Synthetic biology (Oxford, England)最新文献

{"title":"Universal loop assembly: open, efficient and cross-kingdom DNA fabrication.","authors":"Bernardo Pollak, Tamara Matute, Isaac Nuñez, Ariel Cerda, Constanza Lopez, Valentina Vargas, Anton Kan, Vincent Bielinski, Peter von Dassow, Chris L Dupont, Fernán Federici","doi":"10.1093/synbio/ysaa001","DOIUrl":"10.1093/synbio/ysaa001","url":null,"abstract":"<p><p>Standardized type IIS DNA assembly methods are becoming essential for biological engineering and research. These methods are becoming widespread and more accessible due to the proposition of a 'common syntax' that enables higher interoperability between DNA libraries. Currently, Golden Gate (GG)-based assembly systems, originally implemented in host-specific vectors, are being made compatible with multiple organisms. We have recently developed the GG-based Loop assembly system for plants, which uses a small library and an intuitive strategy for hierarchical fabrication of large DNA constructs (>30 kb). Here, we describe 'universal Loop' (uLoop) assembly, a system based on Loop assembly for use in potentially any organism of choice. This design permits the use of a compact number of plasmids (two sets of four odd and even vectors), which are utilized repeatedly in alternating steps. The elements required for transformation/maintenance in target organisms are also assembled as standardized parts, enabling customization of host-specific plasmids. Decoupling of the Loop assembly logic from the host-specific propagation elements enables universal DNA assembly that retains high efficiency regardless of the final host. As a proof-of-concept, we show the engineering of multigene expression vectors in diatoms<i>,</i> yeast, plants and bacteria. These resources are available through the OpenMTA for unrestricted sharing and open access.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"5 1","pages":"ysaa001"},"PeriodicalIF":0.0,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052795/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37729142","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":"Permutational analysis of <i>Saccharomyces cerevisiae</i> regulatory elements.","authors":"Namrita Dhillon,&nbsp;Robert Shelansky,&nbsp;Brent Townshend,&nbsp;Miten Jain,&nbsp;Hinrich Boeger,&nbsp;Drew Endy,&nbsp;Rohinton Kamakaka","doi":"10.1093/synbio/ysaa007","DOIUrl":"https://doi.org/10.1093/synbio/ysaa007","url":null,"abstract":"<p><p>Gene expression in <i>Saccharomyces cerevisiae</i> is regulated at multiple levels. Genomic and epigenomic mapping of transcription factors and chromatin factors has led to the delineation of various modular regulatory elements-enhancers (upstream activating sequences), core promoters, 5' untranslated regions (5' UTRs) and transcription terminators/3' untranslated regions (3' UTRs). However, only a few of these elements have been tested in combinations with other elements and the functional interactions between the different modular regulatory elements remain under explored. We describe a simple and rapid approach to build a combinatorial library of regulatory elements and have used this library to study 26 different enhancers, core promoters, 5' UTRs and transcription terminators/3' UTRs to estimate the contribution of individual regulatory parts in gene expression. Our combinatorial analysis shows that while enhancers initiate gene expression, core promoters modulate the levels of enhancer-mediated expression and can positively or negatively affect expression from even the strongest enhancers. Principal component analysis (PCA) indicates that enhancer and promoter function can be explained by a single principal component while UTR function involves multiple functional components. The PCA also highlights outliers and suggest differences in mechanisms of regulation by individual elements. Our data also identify numerous regulatory cassettes composed of different individual regulatory elements that exhibit equivalent gene expression levels. These data thus provide a catalog of elements that could in future be used in the design of synthetic regulatory circuits.