Synthetic biology (Oxford, England)最新文献

{"title":"Oligonucleotide library assisted sequence mining reveals promoter sequences with distinct temporal expression dynamics for applications in <i>Curvibacter</i> sp. AEP1-3.","authors":"Maurice Mager, Lukas Becker, Nina Schulten, Sebastian Fraune, Ilka M Axmann","doi":"10.1093/synbio/ysaf001","DOIUrl":"10.1093/synbio/ysaf001","url":null,"abstract":"<p><p>The <i>β-proteobacterial</i> species <i>Curvibacter</i> sp. AEP1-3 is a model organism for the study of symbiotic interactions as it is the most abundant colonizer of <i>Hydra vulgaris</i>. Yet, genetic tools for <i>Curvibacter</i> are still in their infancy; few promoters have been characterized so far. Here, we employ an oligonucleotide-based strategy to develop novel expression systems <i>Curvibacter</i>. Potential promoters were systematically mined from the genome <i>in silico</i>. The sequences were cloned as a mixed library into a mCherry reporter vector and positive candidates were selected by Flow Cytometry to be further analysed through plate reader measurements. From 500 candidate sequences, 25 were identified as active promoters of varying expression strength levels. Plate reader measurements revealed unique activity profiles for these sequences across growth phases. The expression levels of these promoters ranged over two orders of magnitudes and showed distinct temporal expression dynamics over the growth phases: while three sequences showed higher expression levels in the exponential phase, we found 12 sequences saturating expression during stationary phase and 10 that showed little discrimination between growth phases. From our library, promoters of the genes <i>dnaK, rpsL</i> and an acyl-homoserine-lactone (AHL) synthase stood out as the most interesting candidates fit for a variety of applications. We identified enriched transcription factor binding motifs among the sorted 33 sequences and genes encoding for homologs of these transcription factors in close proximity to the identified motifs. In this work, we show the value of employing comprehensive high-throughput strategies to establish expression systems for novel model organisms.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"10 1","pages":"ysaf001"},"PeriodicalIF":2.6,"publicationDate":"2025-05-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12094071/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144121646","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Synthetic biology design principles enable efficient bioproduction of Heparosan with low molecular weight and low polydispersion index for the biomedical industry.","authors":"Yadira Boada, Marcelo Flores, Martin Stiebritz, Marco Córdova, Francisco Flores, Alejandro Vignoni","doi":"10.1093/synbio/ysaf006","DOIUrl":"10.1093/synbio/ysaf006","url":null,"abstract":"<p><p>Heparosan is a natural polymer with unique chemical and biological properties, that holds great promise for biomedical applications. The molecular weight (Mw) and polydispersion index (PDI) are critical factors influencing the performance of heparosan-based materials. Achieving precise control over the synthesis process to consistently produce heparosan with low Mw and low PDI can be challenging, as it requires tight regulation of reaction conditions, enzyme activity, and precursor concentrations. We propose a novel approach utilizing synthetic biology principles to precisely control heparosan biosynthesis in bacteria. Our strategy involves designing a biomolecular controller that can regulate the expression of genes involved in heparosan biosynthesis. This controller is activated by biosensors that detect heparosan precursors, allowing for fine-tuned control of the polymerization process. Through this approach, we foresee the implementation of this synthetic device, demonstrating the potential to produce low Mw and low PDI heparosan in the probiotic <i>E. coli</i> Nissle 1917 as a biosafe and biosecure biofactory. This study represents a significant advancement in the field of heparosan production, offering new opportunities for the development and manufacturing of biomaterials with tailored properties for diverse biomedical applications.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"10 1","pages":"ysaf006"},"PeriodicalIF":2.6,"publicationDate":"2025-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12091141/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144113030","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Simulations predict stronger CRISPRi transcriptional repression in plants for identical than heterogeneous gRNA target sites.","authors":"Helen Scott, Alessandro Occhialini, Scott C Lenaghan, Jacob Beal","doi":"10.1093/synbio/ysae020","DOIUrl":"https://doi.org/10.1093/synbio/ysae020","url":null,"abstract":"<p><p>Plant synthetic biologists have been working to adapt the CRISPRa and CRISPRi promoter regulation methods for applications such as improving crops or installing other valuable pathways. With other organisms, strong transcriptional control has typically required multiple gRNA