Synthetic biology (Oxford, England)最新文献

{"title":"Robustness and reproducibility of simple and complex synthetic logic circuit designs using a DBTL loop.","authors":"Breschine Cummins,&nbsp;Justin Vrana,&nbsp;Robert C Moseley,&nbsp;Hamed Eramian,&nbsp;Anastasia Deckard,&nbsp;Pedro Fontanarrosa,&nbsp;Daniel Bryce,&nbsp;Mark Weston,&nbsp;George Zheng,&nbsp;Joshua Nowak,&nbsp;Francis C Motta,&nbsp;Mohammed Eslami,&nbsp;Kara Layne Johnson,&nbsp;Robert P Goldman,&nbsp;Chris J Myers,&nbsp;Tessa Johnson,&nbsp;Matthew W Vaughn,&nbsp;Niall Gaffney,&nbsp;Joshua Urrutia,&nbsp;Shweta Gopaulakrishnan,&nbsp;Vanessa Biggers,&nbsp;Trissha R Higa,&nbsp;Lorraine A Mosqueda,&nbsp;Marcio Gameiro,&nbsp;Tomáš Gedeon,&nbsp;Konstantin Mischaikow,&nbsp;Jacob Beal,&nbsp;Bryan Bartley,&nbsp;Tom Mitchell,&nbsp;Tramy T Nguyen,&nbsp;Nicholas Roehner,&nbsp;Steven B Haase","doi":"10.1093/synbio/ysad005","DOIUrl":"https://doi.org/10.1093/synbio/ysad005","url":null,"abstract":"<p><p>Computational tools addressing various components of design-build-test-learn (DBTL) loops for the construction of synthetic genetic networks exist but do not generally cover the entire DBTL loop. This manuscript introduces an end-to-end sequence of tools that together form a DBTL loop called Design Assemble Round Trip (DART). DART provides rational selection and refinement of genetic parts to construct and test a circuit. Computational support for experimental process, metadata management, standardized data collection and reproducible data analysis is provided via the previously published Round Trip (RT) test-learn loop. The primary focus of this work is on the Design Assemble (DA) part of the tool chain, which improves on previous techniques by screening up to thousands of network topologies for robust performance using a novel robustness score derived from dynamical behavior based on circuit topology only. In addition, novel experimental support software is introduced for the assembly of genetic circuits. A complete design-through-analysis sequence is presented using several OR and NOR circuit designs, with and without structural redundancy, that are implemented in budding yeast. The execution of DART tested the predictions of the design tools, specifically with regard to robust and reproducible performance under different experimental conditions. The data analysis depended on a novel application of machine learning techniques to segment bimodal flow cytometry distributions. Evidence is presented that, in some cases, a more complex build may impart more robustness and reproducibility across experimental conditions. <b>Graphical Abstract</b>.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"8 1","pages":"ysad005"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/5b/51/ysad005.PMC10105856.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9736862","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":"Engineered microbes report levels of freshwater contamination in real time.","authors":"Tea Crnković","doi":"10.1093/synbio/ysad002","DOIUrl":"https://doi.org/10.1093/synbio/ysad002","url":null,"abstract":"","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"8 1","pages":"ysad002"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9969829/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10820814","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":"Variability in genome-engineering source materials: consider your starting point.","authors":"Simona Patange,&nbsp;Sierra D Miller,&nbsp;Samantha D Maragh","doi":"10.1093/synbio/ysad003","DOIUrl":"https://doi.org/10.1093/synbio/ysad003","url":null,"abstract":"<p><p>The presence and impact of variability in cells as the source material for genome engineering are important to consider for the design, execution and interpretation of outcomes of a genome-engineering process. Variability may be present at the genotype and phenotype level, yet the impact of these sources of variability on a genome-engineering experiment may not be regularly considered by researchers. In this perspective, we use clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) genome editing of mammalian cells to provide examples of how variation within or across cell samples may mislead a researcher in their expectations about the cells they are engineering. Furthermore, we highlight the need for understanding the baseline cell genotype and phenotype to appropriately understand the starting cell material and interpret and attribute the impact of engineering on cells. We emphasize that heterogeneity within a cell pool and the inherent variability in the cellular materials used for genome engineering are complex, but of high value to characterize and account for where possible, to move toward the potential of generating desired and predictable engineered products. Provided is a framework cause-and-effect diagram for CRISPR/Cas9 genome editing toward identifying and mitigating potential sources of variability. We encourage researchers to consider the variability of source materials and undertake strategies, which may include those described here, for detecting, attributing and minimizing additional sources of variability where possible toward the aim of fostering greater reliability, confidence and reproducibility in genome-engineering studies. <b>Graphical Abstract</b>.