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bioRxiv2024

Elucidating the metabolic roles of isoflavone synthase-mediated protein-protein interactions in yeast

Transient plant enzyme complexes formed via protein-protein interactions (PPIs) play crucial regulatory roles in secondary metabolism. Complexes assembled on cytochrome P450s (CYPs) are challenging to characterize metabolically due to difficulties in decoupling the PPIs metabolic impacts from the CYPs catalytic activities. Here, we developed a yeast-based synthetic biology approach to elucidate the metabolic roles of PPIs between a soybean-derived CYP, isoflavone synthase (GmIFS2), and other enzymes in isoflavonoid metabolism. By reconstructing multiple complex variants with an inactive GmIFS2 in yeast, we found that GmIFS2-mediated PPIs can regulate metabolic flux between two competing pathways producing deoxyisoflavonoids and isoflavonoids. Specifically, GmIFS2 can recruit chalcone synthase (GmCHS7) and chalcone reductase (GmCHR5) to enhance deoxyisoflavonoid production or GmCHS7 and chalcone isomerase (GmCHI1B1) to enhance isoflavonoid production. Additionally, we identified and characterized two novel isoflavone O-methyltransferases interacting with GmIFS2. This study highlights the potential of yeast synthetic biology for characterizing CYP-mediated complexes.

synthetic biology↗

bioRxiv2021

UNIGEMS: plasmids and parts to facilitate teaching on assembly, gene expression control and logic in E. coli

Synthetic biology is as an excellent vehicle for education, as it enables creative combination of engineering and molecular biology approaches for quantitative characterisations of the assembled constructs. However, there is a limited number of resources available for such applications in the educational context, where straightforward setup, easily measurable phenotypes and extensibility are of particular importance. To expand the availability of education-friendly resources to teach synthetic biology and genetic engineering, we developed Unigems, a set of 10 plasmids that enable out-of-the-box investigations of principles of gene expression control, as well as more complex designs a biological logic gate. The system uses a common high-copy plasmid backbone and a common set of primers to enable Gibson-assembly of PCR-generated or synthesised parts into a target vector. It currently has two reporter genes with either two constitutive (high- or low-level) or two inducible (lactose- or arabinose-) promoters, as well as a single-plasmid implementation of an AND logic gate. The Unigems system has already been employed in undergraduate teaching settings, during outreach events and for training of iGEM teams. All plasmids have been deposited in Addgene.

synthetic biology↗

bioRxiv2024

A Field-Deployable Arsenic Sensor Integrating Bacillus Megaterium with CMOS Technology

Bacteria innately monitor their environment by dynamically regulating gene expression to respond to fluctuating conditions. Through synthetic biology, we can harness this natural capability to design cell-based sensors. Bacillus megaterium, a soil bacterium, stands out due to its remarkable heavy metal tolerance and sporulation ability, making it an ideal candidate for heavy metal detection with low transportation costs. However, challenges persist: the synthetic biology toolkit for this strain is underdeveloped and conventional whole-cell sensors necessitate specialized laboratory equipment to read the output. In our study, we genetically modified B. megaterium for arsenic detection, establishing a detection threshold below the EPA recommendation of 10 ppb for drinking water in both vegetative cell form and spore form. Additionally, we integrated both engineered B. megaterium living cells and spores with CMOS chip for field-deployable arsenic detection. We show that the limit of detection of our integrated sensor is applicable in soil and air arsenic contamination testing. As a proof of concept, this work paves the way for deploying our sensor in resource-limited settings, ensuring real-time arsenic detection in challenging environments. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/604150v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1940e83org.highwire.dtl.DTLVardef@144da0aorg.highwire.dtl.DTLVardef@146e906org.highwire.dtl.DTLVardef@6cb8c1_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2019

