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Results for “Synthetic Biology”

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bioRxiv2024

Development of Modular Expression and Genome Editing Across Phylogenetically Distinct Diazotrophs

Diazotrophic bacteria can reduce atmospheric nitrogen into ammonia enabling bioavailability of the essential element. Many diazotrophs associate closely with plant roots increasing nitrogen availability, acting as plant growth promoters. These associations have the potential to reduce the need for costly synthetic fertilizers if they could be engineered for agricultural applications. However, despite the importance of diazotrophic bacteria, genetic tools are poorly developed in a limited number of species, in turn narrowing the crops and root microbiomes that can be targeted. Here we report optimized protocols and plasmids to manipulate phylogenetically diverse diazotrophs with the goal of enabling synthetic biology and genetic engineering. Three broad-host-range plasmids can be used across multiple diazotrophs, with the identification of one specific plasmid (containing origin of replication RK2 and a kanamycin resistance marker) showing the highest degree of compatibility across bacteria tested. We then demonstrated modular expression testing seven promoters and eleven ribosomal binding sites using proxy fluorescent proteins. Finally, we tested four small molecule inducible systems to report expression in three diazotrophs and demonstrate genome editing in Klebsiella michiganensis M5al. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/595406v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@ea73a4org.highwire.dtl.DTLVardef@4e540eorg.highwire.dtl.DTLVardef@706557org.highwire.dtl.DTLVardef@10d4c9f_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2026

Efficient in vitro refactoring and biosynthetic gene cluster amplification for the overproduction and accelerated discovery of anticancer thioamitides

Thioamitides, a class of highly modified bacterial ribosomally synthesised and post-translationally modified peptides (RiPPs), have potent activities against multiple cancer cell lines. Among these compounds, the structurally divergent thioalbamide combines promising in vivo antiproliferative activity with a superior chemical stability respect to its counterparts. However, thioalbamide is produced in low yields by its genetically intractable native producer and its biosynthetic pathway was initially not productive when transferred into the heterologous host Streptomyces coelicolor M1146. These circumstances substantially hamper to increase the production of this promising compound. Here, we show how in vitro Gibson-like assemblies can be employed for the quick and efficient refactoring of the thioalbamide biosynthetic gene cluster (BGC), leading to substantially increased levels of production in S. coelicolor M1146 through a prioritised selection of promoters. Via this work, PtsrA and PgroEL2 were identified as beneficial additions to the Streptomyces synthetic biology toolbox. We then assessed bacterial genomes for biosynthetic gene clusters (BGCs) predicted to produce thioalbamide-like compounds with improved hydrophilicity. This rational discovery campaign led to the identification a silent thioamitide BGC encoding a thioalbamide-like core peptide but clustered with additional tailoring enzymes, including a previously unknown cupin-fold protein. Applying the refactoring strategy together with the simultaneous expression of multiple BGC copies, we characterised the product of this pathway, thiocupinamide, a polyhydroxylated thioamitide closely related to thioalbamide. We show that thiocupinamide has potent anticancer and antibacterial activities.

synthetic biology↗

bioRxiv2017

Model reduction permits Turing instability analysis of arbitrary reaction-diffusion models

Synthesising a genetic network which generates stable Turing patterns is one of the great challenges of synthetic biology, but a significant obstacle is the disconnect between the mathematical theory and the biological reality. Current mathematical understanding of patterning is typically restricted to systems of 2 or 3 chemical species, for which equations are tractable, but plausible genetic networks typically consist of dozens of interacting species. In this article, we suggest a method for reducing large biochemical systems to systems with 2 or 3 species which can then be studied analytically. We provide conditions to guarantee that the full system forms patterns if the reduced system does, and vice-versa. We confirm our technique with 3 examples: the Brusselator, an example proposed by Turing, and a biochemically plausible patterning system consisting of 17 species. These examples show that our method significantly simplifies the study of pattern formation in large systems.

synthetic biology↗

bioRxiv2026

Engineering auronidin production in Marchantia polymorpha enables a new class of plant-derived textile pigments

