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Results for “synthetic biology”

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At least 541 records · Page 30Linked to original sources

bioRxiv2019

A yeast optogenetic toolkit (yOTK) for gene expression control in Saccharomyces cerevisiae

Optogenetic tools for controlling gene expression are ideal for tuning synthetic biological networks due to the exquisite spatiotemporal control available with light. Here we develop an optogenetic system for gene expression control and integrate it with an existing yeast toolkit allowing for rapid, modular assembly of light-controlled circuits in the important chassis organism Saccharomyces cerevisiae. We reconstitute activity of a split synthetic zinc-finger transcription factor (TF) using light-induced dimerization. We optimize function of this split TF and demonstrate the utility of the toolkit workflow by assembling cassettes expressing the TF activation domain and DNA-binding domain at different levels. Utilizing this TF and a synthetic promoter we demonstrate that light-intensity and duty-cycle can be used to modulate gene expression over the range currently available from natural yeast promoters. This work allows for rapid generation and prototyping of optogenetic circuits to control gene expression in Saccharomyces cerevisiae.

synthetic biology↗

bioRxiv2024

In vitro transcription-based biosensing of glycolate for prototyping of a complex enzyme cascade

In vitro metabolic systems allow the reconstitution of natural and new-to-nature pathways outside of their cellular context and are of increasing interest in bottom-up synthetic biology, cell-free manufacturing and metabolic engineering. Yet, the prototyping of such in vitro networks is very often restricted by time- and cost-intensive analytical methods. To overcome these limitations, we sought to develop an in vitro transcription (IVT)-based biosensing workflow that offers fast results at low-cost, minimal volumes and high-throughput. As a proof-of-concept, we present an IVT biosensor for the so-called CETCH cycle, a complex in vitro metabolic system that converts CO2 into glycolate. To quantify glycolate production, we constructed a sensor module that is based on the glycolate repressor GlcR from Paracoccus denitrificans, and established an IVT biosensing off-line workflow that allows to measure glycolate from CETCH samples from the {micro}M to mM range. We characterized the influence of different cofactors on IVT output and further optimized our IVT biosensor against varying sample conditions. We show that availability of free Mg2+ is a critical factor in IVT biosensing and that IVT output is heavily influenced by ATP, NADPH and other phosphorylated metabolites frequently used in in vitro systems. Our final biosensor is highly robust and shows an excellent correlation between IVT output and classical LC-MS quantification, but notably at [~]10-fold lowered cost and [~]10 times faster turnover time. Our results demonstrate the potential of IVT-based biosensor systems to break current limitations in biological design-build-test cycles for the prototyping of individual enzymes, complex reaction cascades and in vitro metabolic networks.

synthetic biology↗

bioRxiv2018

A broad-host-range event detector: expanding and quantifying performance across bacterial species

Modern microbial biodesign relies on the principle that well-characterized genetic parts can be reused and reconfigured for different functions. However, this paradigm has only been successful in a limited set of hosts, mostly comprised from common lab strains of Escherichia coli. It is clear that new applications - such as chemical sensing and event logging in complex environments - will benefit from new host chassis. This study quantitatively compared how a chemical event logger performed across multiple microbial species. An integrase-based sensor and memory device was operated by two representative soil Pseudomonads - Pseudomonas fluorescens SBW25 and Pseudomonas putida DSM 291. Quantitative comparisons were made between these two non-traditional hosts and two bench-mark Escherichia coli chassis including the probiotic Nissle 1917 and common cloning strain DH5. The performance of sensor and memory components changed according to each host, such that a clear chassis effect was observed and quantified. These results were obtained via fluorescence from reporter proteins that were transcriptionally fused to the integrase and down-stream recombinant region and via data-driven kinetic models. The Pseudomonads proved to be acceptable chassis for the operation of this event logger, which outperformed the common E. coli DH5 in many ways. This study advances an emerging frontier in synthetic biology that aims to build broad-host-range devices and understand the context by which different species can execute programmable genetic operations.

