Synthetic biology (Oxford, England)最新文献

{"title":"Highly efficient libraries design for saturation mutagenesis.","authors":"Gur Pines,&nbsp;Assaf Pines,&nbsp;Carrie A Eckert","doi":"10.1093/synbio/ysac006","DOIUrl":"https://doi.org/10.1093/synbio/ysac006","url":null,"abstract":"<p><p>Saturation mutagenesis is a semi-rational approach for protein engineering where sites are saturated either entirely or partially to include amino acids of interest. We previously reported on a codon compression algorithm, where a set of minimal degenerate codons are selected according to user-defined parameters such as the target organism, type of saturation and usage levels. Here, we communicate an addition to our web tool that considers the distance between the wild-type codon and the library, depending on its purpose. These forms of restricted collections further reduce library size, lowering downstream screening efforts or, in turn, allowing more comprehensive saturation of multiple sites. The library design tool can be accessed via http://www.dynamcc.com/dynamcc_d/. Graphical Abstract.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysac006"},"PeriodicalIF":0.0,"publicationDate":"2022-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/dd/d2/ysac006.PMC9205323.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40240035","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, Johnny Hay, Andrew Romanowski, Anja Nenninger, Alistair McCormick, Andrew J Millar","doi":"10.1093/synbio/ysac002","DOIUrl":"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":2.6,"publicationDate":"2022-01-26","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":"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":"Cofactor-independent RNA editing by a synthetic S-type PPR protein.","authors":"Kalia Bernath-Levin,&nbsp;Jason Schmidberger,&nbsp;Suvi Honkanen,&nbsp;Bernard Gutmann,&nbsp;Yueming Kelly Sun,&nbsp;Anuradha Pullakhandam,&nbsp;Catherine Colas des Francs-Small,&nbsp;Charles S Bond,&nbsp;Ian Small","doi":"10.1093/synbio/ysab034","DOIUrl":"https://doi.org/10.1093/synbio/ysab034","url":null,"abstract":"<p><p>Pentatricopeptide repeat (PPR) proteins are RNA-binding proteins that are attractive tools for RNA processing in synthetic biology applications given their modular structure and ease of design. Several distinct types of motifs have been described from natural PPR proteins, but almost all work so far with synthetic PPR proteins has focused on the most widespread P-type motifs. We have investigated synthetic PPR proteins based on tandem repeats of the more compact S-type PPR motif found in plant organellar RNA editing factors and particularly prevalent in the lycophyte <i>Selaginella</i>. With the aid of a novel plate-based screening method, we show that synthetic S-type PPR proteins are easy to design and bind with high affinity and specificity and are functional in a wide range of pH, salt and temperature conditions. We find that they outperform a synthetic P-type PPR scaffold in many situations. We designed an S-type editing factor to edit an RNA target in <i>E. coli</i> and demonstrate that it edits effectively without requiring any additional cofactors to be added to the system. These qualities make S-type PPR scaffolds ideal for developing new RNA processing tools.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysab034"},"PeriodicalIF":0.0,"publicationDate":"2021-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8809517/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39757488","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":"GoldBricks: an improved cloning strategy that combines features of Golden Gate and BioBricks for better efficiency and usability.","authors":"Vishalsingh R Chaudhari,&nbsp;Maureen R Hanson","doi":"10.1093/synbio/ysab032","DOIUrl":"https://doi.org/10.1093/synbio/ysab032","url":null,"abstract":"<p><p>With increasing complexity of expression studies and the repertoire of characterized sequences, combinatorial cloning has become a common necessity. Techniques like BioBricks and Golden Gate aim to standardize and speed up the process of cloning large constructs while enabling sharing of resources. The BioBricks format provides a simplified and flexible approach to endless assembly with a compact library and useful intermediates but is a slow process, joining only two parts in a cycle. Golden Gate improves upon the speed with use of Type IIS enzymes and joins several parts in a cycle but requires a larger library of parts and logistical inefficiencies scale up significantly in the multigene format. We present here a method that provides improvement over these techniques by combining their features. By using Type IIS enzymes in a format like BioBricks, we have enabled a faster and efficient assembly with reduced scarring, which performs at a similarly fast pace as Golden Gate, but significantly reduces library size and user input. Additionally, this method enables faster assembly of operon-style constructs, a feature requiring extensive workaround in Golden Gate. Our format allows such inclusions resulting in faster and more efficient assembly.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysab032"},"PeriodicalIF":0.0,"publicationDate":"2021-11-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/eb/9e/ysab032.PMC8578713.