Plant-derived pigments offer sustainable alternatives to synthetic colorants, yet their practical deployment in textiles is limited by restricted chemical diversity and low abundance. Liverworts represent a source of diverse chemical compounds, and the model liverworts Marchantia polymorpha is an emerging as chassis for bioengineering and synthetic biology. Here, we report the biotechnological application of auronidins, a rare class of flavonoid pigments, as textile dyes. Using the Marchantia, we engineered enhanced auronidin production through controlled expression of the R2R3-MYB transcription factor MpMYB14. We systematically benchmarked constitutive and inducible gene expression systems, including heat-shock, glucocorticoid receptor, and {beta}-estradiol (XVE) circuits, identifying inducible strategies that decouple biomass accumulation from secondary metabolite production while achieving high pigment yields. Extracted auronidins were used to dye cotton yarn directly, demonstrating the feasibility of auronidins for textile dyeing. Our results establish Marchantia as a versatile plant chassis for programmable secondary metabolite production and introduce auronidins as a promising natural pigment platform for sustainable textile biotechnology.

synthetic biology↗

bioRxiv2024

Design of a self-regulating mRNA gene circuit

Protein expression from mRNA in vivo is predominately controlled via regulatory feedback mechanisms that adjust the level of mRNA transcription. However, for positive sense single-stranded RNA viruses, protein expression is often controlled via secondary structural elements, such as internal ribosomal entry sites, that are encoded within the mRNA. The self-regulation of mRNA translation observed in this class of viruses suggests that it may be possible to design mRNAs that self-regulate their protein expression, enabling the creation of mRNAs for vaccines and other synthetic biology applications where protein levels in the cell can be tightly controlled without feedback to a transcriptional mechanism. As a proof of concept, I design a polycistronic mRNA based on bacteriophage MS2, where the upstream gene is capable of repressing synthesis of the downstream gene. Using a computational tool that simulates ribosome kinetics and the co-translational folding of the mRNA in response, I show that mutations to the mRNA can be identified which enhance the efficiency of the translation and the repression of the downstream gene. The results of this study open up the possibility of designing bespoke mRNA gene circuits in which the amount of protein synthesised in cells are self-regulated for therapeutic or antigenic purposes.

synthetic biology↗

bioRxiv2020

Analysis of Circuits for Dosage Control in Microbial Populations

Designing genetic circuits to control the behaviors of microbial populations is an ongoing challenge in synthetic biology. Here we analyze circuits which implement dosage control by controlling levels of a global signal in a microbial population in face of varying cell density, growth rate, and environmental dilution. We utilize the Lux quorum sensing system to implement dosage control circuits, and we analyze the dynamics of circuits using both simplified analytical analysis and in silico simulations. We demonstrate that strong negative feedback through inhibiting LuxI synthase expression along with AiiA degradase activity results in circuits with fast response times and robustness to cell density and dilution rate. We find that degradase activity yields robustness to variations in population density for large population sizes, while negative feedback to synthase production decreases sensitivity to dilution rates.

synthetic biology↗

bioRxiv2023

Comparison of phage-derived recombinases for genetic manipulation of Pseudomonas species

Several strains in the Pseudomonas genus are categorized as plant growth promoting rhizobacteria (PGPR). Although several of these strains are strong candidates for applications as biofertilizers or biopesticides, known genome editing approaches are generally limited and require further development. Editing genomes in PGPR could enable more robust agricultural applications, persistence and biosafety measures. In this study, we investigate the use of five phage-encoded recombinases to develop a recombineering workflow in 3 PGPR strains: P. protegens Pf-5, P. protegens CHA0, and P. putida KT2440. Using point mutations in the rpoB gene, we reach maximum recombineering efficiencies of 1.5 x 10-4, 3 x 10-4, and 5 x 10-5, respectively, in these strains using {lambda}-Red Beta recombinase from E. coli. We further examine recombineering efficiencies across these strains as a function of selected mutation, editing template concentration, and phosphorothiolate bond protection. This work validates the use of these tools across several environmentally and biotechnologically relevant strains to expand the possibilities of genetic manipulation in the Pseudomonas genus. ImportanceThe Pseudomonas genus contains many members currently being investigated for applications in biodegradation, biopesticides, biocontrol and synthetic biology. Though several strains have been identified with beneficial properties, in situ genetic manipulations to further improve these strains for commercial applications have been limited due to lack of efficient genetic tools that have been tested across this genus. Here we test the recombineering efficiencies of 5 phage-derived recombinases across 3 biotechnologically relevant Pseudomonas strains: P. putida KT2440, P. protegens Pf-5, and P. protegens CHA0. These results demonstrate a method to generate targeted mutations quickly and efficiently across these strains, ideally introducing a method that can be implemented across the Pseudomonas genus and a strategy that may be applied to develop analogous systems in other non-model bacteria.