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"5 1","pages":"ysaa007"},"PeriodicalIF":0.0,"publicationDate":"2020-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysaa007","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38247428","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-based enzyme engineering approach applied to psicose biosynthesis.","authors":"Jeremy Armetta, Rose Berthome, Antonin Cros, Celine Pophillat, Bruno Maria Colombo, Amir Pandi, Ioana Grigoras","doi":"10.1093/synbio/ysz028","DOIUrl":"10.1093/synbio/ysz028","url":null,"abstract":"<p><p>Bioproduction of chemical compounds is of great interest for modern industries, as it reduces their production costs and ecological impact. With the use of synthetic biology, metabolic engineering and enzyme engineering tools, the yield of production can be improved to reach mass production and cost-effectiveness expectations. In this study, we explore the bioproduction of D-psicose, also known as D-allulose, a rare non-toxic sugar and a sweetener present in nature in low amounts. D-psicose has interesting properties and seemingly the ability to fight against obesity and type 2 diabetes. We developed a biosensor-based enzyme screening approach as a tool for enzyme selection that we benchmarked with the <i>Clostridium cellulolyticum</i> D-psicose 3-epimerase for the production of D-psicose from D-fructose. For this purpose, we constructed and characterized seven psicose responsive biosensors based on previously uncharacterized transcription factors and either their predicted promoters or an engineered promoter. In order to standardize our system, we created the Universal Biosensor Chassis, a construct with a highly modular architecture that allows rapid engineering of any transcription factor-based biosensor. Among the seven biosensors, we chose the one displaying the most linear behavior and the highest increase in fluorescence fold change. Next, we generated a library of D-psicose 3-epimerase mutants by error-prone PCR and screened it using the biosensor to select gain of function enzyme mutants, thus demonstrating the framework's efficiency.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"4 1","pages":"ysz028"},"PeriodicalIF":0.0,"publicationDate":"2019-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7445875/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38436590","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":"Rapid generation of sequence-diverse terminator libraries and their parameterization using quantitative Term-Seq.","authors":"Andrew J Hudson,&nbsp;Hans-Joachim Wieden","doi":"10.1093/synbio/ysz026","DOIUrl":"https://doi.org/10.1093/synbio/ysz026","url":null,"abstract":"<p><p>Synthetic biology and the rational design and construction of biological devices require vast numbers of characterized biological parts, as well as reliable design tools to build increasingly complex, multigene architectures. Design principles for intrinsic terminators have been established; however, additional sequence-structure studies are needed to refine parameters for termination-based genetic devices. We report a rapid single-pot method to generate libraries of thousands of randomized bidirectional intrinsic terminators and a modified quantitative Term-Seq (qTerm-Seq) method to simultaneously identify terminator sequences and measure their termination efficiencies (TEs). Using qTerm-Seq, we characterize hundreds of additional strong terminators (TE > 90%) with some terminators reducing transcription read-through by up to 1000-fold in <i>Escherichia coli</i>. Our terminator library and qTerm-Seq pipeline constitute a flexible platform enabling identification of terminator parts that can achieve transcription termination not only over a desired range but also to investigate their sequence-structure features, including for specific genetic and application contexts beyond the common <i>in vivo</i> systems such as <i>E. coli</i>.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"4 1","pages":"ysz026"},"PeriodicalIF":0.0,"publicationDate":"2019-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysz026","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38436589","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":"Highly multiplexed, fast and accurate nanopore sequencing for verification of synthetic DNA constructs and sequence libraries.","authors":"Andrew Currin, Neil Swainston, Mark S Dunstan, Adrian J Jervis, Paul Mulherin, Christopher J Robinson, Sandra Taylor, Pablo Carbonell, Katherine A Hollywood, Cunyu Yan, Eriko Takano, Nigel S Scrutton, Rainer Breitling","doi":"10.1093/synbio/ysz025","DOIUrl":"10.1093/synbio/ysz025","url":null,"abstract":"<p><p>Synthetic biology utilizes the Design-Build-Test-Learn pipeline for the engineering of biological systems. Typically, this requires the construction of specifically designed, large and complex DNA assemblies. The availability of cheap DNA synthesis and automation enables high-throughput assembly approaches, which generates a heavy demand for DNA sequencing to verify correctly assembled constructs. Next-generation sequencing is ideally positioned to perform this task, however with expensive hardware costs and bespoke data analysis requirements few laboratories utilize this technology in-house. Here a workflow for highly multiplexed sequencing is presented, capable of fast and accurate sequence verification of DNA assemblies using nanopore technology. A novel sample barcoding system using polymerase chain reaction is introduced, and sequencing data are analyzed through a bespoke analysis algorithm. Crucially, this algorithm overcomes the problem of high-error rate nanopore data (which typically prevents identification of single nucleotide variants) through statistical analysis of strand bias, permitting accurate sequence analysis with single-base resolution. As an example, 576 constructs (6 × 96 well plates) were processed in a single workflow in 72 h (from <i>Escherichia coli</i> colonies to analyzed data). Given our procedure's low hardware costs and highly multiplexed capability, this provides cost-effective access to powerful DNA sequencing for any laboratory, with applications beyond synthetic biology including directed evolution, single nucleotide polymorphism analysis and gene synthesis.