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bioRxiv2023

Generative artificial intelligence GPT-4 accelerates knowledge mining and machine learning for synthetic biology

Knowledge mining from synthetic biology journal articles for machine learning (ML) applications is a labor-intensive process. The development of natural language processing (NLP) tools, such as GPT-4, can accelerate the extraction of published information related to microbial performance under complex strain engineering and bioreactor conditions. As a proof of concept, we used GPT-4 to extract knowledge from 176 publications on two oleaginous yeasts (Yarrowia lipolytica and Rhodosporidium toruloides). After integration with a molecule inventory database, the outcome is a total of 2037 data instances and 28 features, which serve as machine learning inputs. The structured datasets enabled ML approaches (e.g., a random forest model) to predict Yarrowia fermentation titers with high accuracy (R2 of 0.86 for unseen test data). Via transfer learning, the trained model could also assess the production capability of the non-conventional yeast, R. toruloides, for which there are fewer published reports. This work demonstrated the potential of generative artificial intelligence to speed up information extraction from research articles, thereby improving design-build-test-learn (DBTL) cycles for commercial biomanufacturing development.

bioengineering↗

bioRxiv2024

De novo-designed minibinders expand the synthetic biology sensing repertoire

Synthetic and chimeric receptors capable of recognizing and responding to user-defined antigens have enabled "smart" therapeutics based on engineered cells. These cell engineering tools depend on antigen sensors which are most often derived from antibodies. Advances in the de novo design of proteins have enabled the design of protein binders with the potential to target epitopes with unique properties and faster production timelines compared to antibodies. Building upon our previous work combining a de novo-designed minibinder of the Spike protein of SARS-CoV-2 with the synthetic receptor synNotch (SARSNotch), we investigated whether minibinders can be readily adapted to a diversity of cell engineering tools. We show that the Spike minibinder LCB1 easily generalizes to a next-generation proteolytic receptor SNIPR that performs similarly to our previously reported SARSNotch. LCB1-SNIPR successfully enables the detection of live SARS-CoV-2, an improvement over SARSNotch which can only detect cell-expressed Spike. To test the generalizability of minibinders to diverse applications, we tested LCB1 as an antigen sensor for a chimeric antigen receptor (CAR). LCB1-CAR enabled CD8+ T cells to cytotoxically target Spike-expressing cells. We further demonstrate that two other minibinders directed against the clinically relevant epidermal growth factor receptor are able to drive CAR-dependent cytotoxicity with efficacy similar to or better than an existing antibody-based CAR. Our findings suggest that minibinders represent a novel class of antigen sensors that have the potential to dramatically expand the sensing repertoire of cell engineering tools.

synthetic biology↗

bioRxiv2019

A reversible memory switch for plant synthetic biology based on the phage PhiC31 integration system

Plant synthetic biology aims to contribute to global food security by engineering plants with new or improved functionalities. In recent years, synthetic biology has rapidly advanced from the setup of basic genetic devices to the design of increasingly complex gene circuits able to provide organisms with novel functions. While many bacterial, fungal and mammalian unicellular chassis have been extensively engineered, this progress has been delayed in plants due to their complex multicellular nature and the lack of reliable DNA devices that allow an accurate design of more sophisticated biological circuits. Among these basic devices, gene switches are crucial to deploying new layers of regulation into the engineered organisms. Of special interest are bistable genetic toggle switches, which allow a living organism to exist in two alternative states and switch between them with a minimal metabolic burden. Naturally occurring toggle switches control important decision-making processes such as cell fate and developmental events. We sought to engineer whole plants with an orthogonal genetic toggle switch to be able to regulate artificial functions with minimal interference with their natural pathways. Here we report a bistable toggle memory switch for whole plants based on the phage PhiC31 serine integrase and its cognate recombination directionality factor (RDF). This genetic device was designed to control the transcription of two genes of interest by inversion of a central DNA regulatory element. Each state of the device is defined by one transcriptionally active gene of interest, while the other one remains inactive. The state of the switch can be reversibly modified by the action of the recombination actuators, which were administered externally (e.g. via agroinfiltration), or produced internally in response to an inducible chemical stimulus. We extensively characterized the kinetics, memory, and reversibility of this genetic switch in Nicotiana benthamiana through transient and stable transformation experiments using transgenic plants and hairy roots. Finally, we coupled the integrase expression to an estradiol-inducible promoter as a proof of principle of inducible activation of the switch.