synthetic biology↗

bioRxiv2021

Expression of fatty acyl-CoA ligase drives one-pot de novo synthesis of membrane-bound vesicles in a cell free transcription-translation system

Despite the central importance of lipid membranes in cellular organization, it is challenging to reconstitute their de novo formation from minimal chemical and biological elements. Here we describe a chemoenzymatic route to membrane-forming non-canonical phospholipids in which cysteine-modified lysolipids undergo spontaneous coupling with fatty acyl-CoA thioesters generated enzymatically by a fatty acyl-CoA ligase. Due to the high efficiency of the reaction, we were able to optimize phospholipid membrane formation in a cell-free transcription-translation (TX-TL) system. Combining DNA encoding for the fatty acyl-CoA ligase with suitable lipid precursors, enabled spontaneous one-pot de novo synthesis of membrane-bound vesicles. Non-canonical sphingolipid synthesis was also possible by using a cysteine-modified lysosphingomyelin as a precursor. When the sphingomyelin-interacting protein lysenin is co-expressed alongside the acyl CoA ligase, the in situ assembled membranes were spontaneously modified with protein. Our strategy of coupling gene expression with membrane lipid synthesis in a one-pot fashion could facilitate the generation of proteoliposomes and brings us closer to the bottom-up generation of synthetic cells using recombinant synthetic biology platforms.

synthetic biology↗

bioRxiv2021

Supervised generative design of regulatory DNA forgene expression control

In order to control gene expression, regulatory DNA variants are commonly designed using random synthetic approaches with mutagenesis and screening. This however limits the size of the designed DNA to span merely a part of a single regulatory region, whereas the whole gene regulatory structure including the coding and adjacent non-coding regions is involved in controlling gene expression. Here, we prototype a deep neural network strategy that models whole gene regulatory structures and generates de novo functional regulatory DNA with prespecified expression levels. By learning directly from natural genomic data, without the need for large synthetic DNA libraries, our ExpressionGAN can traverse the whole sequence-expression landscape to produce sequence variants with target mRNA levels as well as natural-like properties, including over 30% dissimilarity to any natural sequence. We experimentally demonstrate that this generative strategy is more efficient than a mutational one when using purely natural genomic data, as 57% of the newly-generated highly-expressed sequences surpass the expression levels of natural controls. We foresee this as a lucrative strategy to expand our knowledge of gene expression regulation as well as increase expression control in any desired organism for synthetic biology and metabolic engineering applications.

synthetic biology↗

bioRxiv2017

Demonstration of de novo chemotaxis in E. coli using a real-time, quantitative, and digital-like approach

Chemotaxis is the movement of an organism in response to an external chemical stimulus. This system enables bacteria to sense their immediate environment and adapt to changes in its chemical composition. Bacterial chemotaxis is mediated by chemoreceptors, membrane proteins that bind an effector and transduce the signal to the downstream proteins. From a synthetic biology perspective, the natural chemotactic repertoire is of little use since bacterial chemoreceptors have evolved to sense specific ligands that either benefit or harm the cell. Here we demonstrate that using a combined computational design approach together with a quantitative, real-time, and digital detection approach, we can rapidly design, manufacture, and characterize a synthetic chemoreceptor in E. coli for histamine (a ligand for which there are no known chemoreceptors). First, we employed a computational protocol that uses the Rosetta bioinformatics software together with high threshold filters to design mutational variants to the native Tar ligand binding domain that target histamine. Second, we tested different ligand-chemoreceptors pairs with a novel chemotaxis assay, based on optical reflectance interferometry of porous silicon (PSi) optical transducers, enabling label-free quantification of chemotaxis by monitoring real-time changes in the optical readout (expressed as the effective optical thickness, EOT). We found that different ligands can be characterized by an individual set of fingerprints in our assay. Namely, a binary, digital-like response in EOT change (i.e. positive or negative) that differentiates between attractants and repellants, the amplitude of change of EOT response, and the rate by which steady state in EOT change is reached. Using this assay, we were able to positively identify and characterize a single mutational chemoreceptor variant for histamine that mediated chemotaxis comparably to the natural Tar-aspartate system. Our results demonstrate the possibility of not only expanding the natural chemotaxis repertoire, but also provide a new quantitative assay by which to characterize the efficacy of the chemotactic response.