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39713192","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":"Data sharing policies: share well and you shall be rewarded.","authors":"Jean Peccoud","doi":"10.1093/synbio/ysab028","DOIUrl":"https://doi.org/10.1093/synbio/ysab028","url":null,"abstract":"<p><p>Sharing research data is an integral part of the scientific publishing process. By sharing data, authors enable their readers to use their results in a way that the textual description of the results does not allow by itself. In order to achieve this objective, data should be shared in a way that makes it as easy as possible for readers to import them in computer software where they can be viewed, manipulated and analyzed. Many authors and reviewers seem to misunderstand the purpose of the data sharing policies developed by journals. Rather than being an administrative burden that authors should comply with to get published, the objective of these policies is to help authors maximize the impact of their work by allowing other members of the scientific community to build upon it. Authors and reviewers need to understand the purpose of data sharing policies to assist editors and publishers in their efforts to ensure that every article published complies with them.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysab028"},"PeriodicalIF":0.0,"publicationDate":"2021-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8482415/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39483587","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":"Contemporary biomedical engineering perspective on volitional evolution for human radiotolerance enhancement beyond low-earth orbit.","authors":"Alexander M Borg,&nbsp;John E Baker","doi":"10.1093/synbio/ysab023","DOIUrl":"https://doi.org/10.1093/synbio/ysab023","url":null,"abstract":"<p><p>A primary objective of the National Aeronautics and Space Administration (NASA) is expansion of humankind's presence outside low-Earth orbit, culminating in permanent interplanetary travel and habitation. Having no inherent means of physiological detection or protection against ionizing radiation, humans incur capricious risk when journeying beyond low-Earth orbit for long periods. NASA has made large investments to analyze pathologies from space radiation exposure, emphasizing the importance of characterizing radiation's physiological effects. Because natural evolution would require many generations to confer resistance against space radiation, immediately pragmatic approaches should be considered. Volitional evolution, defined as humans steering their own heredity, may inevitably retrofit the genome to mitigate resultant pathologies from space radiation exposure. Recently, uniquely radioprotective genes have been identified, conferring local or systemic radiotolerance when overexpressed <i>in vitro</i> and <i>in vivo</i>. Aiding in this process, the CRISPR/Cas9 technique is an inexpensive and reproducible instrument capable of making limited additions and deletions to the genome. Although cohorts can be identified and engineered to protect against radiation, alternative and supplemental strategies should be seriously considered. Advanced propulsion and mild synthetic torpor are perhaps the most likely to be integrated. Interfacing artificial intelligence with genetic engineering using predefined boundary conditions may enable the computational modeling of otherwise overly complex biological networks. The ethical context and boundaries of introducing genetically pioneered humans are considered.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysab023"},"PeriodicalIF":0.0,"publicationDate":"2021-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8434797/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39436532","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":"Evaluation of inducible promoter-riboswitch constructs for heterologous protein expression in the cyanobacterial species Anabaena sp. PCC 7120.","authors":"Jessee Svoboda,&nbsp;Brenda Cisneros,&nbsp;Benjamin Philmus","doi":"10.1093/synbio/ysab019","DOIUrl":"https://doi.org/10.1093/synbio/ysab019","url":null,"abstract":"Abstract Cyanobacteria are promising chassis for synthetic biology applications due to the fact that they are photosynthetic organisms capable of growing in simple, inexpensive media. Given their slower growth rate than other model organisms such as Escherichia coli and Saccharomyces cerevisiae, there are fewer synthetic biology tools and promoters available for use in model cyanobacteria. Here, we compared a small library of promoter–riboswitch constructs for synthetic biology applications in Anabaena sp. PCC 7120, a model filamentous cyanobacterium. These constructs were designed from six cyanobacterial promoters of various strengths, each paired with one of two theophylline-responsive riboswitches. The promoter–riboswitch pairs were cloned upstream of a chloramphenicol acetyltransferase (cat) gene, and CAT activity was quantified using an in vitro assay. Addition of theophylline to cultures increased the CAT activity in almost all cases, allowing inducible protein production with natively constitutive promoters. We found that riboswitch F tended to have a lower induced and uninduced production compared to riboswitch E for the weak and medium promoters, although the difference was larger for the uninduced production, in accord with previous research. The strong promoters yielded a higher baseline CAT activity than medium strength and weak promoters. In addition, we observed no appreciable difference between CAT activity measured from strong promoters cultured in uninduced and induced conditions. The results of this study add to the genetic toolbox for cyanobacteria and allow future natural product and synthetic biology researchers to choose a construct that fits their needs.","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":" ","pages":"ysab019"},"PeriodicalIF":0.0,"publicationDate":"2021-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8546608/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39825393","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}