SEARCH · bioRxiv Science

Results for “Synthetic Biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 757 records · Page 42Linked to original sources

bioRxiv2017

Uncovering cell-free protein expression dynamics by a promoter library with diverse strengths

Cell-free protein expression systems have been widely used for synthetic biology and metabolic engineering applications in recent years. Yet little is known about protein expression in the cell-free systems. Here we take a systems approach to uncover underlying dynamics of cell-free protein expression. We construct a set of T7 promoter variants to express proteins at different transcription rates in a reconstituted and E. coli extract-based cell-free systems. We find that the maximum expression level and the rate of protein synthesis as responses to the transcription rate change are different in the two cell-free systems, suggesting they are driven by different expression dynamics. We confirm this by constructing a simple mathematical model for each cell-free system, which well reproduce the experimental results and also identify different limiting factors for better protein expression in the two cell-free systems. In particular, they revealed there is a negative feedback effect in the mRNA-protein translation by the PURE system and also identified different limiting factors for better protein expression in the two cell-free systems.

synthetic biology↗

bioRxiv2019

Control of ΦC31 integrase-mediated site-specific recombination by protein trans splicing

Serine integrases are emerging as core tools in synthetic biology and have applications in biotechnology and genome engineering. We have designed a split-intein serine integrase-based system for rapid regulation of site-specific recombination events in vivo. The{varphi} C31 integrase was split into two extein domains, and intein sequences (Npu DnaEN and Ssp DnaEC) were attached to the two termini to be fused. Expression of these two components followed by post-translational protein trans-splicing in E. coli generated a fully functional{varphi} C31 integrase. Protein splicing is necessary for recombination activity; no activity was observed when the{varphi} C31 integrase N-and C-terminal extein domains without the intein sequences were co-expressed, nor when a key intein catalytic residue was mutated. As a proof of principle, we used a bistable switch based on an invertible promoter reporter system to demonstrate a potential application of the split intein-regulated site-specific recombination system. We used araC and tet inducible promoters to regulate the expression of the two parts of the split recombinase. Inversion of a DNA segment containing a constitutive promoter, catalyzed by trans-spliced integrase, switches between RFP and GFP expression only when both inducible promoters are ON. We used the same split inteins to regulate the reconstitution of a split integrase-RDF fusion that efficiently catalyzed the reverse attR x attL recombination, demonstrating that our split-intein regulated recombination system can function as a reversible AND gate in which the forward reaction is catalyzed by the integrase, and the reverse reaction by the integrase-RDF fusion. The split-intein integrase is a potentially versatile, regulatable component for building synthetic genetic circuits and devices.

synthetic biology↗

bioRxiv2021

Engineering Bacillus subtilis for the formation of a durable living biocomposite material

Engineered living materials (ELMs) are a fast-growing area of research that combine approaches in synthetic biology and material science. Here, we engineer B. subtilis to become a living component of a silica material composed of self-assembling protein scaffolds for functionalization and cross-linking of cells. B. subtilis was engineered to display SpyTags on polar flagella for cell attachment and cross-linking of SpyCatcher modified secreted scaffolds. Through deletion of the autolysis LytC, endospore limited B. subtilis cells become a structural component of the material with spores for long-term storage of genetic programming. Known silica biomineralization peptides were screened and scaffolds designed for silica polymerization to fabricate biocomposite materials with enhanced mechanical properties. We show that the resulting ELM can be regenerated from a piece of silica material and that new functions can be readily incorporated by co-cultivation of engineered B. subtilis strains. We believe that this work will serve as a framework for the future design of resilient ELMs as functional, self-healing materials for use as responsive coatings and plasters.

synthetic biology↗

bioRxiv2025

iFLinkC-EZ: A scalable and automatable method for the assembly of complex fusion proteins and multi-gene expression constructs based on the iFLinkC framework

