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bioRxiv2026

ro-crate-rs: Development of a Lightweight RO-Crate Rust Library for Automated Synthetic Biology

Advances in laboratory automation and AI-driven experimental design have increased the scale and complexity of data generated in synthetic biology. Whilst biofoundries provide significant resources and infrastructure to execute these experiments, most laboratories rely on isolated automated instruments and software systems that operate as disconnected silos, producing heterogeneous data formats with little structured metadata. This fragmentation hinders data integration, reproducibility, and downstream computational workflows. A potential solution is RO-Crate, which offers a lightweight, extensible framework for packaging research data with machine-readable metadata, but existing tooling remains immature for automation-orientated, cloud-native, or high-throughput laboratory workflows. Here, we introduce ro-crate-rs, a new suite of tools centred on a performant Rust library for constructing, validating and packaging RO-Crates across diverse compute environments and automated hardware. The library enforces RO-Crate 1.1 constraints through strong typing while enabling flexible extensions, and is complemented by a Python API and CLI for interactive use and pipeline integration. We demonstrate this combined approach through a semi-automated Old Yellow Enzyme characterisation workflow, showing how RO-Crates can capture data and metadata across multiple independent instruments. Together, these tools provide a robust foundation for FAIR-compliant, automation-ready data management and enable reproducible reconstruction of experimental workflows even in non-biofoundry settings. Availabilityhttps://github.com/intbio-ncl/ro-crate-rs

synthetic biology↗

bioRxiv2022

Expanding the synthetic biology toolbox with a library of constitutive and repressible promoters

Structured AbstractO_ST_ABSBackgroundC_ST_ABSTo support the increasingly complex circuits needed for plant synthetic biology applications, additional constitutive promoters are essential. Reusing promoter parts can lead to difficulty in cloning, increased heterogeneity between transformants, transgene silencing and trait instability. Moreover, the utility of such promoters could be increased by introducing target sequences not found elsewhere in the Arabidopsis thaliana genome and well-suited for Cas9-associated guide RNAs (gRNAs). MethodsWe have developed a pipeline to identify genes that have stable expression across a wide range of Arabidopsis tissues at different developmental stages, and have identified a number of promoters that are well expressed in both transient (Nicotiana benthamiana) and stable (Arabidopsis) transformation assays. We have also introduced two genome-orthogonal gRNA target-sites in a subset of the screened promoters, converting them into NOR logic gates. ResultsOf twenty-two promoters identified in our bioinformatic screen, sixteen drove detectable reporter expression in N. benthamiana. Only three of these promoters were able to produce visible expression of the RUBY reporter in Arabidopsis despite producing RUBY mRNA that could be readily detected by qPCR. We then modified six of these promoters to be repressible, and five of which functioned as NOR gates ConclusionsOne of the major bottlenecks for the ambitious engineering projects currently under development in plants is the lack of well-characterized constitutive promoters. The work here begins to fill this gap. It can also form the basis of constructing more complex information processing circuits in the future.

synthetic biology↗

bioRxiv2024

Measuring the burden of hundreds of BioBricks defines an evolutionary limit on constructability in synthetic biology

Engineered DNA will slow the growth of a host cell if it redirects limiting resources or otherwise interferes with homeostasis. Populations of engineered cells can rapidly become dominated by "escape mutants" that evolve to alleviate this burden by inactivating the intended function. Synthetic biologists working with bacteria rely on genetic parts and devices encoded on plasmids, but the burden of different engineered DNA sequences is rarely characterized. We measured how 301 BioBricks on high-copy plasmids affected the growth rate of Escherichia coli. Of these, 59 (19.6%) negatively impacted growth. The burden imposed by engineered DNA is commonly associated with diverting ribosomes or other gene expression factors away from producing endogenous genes that are essential for cellular replication. In line with this expectation, BioBricks exhibiting burden were more likely to contain highly active constitutive promoters and strong ribosome binding sites. By monitoring how much each BioBrick reduced expression of a chromosomal GFP reporter, we found that the burden of most, but not all, BioBricks could be wholly explained by diversion of gene expression resources. Overall, no BioBricks reduced the growth rate of E. coli by >45%, which agreed with a population genetic model that predicts such plasmids should be "unclonable" because escape mutants will take over during growth of a bacterial colony or small laboratory culture from a transformed cell. We made this model available as an interactive web tool for synthetic biology education and added our burden measurements to the iGEM Registry descriptions of each BioBrick.