synthetic biology↗

bioRxiv2019

ChassiDex: A microbial database useful for synthetic biology applications

ChassiDex is an open-source, non-profit online host organism database that houses a repository of molecular, biological and genetic data for model organisms with applications in synthetic biology. The structured user-friendly environment makes it easy to browse information. The database consists of a page for each model organism subdivided into sections such as Growth Characteristics, Strain diversity, Culture sources, Maintenance protocol, Transformation protocol, BioBrick parts and commonly used vectors. With tools such as CUTE built for codon usage table generator, it is also easy to generate and download accurate novel codon tables for unconventional hosts in suitable formats. This database was built as a project for the International Genetically Engineered Machine Competition in 2017 with the mission of making it easy to shift from working with one host organism to another unconventional host organism for any researcher in the field of synthetic biology. The code along with other instructions for the usage of the database and tools are publicly available at the GitHub page. We encourage the synthetic biology community to contribute to the database by adding data for any additional or existing host organism.\n\nhttps://chassidex.org; https://github.com/ChassiDex

synthetic biology↗

bioRxiv2025

A high-throughput heterologous expression platform for plant synthetic biology based on Arabidopsis suspension cells

Efficient heterologous expression platforms are essential for plant synthetic biology, particularly for engineering complex multigene pathways. Here, we establish a high-throughput system for both transient and stable transformation of Arabidopsis thaliana suspension cells using plant cell pack infiltration. This method requires no specialized equipment or consumables and is compatible with several cell lines. It enables rapid generation of 100 g of transgenic cells within two weeks and allows expression of at least 6 stacked genes from a single construct. We characterized constitutive promoters for gene expression in Arabidopsis cells and validated plastid targeting peptides. A library of NifB homologs was screened for expression and solubility and several archaeal variants suitable for plant expression were identified. We further engineered stable cell lines expressing up to six genes, encoding the NifB module components NifU, NifS, FdxN, and NifB, demonstrating that the newly developed platform integrates into an established workflow for nitrogenase engineering. The platform accelerates design-build-test cycles and facilitates the production of delicate proteins that require large amounts of transgenic biomass. It thus represents a versatile and scalable tool for advancing synthetic biology and for tackling major biotechnological challenges, such as biological nitrogen fixation. HighlightWe developed a fast and scalable expression platform in Arabidopsis suspension cells, enabling transient and stable multigene expression for applications in plant synthetic biology such as nitrogenase engineering.

plant biology↗

bioRxiv2024

Advancing chloroplast synthetic biology through high-throughput plastome engineering of Chlamydomonas reinhardtii

Chloroplast synthetic biology holds promise for developing improved crops through improving the function of plastids. However, chloroplast engineering efforts face limitations due to the scarcity of genetic tools and the low throughput of plant-based systems. To address these challenges, we here established Chlamydomonas reinhardtii as a prototyping chassis for chloroplast synthetic biology. We developed an automation workflow that enables the generation, handling, and analysis of thousands of transplastomic strains in parallel, expanded the repertoire of selection markers for chloroplast transformation, established new reporter genes, and characterized over 140 regulatory parts, including native and synthetic promoters, UTRs, and intercistronic expression elements. We integrated the system within the Phytobrick cloning standard and demonstrate several applications, including a library-based approach to develop synthetic promoter designs in plastids. Finally, we provide a proof-of-concept for prototyping novel traits in plastids by introducing a chloroplast-based synthetic photorespiration pathway and demonstrating a twofold increase in biomass production. Overall, our study advances chloroplast engineering, and provides a promising platform to rapidly prototype chloroplast manipulations before their transfer into higher plants and crops.