Standardized methods for the assembly of DNA constructs have become indispensable in synthetic biology, biotechnology and basic research. The majority of frameworks - most prominently based on Golden Gate - have been developed and optimized for the assembly of transcriptional units (TUs) and thereof composed multi-gene expression constructs. This is significantly enabled by the modular organization of functional elements underlying any given TU. In contrast, the assembly of protein coding sequences typically necessitates tailored approaches. Addressing this technological gap, iterative functional linker cloning (iFLinkC) was recently developed to provide a standardized framework for assembling protein coding sequences of arbitrary size and complexity from the ground up based on pairwise ligations of any two DNA fragments via a two-base overlap. Yet, a need for physically purifying DNA fragments imposes significant procedural complexity and operator skill. Overcoming these limitations, a new assembly algorithm, termed iFLinkC-EZ, is presented. Crucially, the DNA assembly products are now purified by genetic means which greatly simplifies the underlying assembly process and also renders it compatible with robotic automation. Further, the potency of iFLinkC-EZ is demonstrated in the assembly of mono- and poly-chromatic poly-fluorescent fusion proteins as well as poly-cistronic expression constructs while examining the effect of poly-PT and poly-GGS linkers as well as ribosome binding sites on the functional expression of poly-fluorescent fusion protein constructs. Further, the utility of poly-fluorescent proteins is demonstrated for the enhanced labeling of surface displayed recombinant binders in yeast display. Given its ease and efficiency, iFLinkC-EZ is anticipated to be applicable to the assembly of many different types of fusion proteins and thereof based multi-gene expression constructs including but not limited to synthetic protein switches, sensors and multi-enzyme complexes as well as thereof composed genetic circuits and metabolic pathways.

synthetic biology↗

bioRxiv2025

Modular Engineering of Escherichia coli for Enhanced Nickel Uptake, Survival, Biomineralization, and Hydrogen-Supported Bioremediation

Nickel contamination poses increasing environmental and industrial challenges, demanding effective and sustainable remediation strategies. Here, we present a modular synthetic biology approach to engineer Escherichia coli for efficient nickel enrichment, resilience in toxic environments, intracellular biomineralization, and hydrogen-based co-culture stability. Our system comprises four functional modules: (1) a nickel enrichment module, in which the fusion protein NixA-F1v outperformed existing uptake systems, and a mutant repressor RcnRC35L enhanced retention by suppressing nickel efflux; (2) a survival module where heterologous expression of Helicobacter pylori Hpn conferred tolerance to high nickel concentrations, and YejM increased phage resistance; (3) a nickel microparticle module enabling intracellular bioconversion of nickel into detectable microparticles even without engineered uptake proteins; and (4) a hydrogen supply module supporting co-culture stability through E. coli-cyanobacteria adhesion for sustained hydrogen production. These modules were experimentally validated through comparative uptake assays, survival profiling, and microscopy-based particle detection. Our findings demonstrate a customizable and integrative microbial platform for metal bioremediation and biosensing, with potential applications in environmental engineering and industrial waste treatment.

synthetic biology↗

bioRxiv2019

Microfluidic trapping of vesicles reveals membrane-tension dependent FtsZ cytoskeletal re-organisation

The geometry of reaction compartments can affect the outcome of chemical reactions. Synthetic biology commonly uses giant unilamellar vesicles (GUVs) to generate cell-sized, membrane-bound reaction compartments. However, these liposomes are always spherical due to surface area minimization. Here, we have developed a microfluidic chip to trap and reversibly deform GUVs into rod- or cigar-like shapes, including a constriction site in the trap mimicking the membrane furrow in cell division. When we introduce into these GUVs the bacterial tubulin homologue FtsZ, the primary protein of the bacterial Z ring, we find that FtsZ organization changes from dynamic rings to elongated filaments upon GUV deformation, and that these FtsZ filaments align preferentially with the short GUV axis, in particular at the membrane neck. In contrast, pulsing Min oscillations in GUVs remained largely unaffected. We conclude that microfluidic traps are a useful tool for deforming GUVs into non-spherical membrane shapes, akin to those seen in cell division, and for investigating the effect of confinement geometry on biochemical reactions, such as protein filament self-organization.