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bioRxiv2021

Community science designed ribosomes with beneficial phenotypes

Functional design of ribosomes with mutant ribosomal RNA (rRNA) could expand opportunities for understanding molecular translation, building cells from the bottom-up, and engineering ribosomes with altered capabilities. However, such efforts have been hampered by cell viability constraints, an enormous combinatorial sequence space, and limitations on large-scale, 3D design of RNA structures and functions. To address these challenges, we developed an integrated community science and experimental screening approach for rational design of ribosomes. This approach couples Eterna, an online video game that crowdsources RNA sequence design to community scientists in the form of puzzles, with in vitro ribosome synthesis, assembly, and translation in multiple design-build-test-learn cycles. We applied our framework to discover mutant rRNA sequences that improve protein synthesis in vitro and cell growth in vivo, relative to wild type ribosomes, under diverse environmental conditions. This work provides new insights into ribosome rRNA sequence-function relationships, with implications for synthetic biology.

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bioRxiv2024

Successful establishment of an exogenous gene expression system in Chlamydomnas chloroplast based on viral replication elements

Currently, transformation in unicellular green alga (Chlamydomonas reinhardtii) chloroplast is hindered by a low yield of target products, possibly due to the heterogeneity of the chloroplast genome. In this study, an expression platform was established in C. reinhardtii by leveraging replicative elements from beet curly top geminivirus, which led to a high level of heterologous gene expression, as previously reported in Nicotiana tabacum. Platform performance was verified by the successful expression of a linearized mvenus cassette, achieving a higher expression level than that obtained using the homologous-recombination-based approach. This study provides a new strategy in microalgae synthetic biology and opens new avenue for accumulating valuable bioproducts at high concentrations.

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bioRxiv2025

HP1-enhanced chromatin compaction stabilizes a synthetic metabolic circuit in yeast

Chromatin compaction defines genome topology, evolution, and function. The Saccharomycotina subphylum, including the fermenting yeast Saccharomyces cerevisiae have a decompacted genome, possibly because they lost two genes mediating a specific histone lysine methylation and histone binding protein heterochromatin protein 1 (HP1). This decompaction may result in the higher-than-expected mutation and meiotic recombination rates observed in this species. To test this hypothesis, we retro-engineered S. cerevisiae to compact the genome by expressing the HP1 homologue of Schizosaccharomyces pombe SpSwi6 and H3K9 methyltransferase SpClr4. The resulting strain had significantly more compact chromatin and reduced rates of mutation and meiotic recombination. The increased genomic stability significantly prolongs the optogenetic control of an engineered strain designed to grow only in blue light. This result demonstrates the potential of our approach to enhance the stability of strains for metabolic engineering and other synthetic biology applications, which are prone to lose activities due to genetic instability.

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bioRxiv2019

Harnessing natural modularity of cellular metabolism to design a modular chassis cell for a diverse class of products by using goal attainment optimization

Living cells optimize their fitness against constantly changing environments to survive. Goal attainment optimization is a mathematical framework to describe the simultaneous optimization of multiple conflicting objectives that must all reach a performance above a threshold or goal. In this study, we applied goal attainment optimization to harness natural modularity of cellular metabolism to design a modular chassis cell for optimal production of a diverse class of products, where each goal corresponds to the minimum biosynthesis requirements (e.g., yields and rates) of a target product. This modular cell design approach enables rapid generation of optimal production strains that can be assembled from a modular cell and various exchangeable production modules and hence accelerates the prohibitively slow and costly strain design process. We formulated the modular cell design problem as a blended or goal attainment mixed integer linear program, using mass-balance metabolic models as biological constraints. By applying the modular cell design framework for a genome-scale metabolic model of Escherichia coli, we demonstrated that a library of biochemically diverse products could be effectively synthesized at high yields and rates from a modular (chassis) cell with only a few genetic manipulations. Flux analysis revealed this broad modularity phenotype is supported by the natural modularity and flexible flux capacity of core metabolic pathways. Overall, we envision the developed modular cell design framework provides a powerful tool for synthetic biology and metabolic engineering applications such as industrial biocatalysis to effectively produce fuels, chemicals, and therapeutics from renewable and sustainable feedstocks, bioremediation, and biosensing.