synthetic biology↗

bioRxiv2026

DIPTAR: A synthetic biology platform for functional interrogation of protein degradation

Protein degradation regulates cellular homeostasis, yet many degradation events are difficult to study because they lack a readily selectable phenotype. Here, we develop Degradation-Induced Pyroptosis TArgeting Receptors (DIPTAR), a modular synthetic biology platform that couples protein degradation to CARD8-mediated pyroptosis. Using HIF-1 as a model substrate, we show that DIPTAR faithfully reports oxygen-dependent VHL-mediated degradation and enables pooled CRISPR screening to identify established and previously unrecognized regulators of HIF-1 stability. DIPTAR is functional across multiple cell types and can be programmed with diverse proteins, including BRD4, I{kappa}B, and p53, to convert distinct degradation stimuli into a common pyroptotic output. DIPTAR also detects pathogen-mediated perturbations of host degradation pathways, including both inhibition and induction of degradation-dependent signaling. By converting protein degradation into a robust selectable phenotype, DIPTAR provides a scalable platform for functional genetic discovery, interrogation of degradation pathways, degrader characterization, and investigation of host-pathogen interactions.

synthetic biology↗

bioRxiv2022

Configurable Compartmentation Enables In vitro Reconstitution of Sustained Synthetic Biology Systems

The compartmentalized and communicative nature of biological cells contributes to the complexity and endurance of living organisms. Current in vitro compartmentalization systems such as droplet emulsions reproduce the compartmentalization property of cells yet fail to recapture the configurability of cellular communication with the environment. To mimic biological cells a step further and expand the capabilities of in vitro compartmentalization, we present here a general strategy that inherits the passive transport phenomenon of biology. The strategy incorporates layered, micrometer-sized, hydrogel-based compartments featuring configurability in composition, functionality, and selective permeability of biomolecules. We demonstrated the unique advantage of our strategy in two scenarios of synthetic biology. First, a compartmentalized cell-free protein synthesis system was reconstituted that could support multiple rounds of reactions. Second, we constructed living bacteria-based biosensors in the hydrogel compartments, which could achieve long-lasting functioning with markedly enhanced fitness in complex environments. Looking forward, our strategy should be widely applicable for constructing complex, robust, and sustained in vitro synthetic molecular and cellular systems, paving the way for their practical applications.

synthetic biology↗

bioRxiv2019

A Synthetic Biology Approach to Sequential Stripe Patterning and Somitogenesis

Reaction-diffusion (RD) based clock and wavefront model has long been proposed as the mechanism underlying biological pattern formation of repeated and segmented structures including somitogenesis. However, systematic molecular level understanding of the mechanism remains elusive, largely due to the lack of suitable experimental systems to probe RD quantitatively in vivo. Here we design a synthetic gene circuit that couples gene expression regulation (reaction) with quorum sensing (diffusion) to guide bacterial cells self-organizing into stripe patterns at both microscopic and colony scales. An experimentally verified mathematical model confirms that these periodic spatial structures are emerged from the integration of oscillatory gene expression as the molecular clock and the outward expanding diffusions as the propagating wavefront. Furthermore, our paired model-experiment data illustrate that the RD-based patterning is sensitive to initial conditions and can be modulated by external inducers to generate diverse patterns, including multiple-stripe pattern, target-like pattern and ring patterns with reversed fluorescence. Powered by our synthetic biology setup, we also test different topologies of gene networks and show that network motifs enabling robust oscillations are foundations of sequential stripe pattern formation. These results verified close connections between gene network topology and resulting RD driven pattern formation, offering an engineering approach to help understand biological development.