synthetic biology↗

bioRxiv2024

Identification of cognate recombination directionality factors for large serine recombinases by virtual pulldown

Integrases from the "large serine" family are simple, highly directional site-specific DNA recombinases that have great promise as synthetic biology and genome editing tools. Integrative recombination (mimicking phage or mobile element insertion) requires only integrase and two short ([~]40 - 50) DNA sites. The reverse reaction, excisive recombination, does not occur until it is triggered by the presence of a second protein termed a Recombination Directionality Factor (RDF), which binds specifically to its cognate integrase. Identification of RDFs has been hampered due to their lack of sequence conservation and lack of synteny with the phage integrase gene. Here we use Alphafold2-multimer to identify putative RDFs for more than half of a test set of 98 large serine recombinases, and experimental methods to verify predicted RDFs for 4 of 5 integrases chosen as test cases. We find no universally conserved structural motifs among known and predicted RDFs, yet they are all predicted to bind a similar location on their cognate integrase, suggesting convergent evolution of function. Our methodology greatly expands the available genetic toolkit of cognate integrase - RDF pairs.

synthetic biology↗

bioRxiv2020

The Synthesis Success Calculator: Predicting the Rapid Synthesis of DNA Fragments with Machine Learning

The synthesis and assembly of long DNA fragments has greatly accelerated synthetic biology and biotechnology research. However, long turnaround times or synthesis failures create unpredictable bottlenecks in the design-build-test-learn cycle. We developed a machine learning model, called the Synthesis Success Calculator, to predict whether a long DNA fragment can be readily synthesized with a short turnaround time. The model also identifies the sequence determinants associated with the synthesis outcome. We trained a random forest classifier using biophysical features and a compiled dataset of 1076 DNA fragment sequences to achieve high predictive performance (F1 score of 0.928 on 251 unseen sequences). Feature importance analysis revealed that repetitive DNA sequences were the most important contributor to synthesis failures. We then applied the Synthesis Success Calculator across large sequence datasets and found that 84.9% of the Escherichia coli MG1655 genome, but only 34.4% of sampled plasmids in NCBI, could be readily synthesized. Overall, the Synthesis Success Calculator can be applied on its own to prevent synthesis failures or embedded within optimization algorithms to design large genetic systems that can be rapidly synthesized and assembled.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗

bioRxiv2017

A plasmid-based Escherichia coli gene expression system with cell-to-cell variation below the extrinsic noise limit

Experiments in synthetic biology and microbiology can benefit from protein expression systems with low cell-to-cell variability (noise) and expression levels precisely tunable across a useful dynamic range. Despite advances in understanding the molecular biology of microbial gene regulation, many experiments employ protein-expression systems exhibiting high noise and nearly all-or-none responses to induction. I present an expression system that incorporates elements known to reduce gene expression noise: negative autoregulation and bicistronic transcription. I show by stochastic simulation that while negative autoregulation can produce a more gradual response to induction, bicistronic expression of a repressor and gene of interest can be necessary to reduce noise below the extrinsic limit. I synthesized a plasmid-based system incorporating these principles and studied its properties in Escherichia coli cells, using flow cytometry and fluorescence microscopy to characterize induction dose-response, induction/repression kinetics and gene expression noise. By varying ribosome binding site strengths, expression levels from 55-- 10,740 molecules/cell were achieved with noise below the extrinsic limit. Individual strains are inducible across a dynamic range greater than 20-fold. Experimental comparison of different regulatory networks confirmed that bicistronic autoregulation reduces noise, and revealed unexpectedly high noise for a conventional expression system with a constitutively expressed transcriptional repressor. I suggest a hybrid, low-noise expression system to increase the dynamic range.

synthetic biology↗

bioRxiv2025

Integrated microdroplet workflow for high-throughput cell-free transcription in double emulsion picoreactors

