Synthetic biology (Oxford, England)最新文献

{"title":"Mitigating the deleterious impact of saliva on cell-free expression sensors for point-of-use diagnostics.","authors":"Kathryn Beabout, Svetlana Harbaugh, Jorge L Chávez","doi":"10.1093/synbio/ysag011","DOIUrl":"https://doi.org/10.1093/synbio/ysag011","url":null,"abstract":"<p><p>Point-of-use sensors that detect biomarkers in noninvasive biofluids, such as saliva, could provide individuals with valuable insights into their health and performance status at home or in the field. Cell-free biosensors have the potential to meet these requirements, as these sensors can detect a wide variety of analytes and can be stored at room temperature after freeze-drying. However, saliva inhibits the function of cell-free biosensors, in part due to the presence of interfering proteins and nucleases. Here we show simple strategies to mitigate the inhibitory effects of saliva on the function of cell-free expression systems, including the use of nuclease inhibitors and pretreatment of saliva with centrifugal filtration or heat. We also show successful implementation of our mitigation strategies to detect cortisol spiked into pooled human saliva using a cell-free biosensor. Combined, our results show that simple approaches can be used to enable the function of cell-free biosensors in saliva.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"11 1","pages":"ysag011"},"PeriodicalIF":2.5,"publicationDate":"2026-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13625938/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149119895","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":"A liquid handling platform for standardized quantification of cell-free enzymatic activity encoded by antimicrobial resistance genes.","authors":"Molly Bergum, Sahana Suthakaran, Bethany Martin, J Mark Sutton, Simon J Moore","doi":"10.1093/synbio/ysag010","DOIUrl":"10.1093/synbio/ysag010","url":null,"abstract":"<p><p>Antimicrobial resistance (AMR) is a growing global threat to human health, and rapid methods for characterizing emerging antimicrobial resistance genes (ARGs) are needed. Here, we develop a semi-automated workflow using cell-free gene expression systems to measure the activity of two ARGs encoded on plasmid DNA that produce rifampicin-inactivating and gentamicin-inactivating enzymes. We validated the use of a small benchtop Myra liquid handling system compared to manual pipetting, with no statistical differences observed. After optimizing the pre-incubation time of ARGs and dispensing protocol, expression of <i>aac(3)-IIa</i> increased the half-maximal inhibition concentration (IC₅₀) of gentamicin by over 150-fold, whilst <i>arr-3</i> increased the IC₅₀ of rifampicin by ~20-fold compared to controls. This methodology for rapid, semi-automated ARG characterization offers a strategy to combat AMR by assessing novel ARGs identified through genomic surveillance or profiling activity of new or derivative antibiotics.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"11 1","pages":"ysag010"},"PeriodicalIF":2.5,"publicationDate":"2026-08-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13561876/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148957527","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":"Horseradish peroxidase as an electrochemical reporter protein for cell-free biosensors.","authors":"Aarushi Ruhela, Sahan B W Liyanagedera, Nadanai Laohakunakorn, Jamie R K Marland","doi":"10.1093/synbio/ysag009","DOIUrl":"10.1093/synbio/ysag009","url":null,"abstract":"<p><p>Cell-free biosensor systems offer a promising platform for portable diagnostics. Here, we evaluate electrochemical readout from these systems, using horseradish peroxidase (HRP) as a redox enzyme reporter. HRP was synthesized in an <i>Escherichia coli</i> cell-free transcription-translation system supplemented with hemin, calcium acetate, and commercial disulfide bond enhancers. Electrochemical detection of its activity was established by chronoamperometry, with hydrogen peroxide as a substrate and tetramethylbenzidine as a redox mediator. Cell-free expressed HRP produced a strong steady-state current compared to a catalytically inactive mutant and a no-template control. Kinetic analysis showed a <i>K_m</i> for the cell-free expressed HRP close to that of the native enzyme. To explore the potential of HRP as an electrochemical reporter, we placed it under the control of a tetracycline-responsive regulatory promoter and demonstrated a 2.5-fold current increase in the presence of anhydrotetracycline. These results support HRP as an electrochemical reporter for cell-free biosensors, offering a complementary alternative to existing optical reporters for future use in handheld analytical devices.