Rapid method for generating designer algal mitochondrial genomes

With synthetic biology, we can turn algae into bio-factories that produce high-value molecules (e.g. medicines or biofuels) or tackle global challenges (e.g. malnutrition and climate change). This realization has provoked rapid progress towards the creation of genetic tools for multiple algal species, notably Phaeodactylum tricornutum. The power of synthetic biology to generate more useful or productive organisms is contingent on the ability to produce diverse DNA molecules and rapidly screen them for beneficial variants. However, it is still relatively expensive to synthesize DNA, and delivering large DNA (>50 kbp) to eukaryotic cellular compartments remains challenging. In this study, we establish a robust system for building algal mitochondrial genomes as a practical alternative to DNA synthesis. Our approach permits an inexpensive and rapid generation of mitochondrial derivatives designed for testing targeted DNA delivery. First, we cloned the mitochondrial genome of P. tricornutum into the eukaryotic host organism S. cerevisiae using two different techniques: transformation-associated recombination; and PCR-based cloning. Next, we analyzed the cloned genomes by multiplex PCR, and correct genomes were transferred to prokaryotic host E. coli. Then these genomes were again analyzed by multiplex PCR, followed by diagnostic digest and complete-plasmid sequencing to evaluate the fidelity of each cloning method. Finally, we assessed the burden on eukaryotic and prokaryotic hosts to propagate the cloned genomes. We conclude that our system can reliably generate variants for genome-level engineering of algal mitochondria. HIGHLIGHTSO_LITAR-cloned mitochondrial genome of P. tricornutum in yeast C_LIO_LIDeveloped PCR-based cloning method to create designer algal mitochondrial genomes C_LIO_LIStably propagated algal mitochondrial genomes in S. cerevisiae and E. coli hosts C_LI

synthetic biology↗

bioRxiv2021

Computational characterization of recombinase circuits for periodic behaviors

In nature, recombinases are site-specific proteins capable of rearranging DNA, and they are expanding the repertoire of gene editing tools used in synthetic biology. The on/off response of recombinases, achieved by inverting the direction of a promoter, makes them suitable for Boolean logic computation; however, recombinase-based logic gate circuits are single-use due to the irreversibility of the DNA rearrangement, and it is still unclear how a dynamical circuit, such as an oscillator, could be engineered using recombinases. Preliminary work has demonstrated that recombinase-based circuits can yield periodic behaviors in a deterministic setting. However, since a few molecules of recombinase are enough to perform the inverting function, it is crucial to assess how the inherent stochasticity at low copy number affects the periodic behavior. Here, we propose six different circuit designs for recombinase-based oscillators. We model them in a stochastic setting, leveraging the Gillespie algorithm for extensive simulations, and we show that they can yield periodic behaviors. To evaluate the incoherence of oscillations, we use a metric based on the statistical properties of auto-correlation functions. The main core of our design consists of two self-inhibitory, recombinase-based modules coupled by a common promoter. Since each recombinase inverts its own promoter, the overall circuit can give rise to switching behavior characterized by a regular period. We introduce different molecular mechanisms (transcriptional regulation, degradation, sequestration) to tighten the control of recombinase levels, which slows down the response timescale of the system and thus improves the coherence of oscillations. Our results support the experimental realization of recombinase-based oscillators and, more generally, the use of recombinases to generate dynamic behaviors in synthetic biology.

synthetic biology↗

bioRxiv2021

A ratiometric dual color luciferase reporter for fast characterization of transcriptional regulatory elements

Plant synthetic biology requires precise characterization of genetic elements to construct complex genetic circuits that can improve plant traits or confer them new characteristics. Transcriptional reporter assays are essential to quantify the effect of gene expression regulator elements. Therefore, transcriptional reporter systems are a key tool in understanding control of gene expression in biology. In this work we construct and characterize a dual-color luciferase ratiometric reporter system that possesses several advantages over currently used reporters. It is ratiometric, reducing variability and increasing consistency between experiments; it is fast, as both reporters can be measured at the same time in a single reaction, and it is cheaper to perform than current dual-luciferase reporter assays. We have validated our system quantifying the transcriptional capability of a panel of promoters and terminators commonly used in synthetic biology with a broad range of expression magnitudes, and in a biologically relevant system, nitrate response.

synthetic biology↗

bioRxiv2025

AI-directed gene fusing prolongs the evolutionary half-life of synthetic gene circuits