target sites, which poses a critical engineering choice between heterogeneous sites, which allow each gRNA to target existing locations in a promoter, and identical sites, which typically require modification of the promoter. Here, we investigate the consequences of this choice for CRISPRi plant promoter regulation via simulation-based analysis, using model parameters based on single gRNA regulation and constitutive promoters in <i>Nicotiana benthamiana</i> and <i>Arabidopsis thaliana</i>. Using models of 2-6 gRNA target sites to compare heterogeneous versus identical sites for tunability, sensitivity to parameter values, and sensitivity to cell-to-cell variation, we find that identical gRNA target sites are predicted to yield far more effective transcriptional repression than heterogeneous sites.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"10 1","pages":"ysae020"},"PeriodicalIF":2.6,"publicationDate":"2025-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12007490/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144060307","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cell-free biosensor with automated acoustic liquid handling for rapid and scalable characterization of cellobiohydrolases on microcrystalline cellulose.","authors":"Taeok Kim, Eun Jung Jeon, Kil Koang Kwon, Minji Ko, Ha-Neul Kim, Seong Keun Kim, Eugene Rha, Jonghyeok Shin, Haseong Kim, Dae-Hee Lee, Bong Hyun Sung, Soo-Jung Kim, Hyewon Lee, Seung-Goo Lee","doi":"10.1093/synbio/ysaf005","DOIUrl":"https://doi.org/10.1093/synbio/ysaf005","url":null,"abstract":"<p><p>Engineering enzymes to degrade solid substrates, such as crystalline cellulose from paper sludge or microplastics in sewage sludge, presents challenges for high-throughput screening (HTS), as solid substrates are not readily accessible in cell-based biosensor systems. To address this challenge, we developed a cell-free cellobiose-detectable biosensor (CB-biosensor) for rapid characterization of cellobiohydrolase (CBH) activity, enabling direct detection of hydrolysis products without cellular constraints. The CB-biosensor demonstrates higher sensitivity than conventional assays and distinguishes between CBH subtypes (CBHI and CBHII) based on their modes of action. Integration with the Echo 525 liquid handler enables precise and reproducible sample processing, with fluorescence signals from automated preparations comparable to manual experiments. Furthermore, assay volumes can be reduced to just a few microlitres-impractical with manual methods. This cell-free CB-biosensor with Echo 525 minimizes reagent consumption, accelerates testing, and facilitates reliable large-scale screening. These findings highlight its potential to overcome current HTS limitations, advancing enzyme screening and accelerating the Design-Build-Test-Learn cycle for sustainable biomanufacturing.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"10 1","pages":"ysaf005"},"PeriodicalIF":2.6,"publicationDate":"2025-04-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12006790/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144058581","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"OT-Mation: an open-source code for parsing CSV files into Python scripts for control of OT-2 liquid-handling robotics.","authors":"Alex Laverick, Katherine Convey, Catherine Harrison, Jenny Tomlinson, Jem Stach, Thomas P Howard","doi":"10.1093/synbio/ysaf009","DOIUrl":"https://doi.org/10.1093/synbio/ysaf009","url":null,"abstract":"<p><p>OT-Mation is an open-source Python script designed to automate the programming of OT-2 liquid-handling robots, making combinatorial experiments more accessible to researchers. By parsing user-defined CSV files containing information on labware, reagents, pipettes, and experimental design, OT-Mation generates a bespoke Python script compatible with the OT-2 system. OT-Mation enhances reproducibility, reduces human error, and streamlines workflows, making it a valuable addition to any laboratory utilizing OT-2 robotics for liquid handling. While OT-Mation can be used for setting up any type of experiment on the OT-2, its real utility lies in making the connection between multifactorial experimental design software outputs (i.e. design of experiments arrays) and liquid-handling robot executable code. As such, OT-Mation helps bridge the gap between code-based flexibility and user-friendly operation, allowing researchers with limited programming skills to design and execute complex experiments efficiently. <b>Graphical Abstract</b>.