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"11 1","pages":"ysag009"},"PeriodicalIF":2.5,"publicationDate":"2026-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13455610/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148708767","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":"Integration-coupled activation of promoterless combinatorial pathway libraries in <i>Clostridium</i> avoids burden during DNA assembly.","authors":"Pawel M Mordaka, James J Williamson, John T Heap","doi":"10.1093/synbio/ysag008","DOIUrl":"10.1093/synbio/ysag008","url":null,"abstract":"<p><p>Combinatorial DNA design and assembly is an efficient and pragmatic way to obtain high-performing metabolic pathway designs quickly. However, implementation may require organism-specific technical barriers to be overcome. Firstly, suitable expression control parts such as promoters and ribosome-binding sites (RBSs), which provide a suitable range of expression levels, need to be identified or developed. Secondly, these need to be assembled into pathway-encoding combinatorial libraries of sufficient size, quality, and diversity. For organisms with transformation frequencies too low to allow direct transformation of library assembly reactions, such as many <i>Clostridium spp.</i>, assembly and amplification is typically carried out using <i>Escherichia coli.</i> However, if constructs are deleterious (or 'burdensome') to <i>E. coli</i>, which is often the case when using <i>Clostridium</i> genetic parts, poor libraries may be obtained. Here we develop a new approach called integration-coupled activation of promoterless sequences to overcome this barrier and therefore enable combinatorial assembly in <i>Clostridium</i>. Libraries were designed and assembled as promoterless synthetic operons, preventing expression during DNA assembly, and expression was only activated later, when constructs were integrated into the host genome downstream of a promoter. Variation of expression levels was achieved using a range of context-resistant RBS sequences. This approach was used to produce a <i>Clostridium acetobutylicum</i> library with combinatorial expression variants of an introduced hexanol pathway. This proof of concept provides a generally-applicable approach to implement combinatorial metabolic pathway-encoding libraries in <i>Clostridium spp.</i>, circumventing the excessive strength of <i>Clostridium</i> expression control parts in <i>E. coli</i>, and is applicable to other organisms.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"11 1","pages":"ysag008"},"PeriodicalIF":2.5,"publicationDate":"2026-06-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13499600/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148802639","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":"Chemically synthesized high-fidelity oligos ≤ 600 nt as building blocks to accelerate complex gene construction in synthetic biology.","authors":"Mancang Zhang, Yang Hu, Hao Huang, Yongyong Shi","doi":"10.1093/synbio/ysag005","DOIUrl":"10.1093/synbio/ysag005","url":null,"abstract":"<p><p>Synthetic biology and advanced genetic engineering applications rely heavily on the efficient construction of large and complex DNA sequences. Current DNA synthesis technologies have limited capacity to efficiently generate ultralong oligonucleotides for complex gene construction, particularly those with extensive repetitive motifs and uneven base distribution. Here, we report a novel platform named UCOS (Ultralong Complex Oligonucleotides Synthesis) that enables the efficient synthesis of long, complex, and challenging DNA fragments. This platform employs nonporous silica microspheres as the solid support instead of the traditional controlled pore glass solid support, full-length enrichment based on 5' flank sequence hybridization and an error-removing enzyme for correct sequence selection, substantially enhancing the fidelity of intricate, ultralong oligonucleotides. Using this approach, we successfully synthesized challenging sequences ≤600 nt in length, encompassing tandem repeats and uneven base distributions. Overall, this novel platform demonstrates exceptional efficiency and reliability in handling ultralong DNA fragments with highly repetitive and complex features. This novel platform provides a strong foundation for advancing synthetic biology and metabolic engineering, showing great potential as a powerful tool for constructing challenging genes and enabling the customized synthesis of functional genetic elements for complex genetic programmes and synthetic genomics.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"11 1","pages":"ysag005"},"PeriodicalIF":2.5,"publicationDate":"2026-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13218287/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148140021","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":"A CURE for synthetic regulation of gene expression: rapid screening of guide RNA efficacy as a framework for enabling undergraduate research in plant synthetic biology.","authors":"Tawni Bull, Liz Carlsen, Noah Hoglund, Josh Blarr, Makenzie Ciernia, Hannah Daughtrey, Keana Gulnac, Zane Kathan, Ben Labovitz, Ryan Lonergan, Mira McDermott, Andrew Medina, Zoey Mikol, Zeke Miller, Kyndal Prahl, Chloe Rifai, Emma Schrems, Futa Shinkawa, Jessie Summerfield, Ethan Thevarajah, Skylar Wagner, Trenton Zimmerman, Arjun Khakhar","doi":"10.1093/synbio/ysag007","DOIUrl":"10.1093/synbio/ysag007","url":null,"abstract":"<p><p>Course-based Undergraduate Research Experiences (CUREs) have emerged as a transformative approach to science education, expanding access to authentic research opportunities beyond the