Evolutionary instability is a persistent challenge in synthetic biology, often leading to the loss of heterologous gene expression over time. Here, we present STABLES, a novel gene fusion strategy that links a gene of interest (GOI) to an essential endogenous gene (EG), with a "leaky" stop codon in between. This ensures both selective pressure against deleterious mutations and high expression of the GOI. By leveraging a machine learning (ML) framework, we predict optimal GOI-EG pairs based on bioinformatic and biophysical features, identify linkers likely to minimize protein misfolding, and optimize DNA sequences for stability and expression. Experimental validation in Saccharomyces cerevisiae demonstrated significant improvements in stability and productivity for fluorescent proteins and human proinsulin. The results highlight a scalable, adaptable and organism-agnostic method to enhance the evolutionary stability of engineered strains, with broad implications for industrial biotechnology and synthetic biology.

synthetic biology↗

bioRxiv2023

Manipulation of encapsulated artificial phospholipid membranes using sub-micellar lysolipid concentrations.

Droplet Interface Bilayers (DIBs) constitute a commonly used model of artificial membranes for molecular biology studies with applications in synthetic biology research. However, these model membranes have limited accessibility due to their requirement to be surrounded by an oil environment. Here, we demonstrate in-situ bilayer manipulation of submillimeter, free-standing, encapsulated droplet interface bilayers (eDIBs) in hydrogel capsules formed using dual-material, 3D-printed microfluidic devices. These microfluidic devices required no post-fabrication assembly, nor surface treatment to achieve the high-order emulsification, required for the formation of robust eDIBs. The eDIB capsules were exposed to various concentrations of lysophosphatidylcholine (LPC), in order to investigate the interaction of lysolipids with three-dimensional, encapsulated droplet bilayer networks. Micellar LPC concentrations trigger the bursting of the eDIB droplets, while at concentrations below the critical micelle concentration (CMC), the encapsulated aqueous inner droplet networks endure structural changes, precisely affecting the DIB contact angles and bilayer area. Manipulation of these enclosed, 3D-orchestrated membrane mimics facilitates the exploration of readily accessible compartmentalized artificial cellular machinery. Collectively, the multi-compartmentalized capsules and the lysolipid-mediated membrane modulation, introduce a chemical approach to control the properties and mechanics of artificial cellular membranes, toward responsive soft material developments.

synthetic biology↗

bioRxiv2024

Deletion of HindIIR and HindIIIR improves DNA transfer via electroporation to Haemophilus influenzae Rd

Haemophilus influenzae is a bacterial species of interest for its medical relevance and utility as a model system. Despite its role in several landmark molecular and synthetic biology studies, H. influenzae remains underexplored as a potential chassis organism. The limited availability of reliable and convenient transformation methods and genetic tools for H. influenzae are obstacles to this end. However, a strain of H. influenzae Rd KW20 lacking the type II restriction endonucleases HindII and HindIII has previously been developed. Here, we show that this strain is more readily transformable by electroporation than wild-type Rd KW20. We also developed a series of multi-host plasmids carrying antibiotic selection and fluorescent visual markers based on the pSU20 vector. The availability of H. influenzae {Delta}HindII/III, paired with the electroporation method and plasmids presented here, will promote the exploration of H. influenzae as a host organism for synthetic biology applications.

synthetic biology↗

bioRxiv2022

Manipulating the 3D organization of the largest synthetic yeast chromosome

Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field, especially when the synthetic genomes are extensively modified with thousands of designer features. Here we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp Saccharomyces cerevisiae chromosome resulting from extensive genome streamlining and modification. During the construction of synIV, we developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction and facilitated chromosome debugging. In addition to the drastic sequence changes made to synIV by rewriting it, we further manipulated the three-dimensional structure of synIV in the yeast nucleus to explore spatial gene regulation within the nuclear space. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of the largest synthetic yeast chromosome shed light on higher-order architectural design of the synthetic genomes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/487066v2_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@18bf586org.highwire.dtl.DTLVardef@1a471d5org.highwire.dtl.DTLVardef@12d7690org.highwire.dtl.DTLVardef@1c32510_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDe novo synthesis of the largest eukaryotic chromosome, synIV C_LIO_LISynIV shows similar 3D structure to wild-type IV, despite thousands of changes made to it C_LIO_LI"Inside-out" repositioning of synIV in nucleus shows minor transcriptional changes C_LIO_LIMultipoint tethering synIV to inner nuclear membrane represses transcription of whole chromosome C_LI