synthetic biology↗

bioRxiv2017

Characterization of Azotobacter vinelandii and Kits for Its Synthetic Biology Applications

Azotobacter vinelandii, a Gram-negative aerobic bacterium with an intracellular anaerobic environment that maintains the oxygen-sensitive enzymatic cascades for nitrogen fixation, could be used to express oxygen-sensitive proteins. However, little is known about the properties of A. vinelandii for synthetic biology applications. We therefore first characterized and optimized the conditions for growing and screening BioBrick constructs in A. vinelandii in the presence of 2 antibiotics, ampicillin and chloramphenicol, and then developed two sets of BioBricks for regulated protein expression. The first kit used T7 RNA polymerase, whose expression is under the control of a nitrogen-repressible nifH promoter. The commonly used T7-dependent system in Escherichia coli can then be used in A. vinelandii. Because its intracellular anaerobic environment is favorable for processes such as magnetosome biogenesis, we attempted to migrate the biogenesis machineries from the magnetotactic bacterium Magnetospirillum gryphiswaldense to A. vinelandii. During this undertaking, another insertion kit construct was developed to allow protein conjugation onto magnetosomes. The kit consists of mamC, a gene encoding a transmembrane protein on magnetosomes, and multiple restriction sites downstream of mamC for fusing a gene of interest. This insertion kit allows the attachment of any desired protein onto the magnetosome membrane by fusing with the mamC protein. We demonstrated the function of this kit by fusing mamC to a GFP nanobody. This kit will facilitate the conjugation of any target protein onto magnetosomes for downstream applications in the future.\n\nFinancial DisclosureWe received sponsorship from the 2012-15 Teaching Development Grants Triennium, Faculty of Engineering and Biochemistry Program, School of Life Sciences, The Chinese University of Hong Kong. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\nCompeting InterestsThe authors declare that no competing interests exist.\n\nEthics StatementN/A.\n\nData AvailabilityAll data are fully available without restriction.

synthetic biology↗

bioRxiv2019

DNA-BOT: A low-cost, automated DNA assembly platform for synthetic biology

Multi-part DNA assembly is the physical starting point for many projects in Synthetic and Molecular Biology. The ability to explore a genetic design space by building extensive libraries of DNA constructs is essential for creating programmed biological systems that perform the desired functions. With multiple DNA assembly methods and standards adopted in the Synthetic Biology community, automation of the DNA assembly process has received serious attention in recent years. Importantly, automating DNA assembly enables larger builds using less researcher time, increasing the accessible design space. However, these benefits currently incur high costs for both equipment and consumables. Here, we address this limitation by introducing low-cost DNA assembly with BASIC on OpenTrons (DNA-BOT). For this purpose, we developed an open-source software package dnabot (https://github.com/BASIC-DNA-ASSEMBLY/dnabot). We demonstrate the performance of DNA-BOT by simultaneously assembling 88 constructs composed of 10 genetic parts, exploring the promoter, ribosome binding site (RBS) and gene order design space for a 3-gene operon. All 88 constructs were assembled with high accuracy, at a cost of $1.50 - $5.50 per construct. This illustrates the efficiency, accuracy and affordability of DNA-BOT making it accessible for most labs and democratising automated DNA assembly.

synthetic biology↗

bioRxiv2023

Identifying functional multi-host shuttle plasmids to advance synthetic biology applications in Mesorhizobium and Bradyrhizobium