synthetic biology↗

bioRxiv2026

Ontology-driven software engineering using LLMs for knowledge graphs in engineering biology

Large language models have transformed software engineering practices. However, generated artefacts are not always developer-friendly and may partially meet complex requirements. As the need to standardise, integrate, and develop tools in engineering biology increases, novel approaches are needed to create and maintain intuitive software sustainably. Here, we present an ontology-driven approach using large language models to create user-facing software libraries for knowledge graphs. We introduce an ontology-to-language framework to systematically map domain terms and graph structures. We then demonstrate this approach by creating an ontology for the latest Synthetic Biology Open Language standard and generating the sbol-script software library, which can be used within browsers or to develop applications with native web support. This ontology-driven software engineering approach and these resources are essential for the community and to facilitate the development of sustainable software projects. The SBOL3 Ontology and the sbol-script library are available from https://github.com/SynBioDex/sbol-owl3 and https://github.com/SynBioDex/sbol-script.

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bioRxiv2023

Interrogating the function of bicistronic translational control elements to improve consistency of gene expression

Context independent gene expression is required for genetic circuits to maintain consistent and predicable behavior. Previous efforts to develop context independent translation have leveraged the helicase activity of translating ribosomes via bicistronic design translational control elements (BCDs) located within an efficiently translated leader peptide. We have developed a series of bicistronic translational control elements with strengths that span several orders of magnitude, maintain consistent expression levels across diverse sequence contexts, and are agnostic to common ligation sequences used in modular cloning systems. We have used this series of BCDs to investigate several features of this design, including the spacing of the start and stop codons, the nucleotide identity upstream of the start codon, and factors affecting translation of the leader peptide. To demonstrate the flexibility of this architecture and their value as a generic modular expression control cassette for synthetic biology, we have developed a set of robust BCDs for use in several Rhodococcus species.

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bioRxiv2024

A modular system for programming multistep activation of endogenous genes in stem cells

Although genomes encode instructions for mammalian cell differentiation with rich syntactic relationships, existing methods for genetically programming cells have modest capabilities for stepwise regulation of genes. Here, we developed a sequential genetic system that enables transcriptional activation of endogenous genes in a preprogrammed, stepwise manner. The system relies on the removal of an RNA polymerase III termination signal to induce both the transcriptional activation and the DNA endonuclease activities of a Cas9-VPR protein to effect stepwise progression through cascades of gene activation events. The efficiency of the cascading system enables a new dimension for cellular programming by allowing the manipulation of the sequential order of gene activation for directing the differentiation of human stem cells. One-Sentence SummaryDevelopment of a synthetic biology system for preprogrammed, stepwise activation of endogenous genes.

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bioRxiv2026

Sense-and-response to intracellular signaling via nucleocytoplasmic localization control of a synthetic transcription factor platform

Many therapeutically relevant signaling pathways are sensed inside the cell, yet a modular synthetic biology platform for responding to intracellular signaling at the post-translational level is lacking. Signaling pathways control the nuclear localization and activity of native transcription factors via phosphorylation of nuclear localization/export or cytoplasmic anchor sequences. Here, we apply this concept to develop a platform of nucleocytoplasmic localization tags (NCtags) that control the nuclear translocation of a synthetic transcription factor (synTF) for transgene induction in engineered cells. We identify human-based synTF components that are basally cytoplasmic and induce transgenes under the control of NCtags with minimal background. We demonstrate the modular nature of this platform through transcriptional reporter induction in response to a calcium-inducible NFAT-derived NCtag, and development of novel NCtags responsive to hyperosmolarity, cAMP, or logical combinations of cAMP and calcium. Multiple factors in the tumor microenvironment elevate cAMP in T cells, leading to reduced function. We demonstrate that NCtag-synTF fusions expressed in T cells can respond to endogenous sources of cAMP or calcium in breast cancer spheroids to induce transgene expression. Our use of human components and an all-in-one lentiviral vector encoding the NCtag-synTF-transgene system establishes this as a novel platform for sense-and-response inside therapeutic cells.