synthetic biology↗

bioRxiv2021

Synthetic Biology Tool Development Advances Predictable Gene Expression in the Metabolically Versatile Soil Bacterium Rhodopseudomonas palustris

Harnessing the unique biochemical capabilities of non-model microorganisms would expand the array of biomanufacturing substrates, process conditions, and products. There are non-model microorganisms that fix nitrogen and carbon dioxide, derive energy from light, catabolize methane and lignin-derived aromatics, are tolerant to physiochemical stresses and harsh environmental conditions, store lipids in large quantities, and produce hydrogen. Model microorganisms often only break down simple sugars and require low stress conditions, but they have been engineered for the sustainable manufacture of numerous products, such as fragrances, pharmaceuticals, cosmetics, surfactants, and specialty chemicals, often by using tools from synthetic biology. Transferring complex pathways with all of the needed cofactors, energy sources, and cellular conditions from a non-model microorganism to a common chassis has proven to be exceedingly difficult. Utilization of unique biochemical capabilities could also be achieved by engineering the host; although, synthetic biology tools developed for model microbes often do not perform as designed in other microorganisms. The metabolically versatile Rhodopseudomonas palustris CGA009, a purple non-sulfur bacterium, catabolizes aromatic compounds derived from lignin in both aerobic and anaerobic conditions and can use light, inorganic, and organic compounds for its source of energy. R. palustris utilizes three nitrogenase isozymes to fulfill its nitrogen requirements while also generating hydrogen. Furthermore, the bacterium produces two forms of RuBisCo in response to carbon dioxide/bicarbonate availability. While this potential chassis harbors many beneficial traits, stable heterologous gene expression has been problematic due to its intrinsic resistance to many antibiotics and the lack of synthetic biology parts investigated in this microbe. To address these problems, we have characterized gene expression and plasmid maintenance for different selection markers, started a synthetic biology toolbox specifically for the photosynthetic R. palustris, including origins of replication, fluorescent reporters, terminators, and 5 untranslated regions, and employed the microbes endogenous plasmid for exogenous protein production. This work provides essential synthetic biology tools for engineering R. palustris many unique biochemical processes and has helped define the principles for expressing heterologous genes in this promising microbe through a methodology that could be applied to other non-model microorganisms.

bioengineering↗

bioRxiv2020

Modelling co-translational dimerisation for programmable nonlinearity in synthetic biology

Nonlinearity plays a fundamental role in the performance of both natural and synthetic biological networks. Key functional motifs in living microbial systems, such as the emergence of bistability or oscillations, rely on nonlinear molecular dynamics. Despite its core importance, the rational design of nonlinearity remains an unmet challenge. This is largely due to a lack of mathematical modelling that accounts for the mechanistic basics of nonlinearity. We introduce a model for gene regulatory circuits that explicitly simulates protein dimerization--a well-known source of nonlinear dynamics. Specifically, our approach focusses on modelling co-translational dimerization: the formation of protein dimers during--and not after--translation. This is in contrast to the prevailing assumption that dimer generation is only viable between freely diffusing monomers (i.e., post-translational dimerization). We provide a method for fine-tuning nonlinearity on demand by balancing the impact of co- versus post-translational dimerization. Furthermore, we suggest design rules, such as protein length or physical separation between genes, that may be used to adjust dimerization dynamics in-vivo. The design, build and test of genetic circuits with on-demand nonlinear dynamics will greatly improve the programmability of synthetic biological systems.

synthetic biology↗

bioRxiv2023

Broaden the application of Yarrowia Lipolytica synthetic biology tools to explore the potential of Yarrowia clade biodiversity