synthetic biology↗

bioRxiv2023

Biofoundry-scale DNA assembly validation using cost-effective high-throughput long read sequencing

Biofoundries are automated high-throughput facilities specialising in the design, construction and testing of engineered/synthetic DNA constructs (plasmids), often from genetic parts. A critical step of this process is assessing the fidelity of the assembled DNA construct to the desired design. Current methods utilised for this purpose are restriction digest or PCR followed by fragment analysis, and sequencing. The Edinburgh Genome Foundry (EGF) has recently established a single-molecule sequencing quality control step using the Oxford Nanopore sequencing technology, along with a companion Nextflow pipeline and a Python package to perform in-depth analysis and generate a detailed report. Our software enables biofoundry scientists and end-users to rapidly analyse sequencing data, without specialised bioinformatics knowledge. In conclusion, we have created a laboratory and software protocol that validates assembled, cloned or edited plasmids, using Nanopore long reads, which can serve as a useful resource for the genetics, synthetic biology and sequencing communities. Author informationAll authors contributed to the design of the sequencing quality control step and pipeline, and the preparation of the manuscript. P.V. wrote the manuscript, designed and implemented the bioinformatics pipeline and interpreted results. S.D. wrote the manuscript, implemented the laboratory protocol and interpreted results. G.S. designed the bioinformatics pipeline. R.F. wrote the manuscript and contributed to the design of the laboratory protocol and pipeline. Address: Edinburgh Genome Foundry (University of Edinburgh), Michael Swann Building, Max Born Crescent, Edinburgh, EH9 3BF, United Kingdom Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/558498v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@a819e5org.highwire.dtl.DTLVardef@3c34ceorg.highwire.dtl.DTLVardef@aef5d8org.highwire.dtl.DTLVardef@8e5d2f_HPS_FORMAT_FIGEXP M_FIG C_FIG

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bioRxiv2021

Dichotomous Feedback: A Signal Sequestration-based Feedback Mechanism for Biocontroller Design

We introduce a new design framework for implementing negative feedback regulation in Synthetic Biology, which we term dichotomous feedback. Our approach is different from current methods, in that it sequesters existing fluxes in the process to be controlled, and in this way takes advantage of the processs architecture to design the control law. This signal sequestration mechanism appears in many natural biological systems and can potentially be easier to realise than molecular sequestration and other comparison motifs that are nowadays common in biomolecular feedback control design. The loop is closed by linking the strength of signal sequestration to the process output. Our feedback regulation mechanism is motivated by two-component signalling systems, where we introduce a second response regulator competing with the natural response regulator thus sequestering kinase activity. Here, dichotomous feedback is established by increasing the concentration of the second response regulator as the level of the output of the natural process increases. Extensive analysis demonstrates how this type of feedback shapes the signal response, attenuates intrinsic noise while increasing robustness and reducing crosstalk.

synthetic biology↗

bioRxiv2024

Long-term protein synthesis with PURE in a mesoscale dialysis system

Cell-free systems are powerful tools in synthetic biology with versatile and wide-ranging applications. However, a significant bottleneck for these systems, particularly the PURE cell-free system, is their limited reaction lifespan and yield. Dialysis offers a promising approach to prolong reaction lifetimes and increase yields, yet most custom dialysis systems require access to sophisticated equipment like 3D printers or microfabrication tools. In this study, we utilized an easy-to-assemble, medium-scale dialysis system for cell-free reactions using commercially available components. By employing dialysis with periodic exchange of the feeding solution, we achieved a protein yield of 1.16 mg/mL GFP in the PURE system and extended protein synthesis for at least 12.5 consecutive days, demonstrating the systems excellent stability.

synthetic biology↗

bioRxiv2024

Multi-Layer Autocatalytic Feedback Enables Integral Control Amidst Resource Competition and Across Scales

