanisms unearthed by the study of microbiomes from extreme habitats may be exploited to enhance microbial robustness under the strict conditions needed for different applications. In this work, synthetic biology approaches were used to engineer enhanced acidic tolerance in Escherichia coli under extreme conditions through the characterization of a library of twenty-seven unique operons composed of combinatorial assemblies of three novel genes from an extreme environment and three synthetic ribosome binding sites. The results here presented illustrate the efficacy of combining different metagenomic genes for tolerance in truly synthetic genetic operons, as expression of these gene clusters increased hundred-fold the survival percentage of cells exposed to an acidic shock in minimal media at pH 1.9 under aerobic conditions.

synthetic biology↗

bioRxiv2020

Genetically Engineered Integral Feedback Controllers for Robust Perfect Adaptation in Mammalian Cells

To support their survival, cells adapt to environmental disturbances by maintaining a constant internal milieu. Robust perfect adaptation is a strategy that utilizes integral feedback to promote adaptation by robustly driving regulated physiological variables to their pre-disturbance levels. Present in natural systems, this stringent regulatory strategy promises to enable the engineering of sophisticated genetic programs with diverse applications. Here, we present the first synthetic implementations of integral and proportional-integral feedback controllers in mammalian cells. We show that the integral controller robustly and precisely maintains a desired level of a transcription factor, in spite of induced disturbances and network perturbations. Augmenting proportional feedback reduces stochastic variability while maintaining robust perfect adaptation. We demonstrate the benefits of these controllers in mitigating the impact of resource burden and investigate their use in cell therapy. The synthetic biological realization of robust perfect adaptation holds promise for substantial advances in industrial biotechnology and cell-based therapies.

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bioRxiv2023

Guidelines for Optimizing Type S Non-Ribosomal Peptide Synthetases

Bacterial biosynthetic assembly lines, such as non-ribosomal peptide synthetases (NRPS) and polyketide synthases, are often subject of synthetic biology - because they produce a variety of natural products invaluable for modern pharmacotherapy. Acquiring the ability to engineer these biosynthetic assembly lines allows the production of artificial non-ribosomal peptides (NRP), polyketides, and hybrids thereof with new or improved properties. However, traditional bioengineering approaches have suffered for decades from their very limited applicability and, unlike combinatorial chemistry, are stigmatized as inefficient because they cannot be linked to the high-throughput screening platforms of the pharmaceutical industry. Although combinatorial chemistry can generate new molecules cheaper, faster, and in greater numbers than traditional natural product discovery and bioengineering approaches, it does not meet current medical needs because it covers only a limited biologically relevant chemical space. Hence, methods for high-throughput generation of new natural product-like compound libraries could provide a new avenue towards the identification of new lead compounds. To this end, prior to this work, we introduced an artificial synthetic NRPS type, referred to as type S NRPS, to provide a first-of-its-kind bicombinatorial approach to parallelized high-throughput NRP library generation. However, a bottleneck of these first two generations of type S NRPS was a significant drop in production yields. To address this issue, we applied an iterative optimization process that enabled titer increases of up to 55-fold compared to the non-optimized equivalents, restoring them to wild-type levels and beyond.

synthetic biology↗

bioRxiv2023

A molecular proximity sensor based on an engineered, dual-component guide RNA

One of the goals of synthetic biology is to enable the design of arbitrary molecular circuits with programmable inputs and outputs. Such circuits bridge the properties of electronic and natural circuits, processing information in a predictable manner within living cells. Genome editing is a potentially powerful component of synthetic molecular circuits, whether for modulating the expression of a target gene or for stably recording information to genomic DNA. However, programming molecular events such as protein-protein interactions or induced proximity as triggers for genome editing remains challenging. Here we demonstrate a strategy termed "P3 editing", which links protein-protein proximity to the formation of a functional CRISPR-Cas9 dual-component guide RNA. By engineering the crRNA:tracrRNA interaction, we demonstrate that various known protein-protein interactions, as well as the chemically-induced dimerization of protein domains, can be used to activate prime editing or base editing in human cells. Additionally, we explore how P3 editing can incorporate outputs from ADAR-based RNA sensors, potentially allowing specific RNAs to induce specific genome edits within a larger circuit. Our strategy enhances the controllability of CRISPR-based genome editing, facilitating its use in synthetic molecular circuits deployed in living cells.