synthetic biology↗

bioRxiv2024

Iterative SCRaMbLE for Engineering Synthetic Genome Modules and Chromosomes

Synthetic biology offers the possibility of synthetic genomes with customised gene content and modular organisation. In eukaryotes, building whole custom genomes is still many years away, but work in Saccharomyces cerevisiae yeast is closing-in on the first synthetic eukaryotic genome with genome-wide design changes. A key design change throughout the synthetic yeast genome is the introduction of LoxPsym site sequences. These enable inducible genomic rearrangements in vivo via expression of Cre recombinase via SCRaMbLE (Synthetic Chromosome Recombination and Modification by LoxPsym-mediated Evolution). When paired with selection, SCRaMbLE can quickly generate strains with phenotype improvements by diversifying gene arrangement and content in LoxPsym-containing regions. Here, we demonstrate how iterative cycles of SCRaMbLE can be used to reorganise synthetic genome modules and synthetic chromosomes for improved functional performance under selection. To achieve this, we developed SCOUT (SCRaMbLE Continuous Output and Universal Tracker), a reporter system that allows SCRaMbLEd cells to be sorted into a high diversity pool. When coupled with long-read sequencing, SCOUT enables high-throughput mapping of genotype abundance and correlation of gene content and arrangement with growth-related phenotypes. Iterative SCRaMbLE was applied here to yeast strains with a full synthetic chromosome, and to strains with synthetic genome modules encoding the gene set for histidine biosynthesis. Five synthetic designs for HIS modules were constructed and tested, and we investigated how SCRaMbLE reorganised the poorest performing design to give improved growth under selection. The results of iterative SCRaMbLE serve as a quick route to identify genome module designs with optimised function in a selected condition and offer a powerful tool to generate datasets that can inform the design of modular genomes in the future.

synthetic biology↗

bioRxiv2025

DNA Flipping as Facile Mechanism for Transmembrane Signaling in Synthetic Cells

Transmembrane signaling is essential for cellular communication, yet reconstituting such mechanisms in synthetic systems remains challenging. Here, we report a simple and robust DNA-based mechanism for transmembrane signaling in synthetic cells using cholesterol-modified single-stranded DNA (Chol-ssDNA). We discovered that anchored Chol-ssDNA spontaneously flips across the membrane of giant unilamellar lipid vesicles (GUVs) in a nucleation-driven, defect-mediated process. This flipping enables internal signal processing through hybridization with encapsulated complementary DNA and activation of downstream processes such as RNA transcription. The phenomenon shows a high transduction efficiency, is generic across DNA sequences and lipid compositions, and can be enhanced by glycerol, which modulates membrane dynamics. Mechanistic insights using fluorescence microscopy, nuclease degradation assays, and membrane permeability assays reveal that flipping is dominated by transient membrane pores. Leveraging this facile translocation process, we demonstrate selective transcriptional activation inside synthetic cells, underscoring the potential of Chol-ssDNA flipping as a programmable tool for synthetic biology and bottom-up synthetic cell design.