Integral feedback control strategies have proven effective in regulating protein expression in unpredictable cellular environments. These strategies, grounded in model-based designs and control theory, have advanced synthetic biology applications. Autocatalytic integral feed-back controllers, utilizing positive autoregulation for integral action, are particularly promising due to their similarity to natural behaviors like self-replication and positive feedback seen across biological scales. However, their effectiveness is often hindered by resource competition and context-dependent couplings. This study addresses these challenges with a multi-layer feedback strategy, enabling population-level integral feedback and multicellular integrators. We provide a generalized mathematical framework for modeling resource competition in complex genetic networks, supporting the design of intracellular control circuits. Our controller motif demonstrated precise regulation in tasks ranging from gene expression control to population growth in multi-strain communities. We also explore a variant capable of ratiometric control, proving its effectiveness in managing gene ratios and co-culture compositions in engineered microbial ecosystems. These findings offer a versatile approach to achieving robust adaptation and homeostasis from subcellular to multicellular scales.

synthetic biology↗

bioRxiv2026

Enhanced tRNA array method version 2 for simultaneous in vitro synthesis of 21 tRNAs

Transfer RNAs (tRNAs) play an essential role in translation, and their simultaneous in vitro synthesis remains a key challenge in bottom-up synthetic biology. We previously developed the tRNA array method that enables the simultaneous in vitro synthesis of 21 tRNAs from a single DNA template; however, the translational activity was substantially lower than that achieved using individually prepared 21 tRNAs for some proteins. Here, we identify the tRNA groups (PIEN group) that limit translation in the tRNA array method and improve the translational activities through sequence modification and incorporation of a leader sequence into the array construct. The resulting tRNA array method version 2 produces a tRNA set that allows translation at levels similar to those achieved with individually prepared tRNAs for multiple reporter proteins under both translation-coupled and uncoupled conditions. This tRNA synthesis scheme provides an improved platform for constructing self-reproducible gene expression systems.

synthetic biology↗

bioRxiv2025

Control with Practical Guarantees of Stationary Variance in Stochastic Chemical Reaction Networks

Biomolecular integral feedback controllers offer precise regulation of molecular species copy numbers, making them valuable for synthetic biology applications. Antithetic integral feedback controllers, in particular, can be effective in low-copy-number regimes with stochastic dynamics. In this work, we introduce a modified variant of this controller, called the antithetic dual-rein integral feedback motif, and analyze its performance from a stochastic perspective in the presence of intrinsic dynamic randomness. We demonstrate that our controller enables first-moment control while maintaining a tractable steady-state variance bound under specific parametric regimes. Notably, this variance bound is tunable, as it depends solely on the controller parameters. We derive these results using stochastic model-order reduction and validate them through numerical simulations. Our findings provide new insights into achieving both precise regulation and noise suppression in stochastic genetic circuits.

synthetic biology↗

bioRxiv2022

Engineering stringent genetic biocontainment of yeast with a protein stability switch

Synthetic biology holds immense promise to tackle key problems we are facing, for instance in resource use, environmental health, and human health care. However, comprehensive safety measures are needed to deploy genetically engineered microorganisms in open-environment applications. Here, we describe a genetic biocontainment system based on conditional stability of essential proteins. We used a yeast-adapted destabilizing domain degron, which can be stabilized by estradiol addition (ERdd). Leveraging the yeast GFP collection and lab automation platforms, we ERdd-tagged 775 essential genes and screened for strains with estradiol dependent growth. Three genes, SPC110, DIS3 and RRP46, were found to be particularly suitable targets. Respective strains showed no growth defect in the presence of estradiol and strong growth inhibition in its absence. SPC110-ERdd offered the most stringent containment, with an escape frequency of 7.0x10-8, and full growth restoration at 100 nM estradiol. By systematically analyzing the containment escapees, we identified the non-essential C-terminal region of SPC110 as target for escape mutations. Its removal decreased the escape frequency with a single ERdd tag further to 4.3x10-9. Combining SPC110-ERdd with a second ERdd tag on either DIS3 or RRP46 resulted in escape frequencies below the detection limit of the used assay (<2x10-10). Being based on conditional protein stability, this approach is mechanistically orthogonal to previously reported intrinsic biocontainment systems. It thus can be readily combined with other systems, for instance ones based on transcriptional or translational control of essential gene expression, to achieve multiplexed, extremely stringent control over the survival of engineered organisms. SignificanceSynthetic biology holds enormous potential to tackle key issues humanity is facing and can for instance revolutionize agriculture, bioremediation or health care. In each case, the unchecked spread of engineered organisms in natural environments must be prevented. This is particularly problematic with use cases of engineered microbes in open environments. Intrinsic, genetically encoded biocontainment systems, which control cell survival based on environmental cues, can solve this issue. We have developed such a genetic biocontainment system acting on the stability of essential proteins, leveraging a switchable degron. Through a large-scale screening for suitable essential target genes, we were able to create yeast strains that are strictly dependent on estradiol. Supplied with this small molecule, the engineered cells maintain high fitness and grow as robustly as the unmodified strains.