Ammonia availability has a crucial role in agriculture as it ensures healthy plant growth and increased crop yields. Since diazotrophs are the only organisms capable of reducing dinitrogen to ammonium, they have a great ecological importance and potential to mitigate the environmental and economic costs of synthetic fertilizer use. Rhizobia are especially valuable being that they can engage in nitrogen-fixing symbiotic relationships with legumes, and they demonstrate great diversity and plasticity in genomic and phenotypic traits. However, few rhizobial species have improved genetic tractability for synthetic biology applications. This study established a basic genetic toolbox with antibiotic resistance markers, multi-host shuttle plasmids and a streamlined protocol for biparental conjugation with Mesorhizobium and Bradyrhizobium species. We identified two repABC origins of replication from Sinorhizobium meliloti (pSymB) and Rhizobium etli (p42d) that were stable across all three strains of interest. Furthermore, the NZP2235 genome was sequenced and phylogenetic analysis determined its reclassification to Mesorhizobium huakuii. These tools will enable the use of plasmid-based strategies for more advanced genetic engineering projects and ultimately contribute towards the development of more sustainable agriculture practices by means of novel nitrogen-fixing organelles, elite bioinoculants or symbiotic association with non-legumes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/572671v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@70e377org.highwire.dtl.DTLVardef@165b06corg.highwire.dtl.DTLVardef@12196a9org.highwire.dtl.DTLVardef@2a33e0_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2016

Establishing elements of a synthetic biology platform for Vaccinia virus production: BioBrickTM design, serum-free virus production and microcarrier-based cultivation of CV-1 cells.

Vaccinia virus (VACV) is an established tool for vaccination and is beginning to prove effective as an oncolytic agent. Industrial production of VACV stands to benefit in future from advances made by synthetic biology in genome engineering and standardisation. As a step toward realising these benefits we analysed the Lister Vaccinia virus genome with respect to refactoring options and propose a VACV genome engineering BioBrick. We then used the CV-1 cell line to produce a conventional recombinant Lister strain VACV, VACVL-15 RFP in a serum-free process. CV-1 cells grown in 5% foetal bovine serum (FBS) Dulbeccos Modified Eagle Medium (DMEM) were adapted to growth in OptiPRO and VP-SFM brands of serum-free media. Specific growth rates of 0.047 h-1 and 0.044 h-1 were observed for cells adapted to OptiPRO and VP-SFM respectively, compared to 0.035 h-1 in 5% FBS DMEM. Cells adapted to OptiPRO and to 5% FBS DMEM achieved recovery ratios of over 96%, an indication of their robustness to cryopreservation. Cells adapted to VP-SFM showed a recovery ratio of 82%. VACV production in static culture, measured as plaque forming units (PFU) per propagator cell, was 75 PFU/cell for cells in 5% FBS DMEM. VP-SFM and OptiPRO adaptation increased VACV production to 150 PFU/cell and 350 PFU/cell respectively. Boosted PFU/cell from OptiPRO-adapted cells persisted when 5% FBS DMEM or OptiPRO medium was present during the infection step and when titre was measured using cells adapted to 5% FBS DMEM or OptiPRO medium. Finally, OptiPRO-adapted CV-1 cells were successfully cultivated using Cytodex-1 microcarriers to inform future scale up studies.

Synthetic Biology↗

bioRxiv2023

The CASwitch: a synthetic biology solution for high-performance inducible gene expression systems in biotechnology

Inducible gene expression systems can be used to control the expression of a gene of interest by means of a small-molecule. One of the most common designs involves engineering a small-molecule responsive transcription factor (TF) and its cognate promoter, which often results in a compromise between minimal uninduced background expression (leakiness) and maximal induced expression. Here, we focussed on an alternative strategy using quantitative synthetic biology to mitigate leakiness while maintaining high expression, without modifying neither the TF nor the promoter. Through mathematical modelling and experimental validations, we designed the CASwitch, a mammalian synthetic gene circuit based on combining two well-known network motifs: the Coherent Feed-Forward Loop (CFFL) and the Mutual Inhibition (MI). The CASwitch combines the CRISPR-Cas endoribonuclease CasRx with the state-of-the-art Tet-On3G inducible gene system to achieve high performances. To demonstrate the potentialities of the CASwitch, we applied it to three different scenarios: enhancing a whole-cell biosensor, controlling expression of a toxic gene and inducible production of Adeno-Associated Virus (AAV) vectors.