synthetic biology↗

bioRxiv2018

De novo Design of Translational RNA Repressors

A central goal of synthetic biology is the development of methods for the predictable control of gene expression. RNA is an attractive substrate by which to achieve this goal because the relationship between its sequence, structure, and function is being uncovered with increasing depth. In addition, design approaches that use this relationship are becoming increasingly effective, as evidenced by significant progress in the de novo design of RNA-based gene regulatory mechanisms that activate transcription and translation in bacterial cells. However, the design of synthetic RNA mechanisms that are efficient and versatile repressors of gene expression has lagged, despite their importance for gene regulation and genetic circuit construction. We address this gap by developing two new classes of RNA regulators, toehold repressors and looped antisense oligonucleotides (LASOs), that repress translation of a downstream gene in response to an arbitrary input RNA sequence. Characterization studies show that these designed RNAs robustly repress translation, are highly orthogonal, and can be multiplexed with translational activators. We show that our LASO design can repress endogenous mRNA targets and distinguish between closely-related genes with a high degree of specificity and predictability. These results demonstrate significant yet easy-to-implement improvements in the design of synthetic RNA repressors for synthetic biology, and point more broadly to design principles for repressive RNA interactions relevant to modern drug design.

synthetic biology↗

bioRxiv2024

Rapid generation and screening of transgenic black soldier fly (Hermetia illucens)

BackgroundThe black soldier fly (BSF), Hermetia illucens is a widely used, and mass-produced insect that fulfils an important role in both the management of organic waste and as a component of animal feed formulations. They also have significant potential as a platform for converting organic waste into high-value proteins, and lipids for the production of biofuels. Applying synthetic biology to BSF provides even more potential for improvement through the generation of transgenic BSF to enhance animal feed, produce and fine tune high-value industrial biomolecules, and to expand their waste conversion capabilities. ResultsTo enable the rapid generation and screening of transgenic BSF, we utilised microinjections of piggyBac mRNA with donor plasmids. We have found preliminary screening of G0 BSF to identify mosaics for outcrossing can be completed less than 2 weeks after microinjection. Stable transgenic lines were reliably generated with effective transformation rates of 30-33%, and transmission of the transgene could be confirmed 3 days after outcrossing the G0 adults. We also present a protocol for identifying the location of integrated transgenes. ConclusionsThe methods presented here expedite the screening process for BSF transgenesis and further expand the toolkit for BSF synthetic biology.

synthetic biology↗

bioRxiv2023

A green alternative to fragrant agarwood sesquiterpenoid production

Certain endangered Thymelaeaceous trees are major sources of the fragrant and highly valued resinous agarwood, comprised of hundreds of oxygenated sesquiterpenoids (STPs). Despite growing pressure on natural agarwood sources, the chemical complexity of STPs severely limits synthetic production. Here, we catalogued the chemical diversity in 58 agarwood samples by two-dimensional gas chromatography-mass spectrometry and partially recreated complex STP mixtures through synthetic biology. We improved STP yields in the unicellular alga Chlamydomonas reinhardtii by combinatorial engineering to biosynthesise nine macrocyclic STP backbones found in agarwood. A bioprocess following green-chemistry principles was developed that exploits milking of STPs without cell lysis, solvent-solvent STP extraction, solvent-STP nanofiltration, and bulk STP oxy-functionalisation to obtain terpene mixtures like those of agarwood. This process occurs with total solvent recycling and enables continuous production. Our synthetic-biology approach offers a sustainable alternative to harvesting agarwood trees to obtain mixtures of complex, fragrant, oxygenated STPs.

synthetic biology↗

bioRxiv2020

Contextual dependencies expand the re-usability of genetic inverters

The design and implementation of Boolean logic functions in living cells has become a very active field within synthetic biology. By controlling networks of regulatory proteins, novel genetic circuits are engineered to generate predefined output responses. Although many current implementations focus solely on the genetic components of the circuit, the host context in which the circuit performs is crucial for its outcome. Here, we characterise 20 genetic NOT logic gates (inverters) in up to 7 bacterial-based contexts each, to finally generate 135 different functions. The contexts we focus on are particular combinations of four plasmid backbones and three hosts, two Escherichia coli and one Pseudomonas putida strains. Each NOT logic gate shows seven different logic behaviours, depending on the context. That is, gates can be reconfigured to fit response requirements by changing only contextual parameters. Computational analysis shows that this range of behaviours improves the compatibility between gates, because there are considerably more possibilities for combination than when considering a unique function per genetic construct. Finally, we address the issue of interoperability and portability by measuring, scoring, and comparing gate performance across contexts. Rather than being a limitation, we argue that the effect of the genetic background on synthetic constructs expand the scope of the functions that can be engineered in complex cellular environments, and advocate for considering context as a fundamental design parameter for synthetic biology.