Yeasts have established themselves as prominent microbial cell factories, and the availability of synthetic biology tools has led to breakthroughs in the rapid development of industrial chassis strains. The selection of a suitable microbial host is critical in metabolic engineering applications, but it has been largely limited to a few well-defined strains. However, there is growing consideration for evaluating strain diversity, as a wide range of specific traits and phenotypes have been reported even within a specific yeast genus or species. Moreover, with the advent of synthetic biology tools, non-type strains can now be easily and swiftly reshaped. The yeast Yarrowia lipolytica has been extensively studied for various applications such as fuels, chemicals, and food. Additionally, other members of the Yarrowia clade are currently being evaluated for their industrial potential. In this study, we demonstrate the versatility of synthetic biology tools originally developed for Y. lipolytica by repurposing them for engineering other yeasts belonging to the Yarrowia clade. Leveraging the GoldenGate Y. lipolytica tool kit, we successfully expressed fluorescent proteins as well as the carotenoid pathway in at least six members of the clade, serving as proof of concept. This research lays the foundation for conducting more comprehensive investigations into the under-characterized strains within the Yarrowia clade and exploring their potential applications in biotechnology.

synthetic biology↗

bioRxiv2022

SNAP-tag and HaloTag fused proteins for HaSX8-inducible control over synthetic biological functions in engineered mammalian cells

Drug-inducible systems allow biological processes to be regulated through the administration of exogenous chemical inducers. Such methods can be used to study native biological activities, or to control synthetically engineered ones, with temporal and dose-dependent control. However, the number of existing drug-inducible systems is limited, and there remains a need for synthetic biology components that can be combined with the existing toolset and regulated with independent and orthogonal control. Here, we describe new cell engineering components that can be regulated via a heterodimerization of SNAP-tag and HaloTag domains using a selective small molecule crosslinker termed "HaXS8." The construction and validation of multiple HaXS8-sensitive components are described, including systems for regulating transcription, Cre recombinase activity, and caspase-9 activity in mammalian cells. The systems elaborate the ability to control gene expression, DNA recombination, and apoptosis in cell engineered systems.

synthetic biology↗

bioRxiv2023

At-home, cell-free synthetic biology education modules for transcriptional regulation and environmental water quality monitoring

As the field of synthetic biology expands, the need to grow and train science, technology, engineering, and math (STEM) practitioners is essential. However, the lack of access to hands-on demonstrations has led to inequalities of opportunity and practice. In addition, there is a gap in providing content that enables students to make their own bioengineered systems. To address these challenges, we develop four shelf-stable cell-free biosensing educational modules that work by just-adding-water and DNA to freeze-dried crude extracts of Escherichia coli. We introduce activities and supporting curricula to teach the structure and function of the lac operon, dose-responsive behavior, considerations for biosensor outputs, and a build-your-own activity for monitoring environmental contaminants in water. We piloted these modules with K-12 teachers and 130 high school students in their classrooms - and at home - without professional laboratory equipment or researcher oversight. This work promises to catalyze access to interactive synthetic biology education opportunities.

synthetic biology↗

bioRxiv2025

Characterization and orthogonality assessment of two quorum sensing systems for synthetic biology applications

Quorum sensing systems have a broad range of applications within the field of synthetic biology. However, a bottleneck is the optimization and tuning of these systems due to the lack of standardization and complete characterization. In this research, two quorum sensing systems, namely the LasI/LasR and the EsaI/EsaR system, were fully characterized in the model host organism Escherichia coli. Furthermore, insight was gained in the interplay between the various parts of these systems. To further expand the range of possibilities with these quorum sensing systems, the orthogonality of the two systems was assessed to allow simultaneous use within the same cell without interfering crosstalk. This assessment was performed on three levels: promoter, signal and synthase crosstalk. It was demonstrated that LasR is able to interact with the promoter of the EsaI/EsaR system, albeit to a low extent. Additionally, LasR was able to respond to the autoinducers produced by EsaI. To solve the promoter crosstalk, a nucleotide change was introduced into the binding site of EsaR within the promoter region. Additionally, LasR mutants were created rationally and screened for decreased response to EsaI while retaining functionality. The best performing mutant, LasR(P117S), was further characterized. In conclusion, we have further unlocked the potential of quorum sensing systems for synthetic biology applications by obtaining two functional, characterized and orthogonal quorum sensing systems. Highlights- Characterization of two LuxR-type quorum sensing systems - Assessing the orthogonality of the EsaI/EsaR and LasI/LasR quorum sensing system - Eliminating the crosstalk between the EsaI/EsaR and LasI/LasR quorum sensing system

synthetic biology↗

bioRxiv2020

A geometric and structural approach to the analysis and design of biological circuit dynamics: a theory tailored for synthetic biology