synthetic biology↗

bioRxiv2023

LanTERN: a fluorescent sensor that specifically binds lanthanides

Lanthanides, a series of fifteen f-block elements, are crucial in modern technology, and their purification by conventional chemical means comes at a significant environmental cost. Synthetic biology offers promising solutions. However, the lack of biochemical tools to measure lanthanide binding is a bottleneck to progress. Here, we introduce LanTERN, a lanthanide-responsive fluorescent protein rationally engineered from the lanmodulin-binding protein, LanM. LanTERN was designed based on GCaMP, a genetically encoded calcium indicator that couples the ion binding of four EF hand motifs to increased GFP fluorescence. We engineered seven mutations across the parent constructs four EF hand motifs to switch specificity from calcium to lanthanides. The resulting protein, LanTERN, directly converts the binding of 10 measured lanthanides to 14-fold or greater increased fluorescence. LanTERN development opens new avenues for creating improved lanthanide-binding proteins and bio-sensing systems.

synthetic biology↗

bioRxiv2019

Minimally Complex Nucleic Acid Feedback Control Systems for First Experimental Implementations

Chemical reaction networks based on catalysis, degradation, and annihilation may be used as building blocks to construct a variety of dynamical and feedback control systems in Synthetic Biology. DNA strand-displacement, which is based on DNA hybridisation programmed using Watson-Crick base pairing, is an effective primitive to implement such reactions experimentally. However, experimental construction, validation and scale-up of nucleic acid control systems is still significantly lagging theoretical developments, due to several technical challenges, such as leakage, crosstalk, and toehold sequence design. To help the progress towards experimental implementation, we provide here designs representing two fundamental classes of reference tracking control circuits (integral and state-feedback control), for which the complexity of the chemical reactions required for implementation has been minimised. The supplied minimally complex control circuits should be ideal candidates for first experimental validations of nucleic acid controllers.

synthetic biology↗

bioRxiv2017

Accelerating Evolutionary Hill Climbs in Parallel Turbidostats

Evolution has been used to address many engineering problems. Within the context of metabolic engineering and synthetic biology, directed evolution has natural appli-cations. However, most research concerning optimizing microbial evolution has been focused on library generation and screening, while accelerating evolutionary hill climbs and been largely ignored. Here, we develop a model to explore how population struc-ture can accelerate evolutionary hill climbs. We show that by adjusting the population size, environmental challenge, and meta-population dynamics that the rate of evolution can be accelerated in parallel turbidostats. Our analyses leads to two surprising results: small populations are favored over conventionally large microbial populations, and propagating modest fitness improvements is favored over propagating mutants with large beneficial mutations. When combined with rational design and other optimization techniques our theory can accelerate strain development for applications such as consolidated bioprocessing, and bioremidation systems.

synthetic biology↗

bioRxiv2020

Nanobody-mediated control of gene expression and epigenetic memory

Targeting chromatin regulators to specific genomic locations for gene control is emerging as a powerful method in basic research and synthetic biology. However, many chromatin regulators are large, making them difficult to deliver and combine in mammalian cells. Here, we developed a new strategy for gene control using small nanobodies that bind and recruit endogenous chromatin regulators to a gene. We show that an antiGFP nanobody can be used to simultaneously visualize GFP-tagged chromatin regulators and control gene expression, and that nanobodies against HP1 and DNMT1 can silence a reporter gene. Moreover, combining nanobodies together or with other regulators, such as DNMT3A or KRAB, can enhance silencing speed and epigenetic memory. Finally, we use the slow silencing speed and high memory of antiDNMT1 to build a signal duration timer and recorder. These results set the basis for using nanobodies against chromatin regulators for controlling gene expression and epigenetic memory.

synthetic biology↗