synthetic biology↗

bioRxiv2020

Multiomics data collection, visualization, and utilization for guiding metabolic engineering

Biology has changed radically in the past two decades, growing from a purely descriptive science into also a design science. The availability of tools that enable the precise modification of cells, as well as the ability to collect large amounts of multimodal data, open the possibility of sophisticated bioengineering to produce fuels, specialty and commodity chemicals, materials, and other renewable bioproducts. However, despite new tools and exponentially increasing data volumes, synthetic biology cannot yet fulfill its true potential due to our inability to predict the behavior of biological systems. Here, we present a set of tools that, combined, provide the ability to store, visualize and leverage these data to predict the outcome of bioengineering efforts.

synthetic biology↗

bioRxiv2013

Design and implementation of a synthetic biomolecular concentration tracker

As a field, synthetic biology strives to engineer increasingly complex artificial systems in living cells. Active feedback in closed loop systems offers a dynamic and adaptive way to ensure constant relative activity independent of intrinsic and extrinsic noise. In this work, we design, model, and implement a biomolecular concentration tracker, in which an output protein tracks the concentration of an input protein. Synthetic modular protein scaffold domains are used to colocalize a two-component system, and a single negative feedback loop modulates the production of the output protein. Using a combination of model and experimental work, we show that the circuit achieves real-time protein concentration tracking in Escherichia coli and that steady state outputs can be tuned.

Synthetic Biology↗

bioRxiv2016

Measurements of translation initiation from all 64 codons in E. coli

Our understanding of translation is one cornerstone of molecular biology that underpins our capacity to engineer living matter. The canonical start codon (AUG) and a few near-cognates (GUG, UUG) are typically considered as the \"start codons\" for translation initiation in Escherichia coli (E. coli). Translation is typically not thought to initiate from the 61 remaining codons. Here, we systematically quantified translation initiation in E. coli from all 64 triplet codons. We detected protein synthesis above background initiating from at least 46 codons. Translation initiated from these non-canonical start codons at levels ranging from 0.01% to 2% relative to AUG. Translation initiation from non-canonical start codons may contribute to the synthesis of peptides in both natural and synthetic biological systems

Synthetic Biology↗

bioRxiv2024

CIFR (Clone-Integrate-Flip-out-Repeat): a toolset for iterative genome and pathway engineering of Gram-negative bacteria

Advances in genome engineering enable precise and customizable modifications of bacterial species. Toolsets for metabolic engineering that exhibit broad-host compatibility are particularly valued owing to their portability. Tn5 transposon vectors have been widely used to establish random integrations of desired DNA sequences into bacterial genomes. However, the iteration of the procedure remains challenging because of the limited availability of selection markers. Here, we present CIFR, a mini-Tn5 integration system for iterative genome engineering. The pCIFR vectors incorporate attP and attB sites flanking an antibiotic resistance marker used to select for the insertion. Subsequent removal of these antibiotic determinants is facilitated by the Bxb1 integrase, and a user-friendly counter-selection marker, both encoded in auxiliary plasmids. Hence, CIFR delivers engineered strains harboring stable DNA insertions and free of any antibiotic resistance cassette, allowing for the reusability of the tool. The system was validated in Pseudomonas putida, Escherichia coli, and Cupriavidus necator, underscoring its portability across diverse industrially-relevant hosts. The CIFR toolbox was calibrated through combinatorial integrations of chromoprotein genes in P. putida, generating strains displaying a diverse color palette. Next, we introduced a carotenoid biosynthesis pathway in P. putida in a two-step engineering process, showcasing the potential of the tool for pathway balancing. The broad utility of the toolbox expands the synthetic biology toolkit for metabolic engineering, allowing for the construction of complex phenotypes, while opening new possibilities in bacterial genetic manipulations.

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