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"4 1","pages":"ysz025"},"PeriodicalIF":2.6,"publicationDate":"2019-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7445882/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38436588","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":"Investing in our nation's future military leaders' synthetic biology knowledge to understand and recognize threats and applications.","authors":"J Jordan Steel,&nbsp;Katherine L Bates,&nbsp;Michael D Barnhart","doi":"10.1093/synbio/ysz024","DOIUrl":"https://doi.org/10.1093/synbio/ysz024","url":null,"abstract":"<p><p>Synthetic biology encompasses some of the greatest advancements in biology. With improvements in molecular methods and techniques that allow targeted and highly efficient genome manipulation, the capabilities of engineering biology have significantly increased. These enhancements in biotechnology represent significant potential benefits and risks to the global population. It is important that future leaders are trained and understand the incredible benefits, opportunities and risks associated with synthetic biology. The US Department of Defense (DoD) has issued a technical assessment on the future opportunities of synthetic biology and has encouraged the military institutions to expand and encourage bioengineering research programs. At the US Air Force Academy (USAFA), opportunities are provided for future Air Force officers to recognize the potential and risks associated with synthetic biology by participating in the USAFA Synthetic Biology Education Program (USBEP). Cadets can enroll in synthetic biology courses to learn and master molecular biology techniques and work on independent undergraduate research projects. In addition, cadets have the opportunity to join the USAFA's International Genetically Engineered Machine (iGEM) team and compete in the international synthetic biology competition. This report includes details on how USAFA has recruited, enrolled and encouraged synthetic biology research and education among future leaders in the US Air Force.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"4 1","pages":"ysz024"},"PeriodicalIF":0.0,"publicationDate":"2019-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysz024","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38571065","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":"YouTube resources for synthetic biology education.","authors":"Aaron J Dy,&nbsp;Emily R Aurand,&nbsp;Douglas C Friedman","doi":"10.1093/synbio/ysz022","DOIUrl":"https://doi.org/10.1093/synbio/ysz022","url":null,"abstract":"<p><p>Online video resources have increasingly become a common way to effectively share scientific research ideas and engage viewers at many levels of interest or expertise. While synthetic biology is a comparatively young field, it has accumulated online videos across a spectrum of content and technical depth. Such video content can be used to introduce viewers to synthetic biology, supplement college course content, teach new lab skills and entertain. Here, we compile online videos concerning synthetic biology into public YouTube playlists tailored for six different, though potentially overlapping, audiences: those wanting an introduction to synthetic biology, those wanting to get quick overviews of specific topics within synthetic biology, those wanting teaching or public lectures, those wanting more technical research lectures, those wanting to learn lab protocols and those interested in the International Genetically Engineered Machine competition.