Precise characterization of regulatory sequence performance is fundamental to synthetic biology and next-generation gene therapies, driving the need for scalable and quantitative screening of genetic libraries. While droplet-based microfluidics offers the ultra-high throughput required to scale these assays, it often depends on complex, custom fluorescence-activated droplet sorting platforms. To address this limitation, we introduce an integrated microfluidic workflow that enables cell-free transcription in water-in-oil-in-water double emulsion picoreactors compatible with commercial flow cytometers. The core innovation is an integrated device that combines emulsion reinjection, electric-field-mediated step-injection of in vitro transcription (IVT) reagents, and downstream double emulsification, thereby reducing manual handling and preserving droplet integrity across multistep workflows. We validate the system by coupling isothermal rolling circle amplification (RCA) of DNA templates with on-chip IVT and Mango III aptamer-based fluorescence readout, demonstrating robust detection, binning, and sorting of transcription-active droplets. This workflow provides an accessible and modular platform for quantitative, high-throughput functional screening of regulatory sequences without the need for specialized optical sorting instrumentation.

synthetic biology↗

bioRxiv2020

Reconstitution of contractile actomyosin rings in vesicles

One of the grand challenges of bottom-up synthetic biology is the development of minimal machineries for cell division. The mechanical transformation of large-scale compartments, such as Giant Unilamellar Vesicles (GUVs), requires the geometry-specific coordination of active elements, several orders of magnitude larger than the molecular scale. Of all cytoskeletal structures, large-scale actomyosin rings appear to be the most promising cellular elements to accomplish this task. Here, we have adopted advanced encapsulation methods to study bundled actin filaments in GUVs and compare our results with theoretical modeling. By changing few key parameters, actin polymerization can be differentiated to resemble various types of networks in living cells. Importantly, we find membrane binding to be crucial for the robust condensation into a single actin ring in spherical vesicles, as predicted by theoretical considerations. Upon force generation by ATP-driven myosin motors, these ring-like actin structures contract and locally constrict the vesicle, forming furrow-like deformations. On the other hand, cortex-like actin networks are shown to induce and stabilize deformations from spherical shapes.

synthetic biology↗

bioRxiv2020

Automated Rational Strain Construction Based on High-Throughput Conjugation

Molecular cloning is the core of Synthetic Biology, as it comprises the assembly of DNA and its expression in target hosts. At present, however, cloning is most often a manual, time-consuming and repetitive process that highly benefits from automation. The automation of a complete rational cloning procedure, i.e., from DNA part creation to expression in the target host, involves the integration of different operations and machines. Examples of such workflows are sparse, especially when the design is rational (i.e., the DNA sequence design is fixed, and not based on randomized libraries) and the target host is less genetically tractable (e.g., not sensitive to heat-shock transformation). In this study, an automated workflow for the rational construction of plasmids and their subsequent conjugative transfer into the biotechnological platform organism Corynebacterium glutamicum is presented. The whole workflow is accompanied by a custom-made software tool. As an application example, a rationally designed library of transcription factor biosensors based on the regulator Lrp was constructed and characterized. A sensor with an improved dynamic range was obtained, and insights from the screening provided evidence for a dual regulator function of C. glutamicum Lrp.

synthetic biology↗

bioRxiv2022

Reconstitution and Expression of mcy Gene Cluster in The Model Cyanobacterium Synechococcus 7942 Reveals a Roll of MC-LR in Cell Division