synthetic biology↗

bioRxiv2021

Exploring the impact of terminators on transgene expression in Chlamydomonas reinhardtii with a synthetic biology approach

Chlamydomonas reinhardtii has many attractive features for use as a model organism for both fundamental studies and as a biotechnological platform. Nonetheless, despite the many molecular tools and resources that have been developed, there are challenges for its successful engineering, in particular to obtain reproducible and high levels of transgene expression. Here we describe a synthetic biology approach to screen several hundred independent transformants using standardised parts to explore different parameters that might affect transgene expression. We focused on terminators and, using a standardised workflow and quantitative outputs, tested 9 different elements representing three different size classes of native terminators to determine their ability to support high level expression of a GFP reporter gene. We found that the optimal size reflected the median size of element found in the C. reinhardtii genome. The behaviour of the terminator parts was similar with different promoters, in different host strains and with different transgenes. This approach is applicable to the systematic testing of other genetic elements, facilitating comparison to determine optimal transgene design.

synthetic biology↗

bioRxiv2021

Network visualisation of synthetic biology designs

As genetic circuits become more sophisticated, the size and complexity of data about their designs increases. This data captured goes beyond monolithic genetic sequences and towards circuit modularity and functional details, which are beneficial for analyzing circuit performance and establishing design automation techniques. However, the accessibility, visualisation and usability of design data (and metadata) have received relatively little attention to date. Here, we present a method to turn circuit designs into networks and showcase its potential to enhance the utility of design data. Since networks are dynamic structures, initial graphs can be interactively shaped into sub-networks of relevant information based on requirements such as abstraction, hierarchy and protein interactions. Additionally, several visual changes can be applied, such as colouring or clustering nodes based on types (e.g., genes or promoters), resulting in easier comprehension from a user perspective. This approach allows circuit designs to be coupled to other networks, such as metabolic pathways or implementation protocols captured in graph-like formats. Therefore, we advocate using networks to structure, access and improve synthetic biology information.

synthetic biology↗

bioRxiv2026

Scalable probe-based single-cell transcriptional profiling for virtual cell perturbation mapping and synthetic biology phenotyping

Large-scale single-cell transcriptional phenotyping of genetic perturbations (perturb-seq) links genes to phenotypes and should enable virtual cell predictive modeling and cellular engineering. However, current perturb-seq single-cell methods are costly, information sparse and require barcodes for many applications. We developed ProPer-seq, a perturb-seq method that uses multiplexed custom DNA probe panels to measure and phenotype synthetic biology perturbations at single-cell resolution without barcodes, including multidomain proteins and sgRNAs. ProPer-seq faithfully reproduces gold-standard perturb-seq phenotypes while achieving 4-fold cost reduction and 50% increased gene detection per cell. As a scalable fixed-cell profiling method, ProPer-seq enables atlas-scale profiling for virtual-cell initiatives and demonstrates data quality suitable for training and validating predictive models. Lastly, ProPer-seqs targeted detection of modular transgenes enables library-on-library perturbation profiling of combinatorial synthetic protein design spaces. We applied this to 3,550 sgRNA x dCas9 effector combinations as well as 260 CAR x ORF combinations dynamically profiled in primary T cells, revealing principles of transcriptional control and cell state modulation by multidomain synthetic transgenes.

synthetic biology↗

bioRxiv2020

A Streptomyces venezuelae Cell-Free Toolkit for Synthetic Biology

Prokaryotic cell-free coupled transcription-translation (TX-TL) systems are emerging as a powerful tool to examine natural product biosynthetic pathways in a test-tube. The key advantages of this approach are the reduced experimental timescales and controlled reaction conditions. In order to realise this potential, specialised cell-free systems in organisms enriched for biosynthetic gene clusters, with strong protein production and well-characterised synthetic biology tools, is essential. The Streptomyces genus is a major source of natural products. To study enzymes and pathways from Streptomyces, we originally developed a homologous Streptomyces cell-free system to provide a native protein folding environment, a high G+C (%) tRNA pool and an active background metabolism. However, our initial yields were low (36 g/mL) and showed a high level of batch-to-batch variation. Here, we present an updated high-yield and robust Streptomyces TX-TL protocol, reaching up to yields of 266 g/mL of expressed recombinant protein. To complement this, we rapidly characterise a range of DNA parts with different reporters, express high G+C (%) biosynthetic genes and demonstrate an initial proof of concept for combined transcription, translation and biosynthesis of Streptomyces metabolic pathways in a single one-pot reaction.