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"8 1","pages":"ysad003"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10029982/pdf/ysad003.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9172013","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":"An economy of details: standards and data reusability.","authors":"Ana Delgado","doi":"10.1093/synbio/ysac030","DOIUrl":"https://doi.org/10.1093/synbio/ysac030","url":null,"abstract":"<p><p>Reusability has been a key issue since the origins of the parts-based approach to synthetic biology. Starting with the BioBrick™ standard part, multiple efforts have aimed to make biology more exchangeable. The reusability of parts and other deoxyribonucleic acid-based data has proven over time to be challenging, however. Drawing on a series of qualitative interviews and an international workshop, this article explores the challenges of reusability in real laboratory practice. It shows particular ways that standards are experienced as presenting shortcomings for capturing the kinds of contextual information crucial for scientists to be able to reuse biological parts and data. I argue that researchers in specific laboratories develop a sense of how much circumstantial detail they need to share for others to be able to make sense of their data and possibly reuse it. When choosing particular reporting formats, recharacterizing data to gain closer knowledge or requesting additional information, researchers enact an 'economy of details'. The farther apart two laboratories are in disciplinary, epistemological, technical and geographical terms, the more detailed information needs to be captured for data to be reusable across contexts. In synthetic biology, disciplinary distance between computing science and engineering researchers and experimentalist biologists is reflected in diverging views on standards: what kind of information should be included to enable reusability, what kind of information can be captured by standards at all and how they may serve to produce and circulate knowledge. I argue that such interdisciplinary tensions lie at the core of difficulties in setting standards in synthetic biology.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"8 1","pages":"ysac030"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/e7/47/ysac030.PMC9817096.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10518569","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":"Fighting fire with fire: engineering a microbe into a therapeutic defense against drug-resistant biofilms.","authors":"Charlotte Ayn Cialek","doi":"10.1093/synbio/ysad008","DOIUrl":"https://doi.org/10.1093/synbio/ysad008","url":null,"abstract":"","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"8 1","pages":"ysad008"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10171107/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9839062","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":"Editing <i>Aspergillus terreus</i> using the CRISPR-Cas9 system.","authors":"Sra-Yh Shih,&nbsp;Uffe Hasbro Mortensen,&nbsp;Fang-Rong Chang,&nbsp;HsinYuan Tsai","doi":"10.1093/synbio/ysac031","DOIUrl":"https://doi.org/10.1093/synbio/ysac031","url":null,"abstract":"<p><p>CRISPR-Cas9 technology has been utilized in different organisms for targeted mutagenesis, offering a fast, precise and cheap approach to speed up molecular breeding and study of gene function. Until now, many researchers have established the demonstration of applying the CRISPR/Cas9 system to various fungal model species. However, there are very few guidelines available for CRISPR/Cas9 genome editing in <i>Aspergillus terreus</i>. In this study, we present CRISPR/Cas9 genome editing in <i>A. terreus</i>. To optimize the guide ribonucleic acid (gRNA) expression, we constructed a modified single-guide ribonucleic acid (sgRNA)/Cas9 expression plasmid. By co-transforming an sgRNA/Cas9 expression plasmid along with maker-free donor deoxyribonucleic acid (DNA), we precisely disrupted the <i>lovB</i> and <i>lovR</i> genes, respectively, and created targeted gene insertion (<i>lovF</i> gene) and iterative gene editing in <i>A. terreus</i> (<i>lovF</i> and <i>lovR</i> genes). Furthermore, co-delivering two sgRNA/Cas9 expression plasmids resulted in precise gene deletion (with donor DNA) in the <i>ku70</i> and <i>pyrG</i> genes, respectively, and efficient removal of the DNA between the two gRNA targeting sites (no donor DNA) in the <i>pyrG</i> gene. Our results showed that the CRISPR/Cas9 system is a powerful tool for precise genome editing in <i>A. terreus</i>, and our approach provides a great potential for manipulating targeted genes and contributions to gene functional study of <i>A. terreus</i>.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"7 1","pages":"ysac031"},"PeriodicalIF":0.0,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/9e/24/ysac031.PMC9795164.