Cyanobacterial blooms pose a serious threat to public health due to the presence of cyanotoxins. The most common cyanotoxins, microcystins (MCs), can cause acute poisoning at high concentrations and hepatocellular carcinoma following chronic exposure. Among all MC variants, MC-LR produced by Microcystis aeruginosa PCC 7806 is the most common toxic MC. Although the biosynthetic pathway for MC-LR has been proposed, experimental support of this pathway is lacking. In an effort to experimentally validate this pathway, we expressed the 55 kb microcystin biosynthetic gene cluster (mcy cluster) (mcyA-J) and produced MC-LR in the model cyanobacterium Synechococcus 7942. We designed and constructed the strong bidirectional promoter biPpsbA2 between mcyA and mcyD, reassembled the mcy cluster in yeast by transformation-associated recombination (TAR cloning), transformed the gene cluster into the NSII site of Synechococcus 7942, and successfully expressed MC-LR at a level of 0.006-0.018 fg cell-1 day-1. The expression of MC-LR led to abnormal cell division and the filamentation of Synechococcus 7942 cells, further analysis proved a role of MC-LR in functional assembly of the cell division protein FtsZ, by competing its GTP binding site. These results represent the first synthetic biological expression of the mcy cluster and the autotrophic production of MC-LR in a photosynthetic model organism, which lays the foundation for resolving the MC biosynthesis pathway. The suggested role of MC-LR in cell division reveals a mechanism of how blooming cyanobacteria gain a competitive edge over their non-blooming counterparts. HighlightsO_LIWe expressed the 55 kb mcy cluster and produced MC-LR in the model cyanobacterium Synechococcus 7942. C_LIO_LIThis is the first realized production of MC in the model non-toxin-production cyanobacteria from CO2 by photosynthesis. C_LIO_LICompared with the self-replicating plasmid, the recombination of the mcy cluster into the genome of Synechococcus 7942 is more suitable for the heterologous production of microcystin. C_LIO_LIMC-LR inhibits cell division by irreversibly competing the GTP binding domain of the cell division protein FtsZ. C_LIO_LIThe newly discovered effect of MC-LR on cell division reveals a mechanism of how blooming cyanobacteria gain competitive edge over their non-blooming counterparts. C_LI

synthetic biology↗

bioRxiv2025

Design of a Modular Inducible Circuit for Pulsatile Population Control of Engineered Bacteria

Synthetic biology enables the precise control of bacterial population dynamics through engineered genetic circuits, paving the way for innovative therapeutic and biosensing applications. Here, we present genetic circuit design that leverages AHL (acyl-homoserine lactone)-based quorum sensing and a transcriptional cascade regulated by an external inducer to drive synchronized proliferation and lysis events. In this system, the absence of the inducer arabinose prevents CinR mediated repression, enabling activation of a transcriptional cascade that drives the expression of the lysis protein, fluorescent protein, and a drug resistance gene. The accumulation of AHL acts as a proxy for population density and modulates gene expression through a quorum-sensing mechanism. This inducible circuit provides tunable and pulsatile control over bacterial population dynamics, enabling cycles of population growth and collapse. Such behavior prevents premature population clearance while ensuring functional bacterial densities for extended durations. Our system represents a foundational step toward implementing more complex multi-stable architectures for self-regulation of bacterial population. This dynamic and modular framework offers significant advantages for therapeutic interventions, such as self-regulating drug delivery, inflammation-responsive probiotic therapies, and targeted bacterial therapy.

synthetic biology↗

bioRxiv2022

Computationally-guided design and selection of ribosomal active site mutants with high activity

Understanding how modifications to the ribosome affect function has implications for studying ribosome biogenesis, building minimal cells, and repurposing ribosomes for synthetic biology. However, efforts to design sequence-modified ribosomes have been limited because point mutations in the ribosomal RNA (rRNA), especially in the catalytic active site (peptidyl transferase center; PTC), are often functionally detrimental. Moreover, methods for directed evolution of rRNA are constrained by practical considerations (e.g., library size). Here, to address these limitations, we developed a computational rRNA design approach for screening guided libraries of mutant ribosomes. Our method includes in silico library design and selection using a Rosetta stepwise Monte Carlo method (SWM), library construction and in vitro testing, and functional characterization in vivo. As a model, we apply our method to making modified ribosomes with mutant PTCs. We engineer ribosomes with as many as 30 mutations in their PTCs, highlighting previously unidentified epistatic interactions, and show that SWM helps identify sequences with beneficial phenotypes as compared to random library sequences. We further demonstrate that some variants improve cell growth in vivo, relative to wild type ribosomes. We anticipate that SWM design may serve as a powerful tool for high-resolution rRNA design.