traditional undergraduate research assistant (URA) training. By embedding research into a curriculum, CUREs engage a broad and diverse population of students in a classroom environment that emphasizes experimental design, data analysis, and scientific communication. However, this has been difficult to develop for fields such as plant synthetic biology due to the long timescales of plant transformation. One avenue around this problem is to utilize a recent innovation that enables high throughput and rapid screening of guide RNA (gRNA) efficacy by leveraging viral-based delivery of gRNAs. In this work, we develop and validate a CURE with undergraduate students at Colorado State University (CSU). Students worked in teams to design and test efficacy of gRNAs targeting a Cas9-based transcriptional repressor to different regions of the promoters of the three <i>GIBBERELLIN INSENSITIVE 1</i> genes (<i>GID1a, GID1b,</i> and <i>GID1c</i>) in <i>Arabidopsis thaliana</i>. Over the semester, students generated and analysed gene expression data to understand the efficiency of twelve new gRNAs. We further validated CURE student-identified gRNAs with an URA that assessed target gene expression and phenotypic outcomes in stable transgenic lines expressing synthetic transcription factor (SynTF) constructs with the strongest gRNAs from the class. We further describe the curriculum structure to facilitate adoption at other institutions and present student-generated datasets demonstrating the utility of VipariNama-based screening for identifying effective SynTF gRNAs for plant functional genomics and engineering.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"11 1","pages":"ysag007"},"PeriodicalIF":2.5,"publicationDate":"2026-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13262530/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148254815","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":"Off-target detection of CRISPR-Cas9 nuclease <i>in vitro</i> with CROFT-Seq.","authors":"Paulius Toliusis, Algirdas Grybauskas, Tomas Sinkunas, Tautvydas Karvelis, Giedrius Sasnauskas, Mindaugas Zaremba","doi":"10.1093/synbio/ysag006","DOIUrl":"10.1093/synbio/ysag006","url":null,"abstract":"<p><p>Programmable CRISPR-Cas9 nucleases have become invaluable tools for genome editing. However, off-target cleavage by these nucleases can lead to unintended changes in the edited genome. Detection of off-target sites is critical to make genome editing technology safe and predictable. Although current <i>in vitro</i> methods for off-target detection can identify these sites, they are time-consuming and complex. Here, we present CROFT-Seq (<b>CR</b>ISPR nuclease <b>of</b>f-<b>t</b>arget detection by <b>seq</b>uencing), a sensitive, rapid, and affordable assay for the genome-wide detection of Cas9 off-target sites <i>in vitro</i>. CROFT-Seq performs comparably to the commonly used <i>in vitro</i> methods and serves as a valuable and efficient tool for the rapid assessment of genome-editing nuclease specificity. Notably, a high proportion of the top-ranked off-target sites identified by CROFT-Seq were validated in cells, highlighting its strong predictive performance.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"11 1","pages":"ysag006"},"PeriodicalIF":2.5,"publicationDate":"2026-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13189167/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147990326","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":"Going beyond size: exploring the metabolic burden in <i>Pseudomonas putida</i> during heterologous protein production.","authors":"Marleen Beentjes, Ana-Sofia Ortega-Arbulú, Carina Meiners, Andreas Kremling, Katharina Pflüger-Grau","doi":"10.1093/synbio/ysag004","DOIUrl":"10.1093/synbio/ysag004","url":null,"abstract":"<p><p>In biotechnological applications, it is often necessary to introduce genes or entire pathways into a host cell, which can create a significant metabolic burden on the host, limiting productivity. In this study, we systematically investigated the physiological stress responses of <i>Pseudomonas putida</i> during heterologous protein production using a modular monitoring system consisting of a plasmid encoding a heterologous protein fused to eGFP and a chromosomally integrated capacity reporter. Our findings reveal that translation is the main bottleneck, with translational capacity becoming saturated under high expression loads. While increasing the strength of the ribosome binding site improved protein production for non-burdensome proteins, this effect was not observed for larger fusion proteins. Variations in fusion protein size suggested that translational demand, rather than the overall mass of protein produced, determines metabolic burden. We further evaluated how resource availability affects protein expression by modifying the metabolic regime or supplementing with amino acids. While the carbon source affected cellular capacity, it did not significantly alter heterologous protein production. Amino acid supplementation alleviated the growth defects of MBPeGFP-producing cells and modestly improved protein production rates. Together, these findings emphasize that metabolic burden is influenced not only by the size of the produced protein but also by transcript architecture, resource allocation, and the physiological state of the host. Therefore, successful optimization of heterologous protein production requires a holistic approach integrating construct design with host physiology and cultivation strategies.