Much of the progress in developing our ability to successfully design genetic circuits with predictable dynamics has followed the strategy of molding biological systems to fit into conceptual frameworks used in other disciplines, most notably the engineering sciences. Because biological systems have fundamental differences from systems in these other disciplines, this approach is challenging and the insights obtained from such analyses are often not framed in a biologically-intuitive way. Here, we present a new theoretical framework for analyzing the dynamics of genetic circuits that is tailored towards the unique properties associated with biological systems and experiments. Our framework approximates a complex circuit as a set of simpler circuits, which the system can transition between by saturating its various internal components. These approximations are connected to the intrinsic structure of the system, so this representation allows the analysis of dynamics which emerge solely from the systems structure. Using our framework, we analyze the presence of structural bistability in a leaky autoactivation motif and the presence of structural oscillations in the Repressilator.

synthetic biology↗

bioRxiv2025

A synthetic biology toolkit for interrogating plasmid-dependent methylotrophy and enhancing methanol-based biosynthesis of Bacillus methanolicus

Bacillus methanolicus represents a thermophilic methylotroph whose methanol utilization depends on plasmid-encoded genes. It serves as a unique model for deciphering plasmid-dependent methylotrophy and an ideal chassis for low-carbon biomanufacturing using CO2-derived C1 substrates. Despite its evolutionary uniqueness and industrial potential, the lack of synthetic biology tools has hindered both mechanistic understanding and strain engineering. Here, we present a comprehensive synthetic biology platform comprising a high-efficiency electroporation protocol, a CRISPR method enabling robust and multiplex genome editing, diverse neutral loci for gene integration and overexpression, and a cloud-based genome-scale metabolic model iBM822 for user-friendly biodesign. Leveraging this toolkit, we systematically dissected plasmid-dependent methylotrophy, host restriction-modification systems, and functional significance of the chromosomal methylotrophic genes through targeted deletion. To address plasmid loss-induced strain degeneration, we integrated the large endogenous plasmid pBM19 into the chromosome for stable and intact methylotrophic growth. Finally, by integrating metabolic modeling with CRISPR editing, we engineered L-arginine feedback regulation to achieve the first L-arginine biosynthesis from methanol. This study establishes a synthetic biology framework for B. methanolicus, promoting mechanistic exploration of methylotrophy and low-carbon biomanufacturing.

synthetic biology↗

bioRxiv2023

A synthetic biology approach to transgene expression

The ability to control gene expression is pivotal in genetic engineering and synthetic biology. However, in most non-model and pest insect species, empirical evidence for predictable modulation of gene expression levels is lacking. This knowledge gap is critical for genetic control systems, particularly in mosquitoes, where transgenic methods offer novel routes for pest control. Commonly, the choice of RNA polymerase II promoter (Pol II) is the primary method for controlling gene expression, but the options are limited. To address this, we developed a systematic approach to characterize modifications in translation initiation sequences (TIS) and 3 untranslated regions (UTR) of transgenes, enabling the creation of a toolbox for gene expression modulation in mosquitoes and potentially other insects. The approach demonstrated highly predictable gene expression changes across various cell lines and promoter sequences, representing a significant advancement in mosquito synthetic biology gene expression. tools. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/555539v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1db954forg.highwire.dtl.DTLVardef@1279543org.highwire.dtl.DTLVardef@1b86fa2org.highwire.dtl.DTLVardef@1a9176a_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