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10454941","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":"Development of an expression-tunable multiple protein synthesis system in cell-free reactions using T7-promoter-variant series.","authors":"Naoko Senda,&nbsp;Toshihiko Enomoto,&nbsp;Kenta Kihara,&nbsp;Naoki Yamashiro,&nbsp;Naosato Takagi,&nbsp;Daisuke Kiga,&nbsp;Hirokazu Nishida","doi":"10.1093/synbio/ysac029","DOIUrl":"https://doi.org/10.1093/synbio/ysac029","url":null,"abstract":"<p><p>New materials with a low environmental load are expected to be generated through synthetic biology. To widely utilize this technology, it is important to create cells with designed biological functions and to control the expression of multiple enzymes. In this study, we constructed a cell-free evaluation system for multiple protein expression, in which synthesis is controlled by T7 promoter variants. The expression of a single protein using the T7 promoter variants showed the expected variety in expression levels, as previously reported. We then examined the expression levels of multiple proteins that are simultaneously produced in a single well to determine whether they can be predicted from the promoter activity values, which were defined from the isolated protein expression levels. When the sum of messenger ribonucleic acid (mRNA) species is small, the experimental protein expression levels can be predicted from the promoter activities (graphical abstract (a)) due to low competition for ribosomes. In other words, by using combinations of T7 promoter variants, we successfully developed a cell-free multiple protein synthesis system with tunable expression. In the presence of large amounts of mRNA, competition for ribosomes becomes an issue (graphical abstract (b)). Accordingly, the translation level of each protein cannot be directly predicted from the promoter activities and is biased by the strength of the ribosome binding site (RBS); a weaker RBS is more affected by competition. Our study provides information regarding the regulated expression of multiple enzymes in synthetic biology.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"7 1","pages":"ysac029"},"PeriodicalIF":0.0,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/fc/14/ysac029.PMC9791696.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10459894","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":"Self-growing environmentally responsive houses made from agricultural waste and fungal mycelia.","authors":"Sonja Billerbeck","doi":"10.1093/synbio/ysac003","DOIUrl":"https://doi.org/10.1093/synbio/ysac003","url":null,"abstract":"Mix the ingredients, pour them into a tin, and ‘bake’ at ambient temperature for 5days. What sounds like instructions for a ready-made baking mix could soon become a way to grow your own home—or emergency shelter needed after a natural disaster (1). While synthetic biology often focuses on using cells as factories to make molecules and nano-structures of interest, Rodrigo–Navarro et al focused on the cells themselves as the building blocks of macro-structure materials suitable for houses and shelters. This “engineered living material” (ELM) could be grown on demand, they are self-healing, responsive to environmental cues, and recyclable into new structures (2). This macro-scale ELM was developed in a collaboration between the New York-based biomaterial company Ecovative Design and the laboratories of Prof. Harris Wang (Columbia) and Prof. Chris Voigt. The ‘recipe’ for the team’s ELM requires a mix of agricultural byproducts, water, flour and calcium sulfate, and the tree fungus Ganoderma spec. The fungus uses the agricultural waste for nutrition and structural support. Once mixed and cast into brick-shaped foldable paper moulds, the fungal mycelia glue the agricultural waste together into a dense material. In contrast to Ecovative’s standard process of ‘baking’ the ingredients at high temperature, which kills the fungus, McBee et al were able to desiccate the material at ambient temperature. In this state, the fungus rests but can be revived by moisturization. This allows casting of modular bricks that can later be grown together into larger 3D structures—like walls or shelters—without additional mortar. It also allows the material to self-heal if broken. The authors show that a broken brick could be regrown by placing the broken halves close to each other with the healed material retaining most of its original mechanical properties. Further, the material could be fully recycled by grinding it down and using it as inoculum to grow new bricks. After developing this core living material, the team went one step further and equipped it with additional functions by adding an engineered bacterium that carries user-defined synthetic circuitry to the material mix. Instead of using an established, laboratory-tamed synthetic biology chassis such as Escherichia coli, which might have been outcompeted by the fungus, the authors performed a detailed microbiome analysis of the material, identifying and isolating a prevalent member, Pantoea agglomerans. They turned P. agglomerans into an engineerable chassis that could be reintroduced and maintained within the material. The