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"11 1","pages":"ysag004"},"PeriodicalIF":2.5,"publicationDate":"2026-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12998221/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147488974","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":"A protein-fragment complementation assay to quantify synthetic protein scaffold efficiency.","authors":"Pascale Lemieux, Alexandre K Dubé, Christian R Landry","doi":"10.1093/synbio/ysag003","DOIUrl":"10.1093/synbio/ysag003","url":null,"abstract":"<p><p>Scaffolds are powerful tools in synthetic biology used for various applications, from increasing yield to optimizing signalling specificity. Protein scaffolds can be built by fusing peptide binding domains (PBD) and attaching the peptide they bind to enzymes, inducing spatial proximity. Only a few PBD-peptide combinations have been tested in this context, and no combination produced a high yield in yeast, an important chassis in biotechnology. Therefore, there is a need for more exploration of PBD-peptide pairs to be used in this model. Scaffold characterization is challenging because it is often dependent on a model pathway with an output that is difficult to measure quantitatively. Here, we use the dihydrofolate reductase protein-fragment complementation assay (DHFR PCA) to study scaffolding efficiency in yeast, which allows to couple scaffolding efficiency with growth rate. First, we characterize the strength of PBD-peptide interactions (PPI) and the binding availability of the PBDs and peptides. Then, we test different scaffold architectures and expression levels to quantify the simultaneous binding of peptide pairs to the scaffold. We show that PPI strength of the weakest binding PBD-peptide pair is critical for scaffolding efficiency and that PPI strength is limited by low binding availability of some domains and peptides <i>in vivo</i>. Also, we find that slight architectural variations and expression levels have a significant impact on scaffolding efficiency detected by DHFR PCA. Finally, we used DHFR PCA approaches to characterize novel PBD-peptide pairs and we identified pairs to expand the sequence toolbox for scaffold design in yeast through DHFR PCA easy-to-read signal.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"11 1","pages":"ysag003"},"PeriodicalIF":2.5,"publicationDate":"2026-01-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12981675/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147470491","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":"EASY-C: Extraction and Analysis of Small Yeast Chromosomes-A rapid and universal platform for recovering artificial mini-chromosomes from synthetic Sc2.0 yeast and large plasmids from <i>Saccharomyces cerevisiae</i> and nonconventional yeast species.","authors":"Reem Swidah, Marco Monti, Daniela Delneri","doi":"10.1093/synbio/ysag002","DOIUrl":"10.1093/synbio/ysag002","url":null,"abstract":"<p><p>The protocol for Extraction and Analysis of Small Yeast-Chromosomes (EASY-C) is a transformative, rapid, cost-effective, and user-friendly method designed for the efficient isolation of artificial synthetic mini-chromosomes (~42-52 kb) and large plasmids (~12 kb) from <i>Saccharomyces cerevisiae</i> wild-type and synthetic yeast strains (Sc2.0), and a range of nonconventional yeast (NCY) species. In this two-step workflow, the DNA from yeast is first extracted and transferred into bacteria, and then the circular DNA is recovered from the bacteria and subjected to downstream analysis, including long-read sequencing. The EASY-C protocol operates at small volumes (~1 mL) and requires less than 2.5 hours, allowing the use of standard commercial plasmid purification kits for bacterial plasmids. Under the tested conditions, the EASY-C methodology yielded clean DNA that could be digested and linearized prior to sequencing, resulting in a higher number of high-quality reads (~2000). The EASY-C protocol worked successfully for the extraction of a variety of constructs, including low-copy centromeric vectors (CEN/ARS), high-copy plasmids (pan/ARS), and artificial mini-chromosomes harbouring (CEN/ARS). It is also applicable to a variety of yeast species, including NCY such as <i>Starmerella</i> sp.<i>, Maudiozyma</i> sp.<i>, and Kazachstania</i> sp<i>.</i> Thanks to its precision, robustness, and simplicity, EASY-C equips researchers with a powerful, time-saving tool and cost-effective approach to accelerate the validation of a wide array of synthetic genetic and metabolic constructs engineered <i>in vivo</i> across diverse yeast species.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"11 1","pages":"ysag002"},"PeriodicalIF":2.5,"publicationDate":"2026-01-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12922780/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147273554","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}