synthetic biology↗

bioRxiv2016

Tellurium: A Python Based Modeling and Reproducibility Platform for Systems Biology

In this article, we present Tellurium, a powerful Python-based integrated environment designed for model building, analysis, simulation and reproducibility in systems and synthetic biology. Tellurium is a modular, cross-platform, and open-source integrated development environment (IDE) composed of multiple libraries, plugins, and specialized modules and methods. Tellurium ensures exchangeability and reproducibility of computational models by supporting SBML (Systems Biology Markup Language), SED-ML (Simulation Experiment Description Markup Language), the COMBINE archive, and SBOL (Synthetic Biology Open Language). Tellurium is a self-contained modeling platform which comes with a fully configured Python distribution independent of other local Python installations on the target machine. The main interface is based on the Spyder IDE which has a highly accessible user interface akin to MATLAB (https://www.mathworks.com/). Tellurium uses libRoadRunner as the default SBML simulation engine due to its superior performance, scalability and ease of integration. libRoadRunner supports deterministic simulations, stochastic simulations and steady state analyses. Tellurium also includes Antimony, a human-readable model definition language which can be converted to and from SBML. Other standard Python scientific libraries such as NumPy, SciPy, and matplotlib are included by default. Additionally, we include several user-friendly plugins and advanced modules for a wide-variety of applications, ranging from visualization tools to complex algorithms for bifurcation analysis and multi-dimensional parameter scanning. By combining multiple libraries, plugins, and modules into a single package, Tellurium provides a unified but extensible solution for biological modeling and simulation.

Systems Biology↗

bioRxiv2022

Developing a temperature-inducible transcriptional rheostat in Neurospora crassa

Heat shock protein (hsp) encoding genes, part of the highly conserved Heat Shock Response (HSR), are known to be induced by thermal stress in several organisms. In Neurospora crassa, three hsp genes, hsp30, hsp70, and hsp80, have been characterized; however, the role of defined cis-elements in their response to discrete changes in temperature remains largely unexplored. To fill this gap, while also aiming to obtain a reliable fungal heat-shock inducible system, we analyzed different sections of each hsp promoter, by assessing the expression of real-time transcriptional reporters. Whereas all three promoters, and their resected versions, were acutely induced by high temperatures, only hsp30 displayed a broad range of expression and high tunability amply exciding other inducible promoter systems existing in Neurospora, such as Quinic acid- or light-inducible ones. As proof of concept, we employed one of these promoters to control the expression of clr-2, which encodes for the master regulator of Neurospora cellulolytic capabilities. The resulting strain fails to grow on cellulose at 25{degrees}C, whereas it robustly grows if heat shock pulses are delivered daily. Additionally, we designed two hsp30 synthetic promoters and characterized these, as well as the native promoters, to a gradient of high temperatures, yielding a wide range of responses to thermal stimuli. Thus, Neurospora hsp30-based promoters represent a new set of modular elements that can be used as a transcriptional rheostat to adjust the expression of a gene of interest or for the implementation of regulated circuitries for synthetic biology and biotechnological strategies. ImportanceTimely and dynamic response to strong temperature rises is paramount for organismal biology. At the same time, inducible promoters are a powerful tool for fungal biotechnological and synthetic biology endeavors. In this work, we analyzed the activity of several N. crassa heat shock protein (hsp) promoters upon a wide range of temperatures, observing that hsp30 exhibits remarkable sensitivity and dynamic range of expression as we chartered the response of this promoter to subtle increases in temperature, while also building synthetic promoters based on hsp30 cis-elements. As proof of concept, we analyzed the ability of hsp30 to provide tight control of a central process such as cellulose degradation. While this study provides an unprecedented description of the regulation of the N. crassa hsp genes it also contributes with a noteworthy addition to the molecular toolset of transcriptional controllers in filamentous fungi.

molecular biology↗