authors then implemented a toy circuit distributed over two engineered strains of P. agglomerans. The first strain generated a volatile quorum sensing molecule (sender strain) that could be sensed and propagated through the material by a second strain (responderpropagator strain) that also created a fluorescent output that could be visualized under the microscope. As such, individual bricks ","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"7 1","pages":"ysac003"},"PeriodicalIF":0.0,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9845837/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10579566","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":"SynBio2Easy-a biologist-friendly tool for batch operations on SBOL designs with Excel inputs.","authors":"Tomasz Zieliński,&nbsp;Johnny Hay,&nbsp;Andrew Romanowski,&nbsp;Anja Nenninger,&nbsp;Alistair McCormick,&nbsp;Andrew J Millar","doi":"10.1093/synbio/ysac002","DOIUrl":"https://doi.org/10.1093/synbio/ysac002","url":null,"abstract":"<p><p>Practical delivery of Findable, Accessible, Reusable and Interoperable principles for research data management requires expertise, time resource, (meta)data standards and formats, software tools and public repositories. The Synthetic Biology Open Language (SBOL2) metadata standard enables FAIR sharing of the designs of synthetic biology constructs, notably in the repository of the SynBioHub platform. Large libraries of such constructs are increasingly easy to produce in practice, for example, in DNA foundries. However, manual curation of the equivalent libraries of designs remains cumbersome for a typical lab researcher, creating a barrier to data sharing. Here, we present a simple tool SynBio2Easy, which streamlines and automates operations on multiple Synthetic Biology Open Language (SBOL) designs using <i>Microsoft Excel®</i> tables as metadata inputs. The tool provides several utilities for manipulation of SBOL documents and interaction with SynBioHub: for example, generation of a library of plasmids based on an original design template, bulk deposition into SynBioHub, or annotation of existing SBOL component definitions with notes and authorship information. The tool was used to generate and deposit a collection of 3661 cyanobacterium <i>Synechocystis</i> plasmids into the public SynBioHub repository. In the process of developing the software and uploading these data, we evaluated some aspects of the SynBioHub platform and SBOL ecosystem, and we discuss proposals for improvement that could benefit the user community. With software such as SynBio2Easy, we aim to deliver a user-driven tooling to make FAIR a reality at all stages of the project lifecycle in synthetic biology research. Graphical Abstract.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"7 1","pages":"ysac002"},"PeriodicalIF":0.0,"publicationDate":"2021-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8944294/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10841703","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":"Synthesis of libraries and multi-site mutagenesis using a PCR-derived, dU-containing template.","authors":"Gretchen Meinke,&nbsp;Nahide Dalda,&nbsp;Benjamin S Brigham,&nbsp;Andrew Bohm","doi":"10.1093/synbio/ysaa030","DOIUrl":"https://doi.org/10.1093/synbio/ysaa030","url":null,"abstract":"<p><p>Directed DNA libraries are useful because they focus genetic diversity in the most important regions within a sequence. Ideally, all sequences in such libraries should appear with the same frequency and there should be no significant background from the starting sequence. These properties maximize the number of different sequences that can be screened. Described herein is a method termed SLUPT (Synthesis of Libraries via a dU-containing PCR-derived Template) for generating highly targeted DNA libraries and/or multi-site mutations wherein the altered bases may be widely distributed within a target sequence. This method is highly efficient and modular. Moreover, multiple distinct sites, each with one or more base changes, can be altered in a single reaction. There is very low background from the starting sequence, and SLUPT libraries have similar representation of each base at the positions selected for variation. The SLUPT method utilizes a single-stranded dU-containing DNA template that is made by polymerase chain reaction (PCR). Synthesis of the template in this way is significantly easier than has been described earlier. A series of oligonucleotide primers that are homologous to the template and encode the desired genetic diversity are extended and ligated in a single reaction to form the mutated product sequence or library. After selective inactivation of the template, only the product library is amplified. There are no restrictions on the spacing of the mutagenic primers except that they cannot overlap.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"6 1","pages":"ysaa030"},"PeriodicalIF":0.0,"publicationDate":"2021-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8260824/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9770036","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}