synthetic biology↗

bioRxiv2023

Exploiting Intrinsic Noise for Heterogeneous Cell Control Under Time Delays and Model Uncertainties

The majority of previous research in synthetic biology has focused on enabling robust control performance despite the presence of noise, while the understanding for how controllers may exploit that noise remains incomplete. Motivated by Maxwells Demon, we previously proposed a cellular control regime in which the exploitation of stochastic noise can break symmetry between and allow for specific control of multiple cells using a single input signal (i.e., single-input-multiple-output or SIMO control). The current work extends that analysis to include uncertain stochastic systems where system dynamics are are affected by time delays, intrinsic noises, and model uncertainty. We find that noise-exploiting controllers can remain highly effective despite coarse approximations to the models scale or incorrect estimations or extrinsic noise in key model parameters, and these controllers can even retain performance under substantial observer or actuator time delays. We also demonstrate how SIMO controllers could drive multi-cell systems to follow different trajectories with different phases and frequencies. Together, these findings suggest that noise-exploiting control should be possible even in the practical case where models are always approximate, where parameters are always uncertain, and where observations are corrupted by errors.

synthetic biology↗

bioRxiv2025

An engineered multi-step differentiation program in Escherichia coli for self-organized spatial patterning

In nature, complex multicellular structures originate from individual cells containing all essential information for differentiation, patterning and morphogenesis. Synthetic biology enables a bottom-up approach to study these processes by engineering and combining individual modules to progressively increase the systems complexity. Here, we engineered a multi-step program mimicking cell differentiation in the model prokaryote Escherichia coli. Starting from genetically identical cells and without providing any external positional information, we generated autonomous spatial patterns of colonies on a solid surface. We first employed a toggle switch to break population homogeneity (symmetry breaking), stochastically differentiating cells into two subpopulations: senders and receivers. Next, we activated expression of a third reporter in receiver colonies located in close proximity to sender colonies via quorum-sensing based communication (paracrine signaling). Finally, we mimic maturation of the newly emerged population by expressing a fourth reporter via an orthogonal, self-activating, quorum sensing signal (autocrine signaling). The diversity of spatial patterns generated by this multi-step program was accurately captured by simulations of a corresponding mathematical model. Together, these results demonstrate that multi-step differentiation programs can be engineered in unicellular bacteria to drive fully self-organized spatial pattern formation.

synthetic biology↗

bioRxiv2023

Stoichiometric expression of messenger polycistrons by eukaryotic ribosomes (SEMPER) for compact, ratio-tunable multi-gene expression from single mRNAs

Applications of mammalian synthetic biology increasingly require the ability to express multiple proteins at user-determined stoichiometries from single, compactly encoded transcripts. Here we present an approach for expressing multiple open reading frames (ORFs) from a single transcript, taking advantage of the leaky scanning model of translation initiation. In this method, adjacent ORFs are translated from a single messenger RNA at tunable ratios determined by their order in the sequence and the strength of their translation initiation sites. We call this approach Stoichiometric Expression of Messenger Polycistrons by Eukaryotic Ribosomes (SEMPER). We demonstrate the principles of this approach by expressing up to three fluorescent proteins from one plasmid in two different cell lines. We then use it to encode a stoichiometrically tuned polycistronic construct encoding gas vesicle acoustic reporter genes, showing that enforcing the optimal ratio in every cell enables efficient formation of the multi-protein complex while minimizing cellular toxicity. Finally, we demonstrate the polycistronic expression of two fluorescent proteins from single mRNAs made through in vitro transcription and delivered to cells. SEMPER will enable a broad range of applications requiring tunable expression from compact eukaryotic constructs.

synthetic biology↗