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bioRxiv2019

Linking Engineered Cells to Their Digital Twins: a Version Control System for Strain Engineering

1.As DNA sequencing and synthesis become cheaper and more easily accessible, the scale and complexity of biological engineering projects is set to grow. Yet, although there is an accelerating convergence between biotechnology and computing science, a deficit in software and laboratory techniques diminishes the ability to make biotechnology more agile, reproducible and transparent while, at the same time, limiting the security and safety of synthetic biology constructs. To partially address some of these problems, this paper presents an approach for physically linking engineered cells to their digital footprint - we called it digital twinning. This enables the tracking of the entire engineering history of a cell line in a specialised version control system for collaborative strain engineering.

synthetic biology↗

bioRxiv2020

De novo designed receptor transmembrane domains enhance CAR-T cell cytotoxicity and attenuate cytokine release

De novo designed receptor transmembrane domains (TMDs) present opportunities for precise control of cellular receptor functions. We developed a de novo design strategy for generating programmed membrane proteins (proMPs): single-pass -helical TMDs that self-assemble through computationally defined and crystallographically validated interfaces. We used these proMPs to program specific oligomeric interactions into a chimeric antigen receptor (CAR) and found that both in vitro CAR T cell cytokine release and in vivo antitumor activity scaled linearly with the oligomeric state encoded by the receptor TMD, from monomers up to tetramers. All programmed CARs (proCARs) stimulated substantially lower T cell cytokine release relative to the commonly used CD28 TMD, which we show elevated cytokine release through lateral recruitment of the endogenous T cell costimulatory receptor CD28. Precise design using orthogonal and modular TMDs thus provides a new way to program receptor structure and predictably tune activity for basic or applied synthetic biology.

synthetic biology↗

bioRxiv2024

STRAIGHT-IN Dual: a platform for dual, single-copyintegrations of DNA payloads and gene circuits into human induced pluripotent stem cells

Targeting DNA payloads into human (h)iPSCs involves multiple time-consuming, inefficient steps that must be repeated for each construct. Here, we present STRAIGHT-IN Dual, which enables simultaneous, allele-specific, single-copy integration of two DNA payloads with 100% efficiency within one week. Notably, STRAIGHT-IN Dual leverages the STRAIGHT-IN platform to allow near-scarless cargo integration, facilitating the recycling of components for subsequent cellular modifications. Using STRAIGHT-IN Dual, we investigated how promoter choice and gene syntax influence transgene silencing, demonstrating the impact these design features have on reporter gene expression and forward programming of hiPSCs into neurons, motor neurons, and endothelial cells. Furthermore, we designed a grazoprevir-inducible synZiFTR system to complement the widely used tetracycline-inducible system, providing independent, tunable, and temporally controlled expression of different transcription factors within the same cell. The unprecedented efficiency and speed with which STRAIGHT-IN Dual generates homogenous genetically engineered hiPSC populations represents a major advancement for synthetic biology in stem cell applications and opens opportunities for precision cell engineering.

synthetic biology↗

bioRxiv2024

Creating overlapping genes by alternate-frame insertion

Overlapping genes-wherein two different proteins are translated from alternative frames of the same DNA sequence-provide a means to stabilize an engineered gene by directly linking its evolutionary fate with that of an overlapped gene. However, creating overlapping gene pairs is challenging as it requires redesign of both protein products to accommodate overlap constraints. Here, we present a new "overlapping, alternate-frame insertion" (OAFI) method for creating overlapping genes by insertion of an "inner" gene, encoded in an alternate frame, into a flexible region of an "outer" gene. Using OAFI, we create new overlapping gene pairs of bacterial toxins within an antibiotic resistance gene. We show that both the inner and outer genes retain functionality despite redesign, with translation of the inner gene is influenced by its overlap position in the outer gene. Additionally, we show that selection for the outer gene alters the permitted inactivating mutations in the inner gene and that overlapping toxins can restrict horizontal gene transfer of the antibiotic resistance gene. Overall, OAFI offers a versatile tool for synthetic biology, expanding the applications of overlapping genes in gene stabilization and biocontainment.