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"10 1","pages":"ysaf009"},"PeriodicalIF":2.6,"publicationDate":"2025-04-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12063526/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144063349","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Engineering a custom-sized DNA scaffold for more efficient DNA origami-based nucleic acid data storage.","authors":"Sarah E Kobernat, Maryna Lazouskaya, Benjamin C Balzer, Amanda Wolf, Golam M Mortuza, George D Dickinson, Tim Andersen, William L Hughes, Luca Piantanida, Eric J Hayden","doi":"10.1093/synbio/ysaf008","DOIUrl":"https://doi.org/10.1093/synbio/ysaf008","url":null,"abstract":"<p><p>DNA has emerged as a promising material to address growing data storage demands. We recently demonstrated a structure-based DNA data storage approach where DNA probes are spatially oriented on the surface of DNA origami and decoded using DNA-PAINT. In this approach, larger origami structures could improve the efficiency of reading and writing data. However, larger origami require long single-stranded DNA scaffolds that are not commonly available. Here, we report the engineering of a novel longer DNA scaffold designed to produce a larger rectangle origami needed to expand the origami-based digital nucleic acid memory (dNAM) approach. We confirmed that this scaffold self-assembled into the correct origami platform and correctly positioned DNA data strands using atomic force microscopy and DNA-PAINT super-resolution microscopy. This larger structure enables a 67% increase in the number of data points per origami and will support efforts to efficiently scale up origami-based dNAM.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"10 1","pages":"ysaf008"},"PeriodicalIF":2.6,"publicationDate":"2025-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12047451/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144063405","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Two highly specific growth-coupled biosensor for glycolaldehyde detection across micromolar and millimolar concentrations.","authors":"Paul A Gómez-Coronado, Armin Kubis, Maria Kowald, Rahma Ute, Charlie Cotton, Steffen N Lindner, Arren Bar-Even, Tobias J Erb","doi":"10.1093/synbio/ysaf004","DOIUrl":"https://doi.org/10.1093/synbio/ysaf004","url":null,"abstract":"<p><p>Glycolaldehyde (GA), the smallest sugar, has significant potential as a biomass-derived platform chemical and is a key metabolite in several synthetic pathways for one-carbon metabolism and new-to-nature photorespiration. This study introduces two metabolic schemes for engineering <i>Escherichia coli</i> into GA biosensors. Through creating GA-dependent auxotrophies, we link growth of these strains to GA-dependent biosynthesis of the essential vitamin pyridoxal-5-phosphate, and 2-ketoglutarate, respectively. We characterized and optimized these strains for the quantification of externally added GA from 2 µM to 1.5 mM. We also demonstrate the capability of these strains to detect GA that is produced intracellularly through different metabolic routes and from different substrates such as xylose, ethylene glycol, and glycolate. Our biosensors offer complementary sensitivities and features, opening up different applications in metabolic engineering and synthetic biology, which we demonstrate in a proof-of-principle by providing the first <i>in vivo</i> demonstration of the reduction of glycolate to GA by a new-to-nature route using engineered enzymes.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"10 1","pages":"ysaf004"},"PeriodicalIF":2.6,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12022395/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144053197","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"<i>In silico</i> prediction of loop-mediated isothermal amplification using a generalized linear model.","authors":"Kenshiro Taguchi, Satoru Michiyuki, Takumasa Tsuji, Jun'ichi Kotoku","doi":"10.1093/synbio/ysaf007","DOIUrl":"https://doi.org/10.1093/synbio/ysaf007","url":null,"abstract":"<p><p>Loop-mediated isothermal amplification (LAMP), a DNA amplification technique under isothermal conditions, provides the important benefits of high sensitivity, specificity, rapidity, and simplicity. Maximizing LAMP features necessitates the design of a complex LAMP primer set (LPS) consisting of four primers for six regions of a given target DNA. Furthermore, the LPS of a given target DNA is designed with LPS design support software such as Primer Explorer. However, even if the design is completed, we still must do many <i>in vitro</i> experiments and evaluations. Consequently, designing LPS often fails to achieve high performance, including efficient amplification. For this study, we examined <i>in silico</i> LAMP: a generalized linear model to predict DNA amplification from LPS. Using logistic regression with elastic net regularization, we identified factors that strongly affect LPS design. These factors, combined with domain knowledge for LPS design, led to the creation of LAMP kernel variables that are highly essential for high LAMP reaction. <i>In silico</i> LAMP, constructed using logistic regression with LAMP kernel variables, allows classification and performance prediction of LPS with an area under the curve of 0.86. These results suggest that a high LAMP reaction can be predicted using LAMP kernel variables and generalized linear regression model. Moreover, an LPS with high performance can be constructed without <i>in vitro</i> experimentation.