synthetic biology↗

bioRxiv2026

A CURE for synthetic regulation of gene expression: Rapid screening of guide RNA efficacy as a framework for enabling undergraduate research in plant synthetic biology

Course-based Undergraduate Research Experiences (CUREs) have emerged as a transformative approach to science education, expanding access to authentic research opportunities beyond the traditional undergraduate research assistant (URA) training. By embedding research into a curriculum, CUREs engage a broad and diverse population of students in a classroom environment that emphasizes experimental design, data analysis, and scientific communication. However, this has been difficult to develop for fields such as plant synthetic biology due to the long timescales of plant transformation. One avenue around this problem is to utilize a recent innovation that enables high throughput and rapid screening of gRNA efficacy by leveraging viral-based delivery of guide RNAs (gRNAs). In this work, we develop and validate a CURE with undergraduate students at Colorado State University (CSU). Students worked in teams to design and test efficacy of gRNAs targeting a Cas9-based transcriptional repressor to different regions of the promoters of the three GIBBERELLIN INSENSITIVE 1 genes (GID1a, GID1b, and GID1c) in Arabidopsis thaliana. Over the semester, students generated and analyzed gene expression data to understand the efficiency of twelve new gRNAs. We further validated CURE student-identified gRNAs with an undergraduate research assistant (URA) that assessed target gene expression and phenotypic outcomes in stable transgenic lines expressing SynTF constructs with the strongest gRNAs from the class. We further describe the curriculum structure to facilitate adoption at other institutions and present student-generated datasets demonstrating the utility of ViN-based screening for identifying effective SynTF gRNAs for plant functional genomics and engineering.

synthetic biology↗

bioRxiv2026

Caffeinated Coli: Inspiring the Next Generation of Scientists through Synthetic Biology

The Caffeinated Coli Educational Module brings inquiry-driven learning to high schools, introducing students to scientific research, synthetic biology, and genetic engineering. The module focuses on the exploration of a genetically engineered strain of E. coli modified to grow exclusively on caffeine and, as such, can be used as a measurement device to determine the amount of caffeine in a liquid or beverage. Students conduct two bioassay experiments using these bacteria. During this process, they learn to create cultures and then measure bacterial growth followed by calculating the caffeine concentrations of unknown samples using their own data. This flexible module contains five weeks of original lectures and student learning materials that follow Next Generation Science Standards (NGSS), allowing the content to be adapted for basic, intermediate, or advanced biology courses. Upon completion of the module, students and teachers expressed that the most memorable aspects of the module include collaboration with peers, hands-on learning of content, and the opportunity to interact with the professor/mentors through office hours. Already implemented in 8 Texas high schools over the past two academic years, our module inspires STEM learning while bringing 21st century biology research to new audiences.

scientific communication and education↗

bioRxiv2019

An automated model reduction tool to guide the design and analysis of synthetic biological circuits

We present an automated model reduction algorithm that uses quasi-steady state approximation to minimize the error between the desired outputs. Additionally, the algorithm minimizes the sensitivity of the error with respect to parameters to ensure robust performance of the reduced model in the presence of parametric uncertainties. We develop the theory for this model reduction algorithm and present the implementation of the algorithm that can be used to perform model reduction of given SBML models. To demonstrate the utility of this algorithm, we consider the design of a synthetic biological circuit to control the population density and composition of a consortium consisting of two different cell strains. We show how the model reduction algorithm can be used to guide the design and analysis of this circuit.

synthetic biology↗