synthetic biology↗

bioRxiv2017

A genetically encoded adhesin toolbox for programming multicellular morphologies and patterns

Synthetic multicellular systems hold promise for understanding natural development of biofilms and higher organisms1,2, as well as for engineering complex multi-component metabolic pathways2,3 and materials4. However, such efforts will require tools to adhere cells into defined morphologies and patterns, and these tools are currently lacking1,5,6. Here we report the first 100% genetically encoded synthetic platform for modular cell-cell adhesion in Escherichia coli, which provides control over multicellular self-assembly. Adhesive selectivity is provided by a library of outer membrane-displayed nanobody7,8 and antigen peptides with orthogonal intra-library specificities, while affinity is controlled by intrinsic adhesin affinity, competitive inhibition, and inducible expression. We demonstrate the resulting capabilities for rational design of well-defined morphologies and patterns through homophilic and heterophilic interactions, lattice-like self-assembly, phase separation, differential adhesion, and sequential layering. This adhesion toolbox, compatible with synthetic biology standards9, will enable construction of high-level multicellular designs and shed light on the evolutionary transition to multicellularity6,10.

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bioRxiv2019

Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function in Bacteria via Dynamic RNA Nanotechnology

A guide RNA (gRNA) directs the function of a CRISPR protein effector to a target gene of choice, providing a versatile programmable platform for engineering diverse modes of synthetic regulation (edit, silence, induce, bind). However, the fact that gRNAs are constitutively active places limitations on the ability to confine gRNA activity to a desired location and time. To achieve programmable control over the scope of gRNA activity, here we apply principles from dynamic RNA nanotechnology to engineer conditional guide RNAs (cgRNAs) whose activity is dependent on the presence or absence of an RNA trigger. These cgRNAs are programmable at two levels, with the trigger-binding sequence controlling the scope of the effector activity and the target-binding sequence determining the subject of the effector activity. We demonstrate molecular mechanisms for both constitutively active cgRNAs that are conditionally inactivated by an RNA trigger (ON[->]OFF logic) and constitutively inactive cgRNAs that are conditionally activated by an RNA trigger (OFF[->]ON logic). For each mechanism, automated sequence design is performed using the reaction pathway designer within NUPACK to design an orthogonal library of three cgRNAs that respond to different RNA triggers. In E. coli expressing cgRNAs, triggers, and silencing dCas9 as the protein effector, we observe programmable conditional gene silencing with a median dynamic range of {approx}6-fold for an ON[->]OFF \"terminator switch\" mechanism, {approx}15-fold for an ON[->]OFF \"splinted switch\" mechanism, and {approx}3.6-fold for an OFF[->]ON \"toehold switch\" mechanism; the median crosstalk within each cgRNA library is <2%, <2%, and {approx}20% for the three mechanisms. By providing programmable control over both the scope and target of protein effector function, cgRNA regulators offer a promising platform for synthetic biology.\n\nO_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

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bioRxiv2013

Resource usage and gene circuit performance characterization in a cell-free ?breadboard?

The many successes of synthetic biology have come in a manner largely different from those in other engineering disciplines; in particular, without well-characterized and simplified prototyping environments to play a role analogous to wind-tunnels in aerodynamics and breadboards in electrical engineering. However, as the complexity of synthetic circuits increases, the benefits--in cost savings and design cycle time--of a more traditional engineering approach can be significant. We have recently developed an in vitro breadboard prototyping platform based on E. coli cell extract that allows biocircuits to operate in an environment considerably simpler than but functionally similar to in vivo. The simplicity of this system makes it a promising tool for rapid biocircuit design and testing, as well as for probing fundamental aspects of gene circuit operation normally masked by cellular complexity. In this work we characterize the cell-free breadboard using real-time and simultaneous measurements of transcriptional and translational activities of a small set of reporter genes and a transcriptional activation cascade. We determine the effects of promoter strength, gene concentration, and nucleoside triphosphate concentration on biocircuit properties, and we isolate the specific contributions of essential biomolecular resources--core RNA polymerase and ribosomes--to overall performance. Importantly, we show how limits on resources, particularly those involved in translation, are manifested as reduced expression in the presence of orthogonal genes that serve as additional loads on the system.

Synthetic Biology↗