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"10 1","pages":"ysaf007"},"PeriodicalIF":2.6,"publicationDate":"2025-03-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12032545/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144058704","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"biGMamAct: efficient CRISPR/Cas9-mediated docking of large functional DNA cargoes at the <i>ACTB</i> locus.","authors":"Martin Pelosse, Marco Marcia","doi":"10.1093/synbio/ysaf003","DOIUrl":"10.1093/synbio/ysaf003","url":null,"abstract":"<p><p>Recent advances in molecular and cell biology and imaging have unprecedentedly enabled multiscale structure-functional studies of entire metabolic pathways from atomic to micrometer resolution and the visualization of macromolecular complexes <i>in situ</i>, especially if these molecules are expressed with appropriately engineered and easily detectable tags. However, genome editing in eukaryotic cells is challenging when generating stable cell lines loaded with large DNA cargoes. To address this limitation, here, we have conceived biGMamAct, a system that allows the straightforward assembly of a multitude of genetic modules and their subsequent integration in the genome at the <i>ACTB</i> locus with high efficacy, through standardized cloning steps. Our system comprises a set of modular plasmids for mammalian expression, which can be efficiently docked into the genome in tandem with a validated Cas9/sgRNA pair through homologous-independent targeted insertion. As a proof of concept, we have generated a stable cell line loaded with an 18.3-kilobase-long DNA cargo to express six fluorescently tagged proteins and simultaneously visualize five different subcellular compartments. Our protocol leads from the <i>in silico</i> design to the genetic and functional characterization of single clones within 6 weeks and can be implemented by any researcher with familiarity with molecular biology and access to mammalian cell culturing infrastructure.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"10 1","pages":"ysaf003"},"PeriodicalIF":2.6,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11891445/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143598380","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biosensor characterization: formal methods from the perspective of proteome fractions.","authors":"Nicolás A Vaccari, Dahlin Zevallos-Aliaga, Tom Peeters, Daniel G Guerra","doi":"10.1093/synbio/ysaf002","DOIUrl":"10.1093/synbio/ysaf002","url":null,"abstract":"<p><p>Many studies characterize transcription factors and other regulatory elements to control gene expression in recombinant systems. However, most lack a formal approach to analyse the inherent and context-specific variations of these regulatory components. This study addresses this gap by establishing a formal framework from which convenient methods are inferred to characterize regulatory circuits. We modelled the bacterial cell as a collection of proteome fractions. Deriving the time-dependent proteome fraction, we obtained a general theorem that describes its change as a function of its expression fraction, a specific portion of the total biosynthesis flux of the cell. Formal deduction reveals that when the proteome fraction reaches a maximum, it becomes equivalent to its expression fraction. This equation enables the reliable measurement of the expression fraction through direct protein quantification. In addition, the experimental data demonstrate a linear correlation between protein production rate and specific growth rate over a significant time period. This suggests a constant expression fraction within this window. For an Isopropyl β- d-1-thiogalactopyranoside (IPTG) biosensor, in five cellular contexts, expression fractions determined by the maximum method and the slope method produced strikingly similar dose-response parameters when independently fit to a Hill function. Furthermore, by analysing two more biosensors, for mercury and cumate detection, we demonstrate that the slope method can be applied effectively to various systems. Therefore, the concepts presented here provide convenient methods for obtaining dose-response parameters, clearly defining the time interval of their validity and offering a framework for interpreting typical biosensor outputs in terms of bacterial physiology. Graphical Abstract Nutrients, transformed by the action of the Nutrient Fixators (purple arrow), are used at a rate of ρ for Protein biosynthesis. The total rate ρ is multiplied by expression fractions f_R, f_C, f_H, and f_Q to obtain the biosynthesis rate (black arrows) of each proteome fraction Φ_R, Φ_C, Φ_H, Φ_Q, respectively. In a graph of Growth rate versus Proteome Fraction Production Rate, a linear function (green lines) can be observed, and its slope is equal to the expression fraction at each condition.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"10 1","pages":"ysaf002"},"PeriodicalIF":2.6,"publicationDate":"2025-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11826058/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143434472","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}