A synthetic biology approach to assemble and reboot clinically-relevant Pseudomonas aeruginosa tailed phages

The rise in frequency of antibiotic resistance has made bacterial infections, specifically Pseudomonas aeruginosa, a cause for greater concern. Phage therapy is a promising solution that uses naturally isolated phages to treat bacterial infections. Ecological limitations, which stipulate a discrete host range and the inevitable evolution of resistance, may be overcome through a better understanding of phage biology and the utilization of engineered phages. In this study, we developed a synthetic biology approach to construct tailed phages that naturally target clinically-relevant strains of Pseudomonas aeruginosa. As proof of concept, we successfully cloned and assembled the JG024 and DMS3 phage genomes in yeast using transformation-associated recombination (TAR) cloning and rebooted these two phage genomes in two different strains of P. aeruginosa. We identified factors that affected phage reboot efficiency like the phage species or the presence of antiviral defense systems in the bacterial strain. We have successfully extended this method to two other phage species and observed that the method enables the reboot of phages that are naturally unable to infect the strain used for reboot. This research represents a critical step towards the construction of clinically-relevant, engineered P. aeruginosa phages. ImportancePseudomonas aeruginosa is a bacterium responsible for severe infections and a common major complication in cystic fibrosis. The use of antibiotics to treat bacterial infections has become increasingly difficult as antibiotic resistance has become more prevalent. Phage therapy is an alternative solution that is already being used in some European countries, but its use is limited by narrow host range due to the phage receptor specificity, the presence of antiviral defense systems in the bacterial strain, and the possible emergence of phage resistance. In this study, we demonstrate the use of a synthetic biology approach to construct and reboot clinically-relevant P. aeruginosa tailed phages. This method enables a significant expansion of possibilities through the construction of engineered phages for therapy applications.

synthetic biology↗

bioRxiv2024

Do the shuffle: Expanding the synthetic biology toolkit for shufflon-like recombination systems

Naturally occurring DNA inversion systems play an important role in the generation of genetic variation and adaptation in prokaryotes. Shufflon invertase (SI) Rci from plasmid R64, recognizing asymmetric sfx sites, has been adopted as a tool for synthetic biology. However, the availability of a single enzyme with moderate rates of recombination has hampered the more widespread use of SIs. We identified 14 previously untested SI genes and their sfx sites in public databases. We established an assay based on single-molecule sequencing that allows the quantification of the inversion rates of these enzymes and determined cross-recognition to identify orthogonal SI/sfx pairs. We describe SI enzymes with substantially improved shuffling rates when expressed in an inducible manner in E. coli. Our findings will facilitate the use of SIs in engineering biology where synthetic shufflons enable the generation of millions of sequence variants in vivo for applications such as barcoding or experimental selection.

synthetic biology↗

bioRxiv2023

Expanding the toolbox of probiotic Escherichia coli Nissle 1917 for synthetic biology

Escherichia coli Nissle 1917 (EcN) is a probiotic microbe that has the potential to be developed as a promising chassis for synthetic biology applications. However, the molecular tools and techniques for utilizing EcN have not been fully explored. To address this opportunity, we systematically expanded the EcN-based toolbox, enabling EcN as a powerful platform for more applications. First, two EcN cryptic plasmids and other compatible plasmids were genetically engineered to enrich the manipulable plasmid toolbox for multiple gene coexpression. Next, we developed two EcN-based enabling technologies, including the conjugation strategy for DNA transfer, and quantification of protein expression capability. Finally, we expanded the EcN-based applications by developing EcN native integrase-mediated genetic engineering capabilities and establishing an in vitro cell-free protein synthesis (CFPS) system. Overall, this study expanded the toolbox for manipulating EcN as a commonly used probiotic chassis, providing several simplified, dependable, and predictable strategies for researchers working in synthetic biology fields. For Table of Contents Use Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/543671v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1f8e91forg.highwire.dtl.DTLVardef@90fb70org.highwire.dtl.DTLVardef@6badbforg.highwire.dtl.DTLVardef@15be840_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