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"4 1","pages":"ysz022"},"PeriodicalIF":0.0,"publicationDate":"2019-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysz022","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38436586","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 bacterial stem cells and their multicellularity for synthetic biology and beyond.","authors":"Daniel Bojar","doi":"10.1093/synbio/ysz023","DOIUrl":"https://doi.org/10.1093/synbio/ysz023","url":null,"abstract":"Differentiation produces the plethora of different cell types in any multicellular organism. One of the core principles allowing stem cells to produce differentiated daughter cells is asymmetric cell division, creating two cells with different cellular content (1). The transcription factors and signaling complexes which remain in one of these cells then commit it to differentiate into a specific lineage whereas the other daughter cell replenishes the stem cell population. Being a paradigm of multicellular eukaryotic organisms, asymmetric cell division to kickstart differentiation is largely absent in bacteria and prokaryotes in general. In a recent publication, the group around Matthew R. Bennett at Rice University (with Sara Molinari and David L. Shis in the lead) described the introduction of asymmetric cell division into the bacterium Escherichia coli using principles from synthetic biology (2). Implementing guiding ideas from engineering into biology, synthetic biology aims to modify biological systems in a rational and predictable manner, mainly through genetic modification. One of these engineering principles is the usage of modular parts when constructing a system. Bennett and colleagues used the chromosome partitioning system of another bacterium, Caulobacter crescentus, as a unit in their design. Consisting of the DNA-binding protein ParB and the DNA element parS, the chromosome partitioning system is modular enough to be transferable to E. coli. Integrating the parS DNA sequence into a plasmid which additionally carries a gene expression cassette, for instance for a fluorescent protein, then causes ParB to bind to the parS element. Forming a cluster, parS-containing plasmid is then exclusively and asymmetrically present in one of the two daughter cells after the process of cell division. And now here comes the trick: by making the production of ParB contingent on the presence of a small molecule (for instance by using arabinose-inducible promoters), the researchers can control when exactly they want to initiate asymmetric cell division. This way, a sustainable population of stem cell-like bacterial cells containing the parS-marked plasmid can be replenished at every cell division event, spawning descendant, differentiated cells in the process. To further build on their approach, the Bennett lab then added an orthogonal chromosome partitioning system (this time consisting of the DNA-sequestering SopB and the DNA-element sopC from the F plasmid of E. coli). Controlled by a different small molecule, isopropyl b-D-1-thiogalactopyranoside, their final system now had three differentiated and one pluripotent stem cell-like state. Adding each of the inducers (or both) led to distinct differentiated states, differing in the genecarrying plasmids with the parS or sopC DNA elements. Establishing a bacterial stem cell population able to differentiate into multiple states, this publication lays the groundwork for prokaryotic multicellular organisms,","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"4 1","pages":"ysz023"},"PeriodicalIF":0.0,"publicationDate":"2019-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysz023","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38436587","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":"Optimization of the experimental parameters of the ligase cycling reaction.","authors":"Niels Schlichting,&nbsp;Felix Reinhardt,&nbsp;Sven Jager,&nbsp;Michael Schmidt,&nbsp;Johannes Kabisch","doi":"10.1093/synbio/ysz020","DOIUrl":"https://doi.org/10.1093/synbio/ysz020","url":null,"abstract":"<p><p>The ligase cycling reaction (LCR) is a scarless and efficient method to assemble plasmids from fragments of DNA. This assembly method is based on the hybridization of DNA fragments with complementary oligonucleotides, so-called bridging oligos (BOs), and an experimental procedure of thermal denaturation, annealing and ligation. In this study, we explore the effect of molecular crosstalk of BOs and various experimental parameters on the LCR by utilizing a fluorescence-based screening system. The results indicate an impact of the melting temperatures of BOs on the overall success of the LCR assembly. Secondary structure inhibitors, such as dimethyl sulfoxide and betaine, are shown to negatively impact the number of correctly assembled plasmids. Adjustments of the annealing, ligation and BO-melting temperature further improved the LCR. The optimized LCR was confirmed by validation experiments. Based on these findings, a step-by-step protocol is offered within this study to ensure a routine for high efficient LCR assemblies.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"4 1","pages":"ysz020"},"PeriodicalIF":0.0,"publicationDate":"2019-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysz020","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38439149","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":"Riboswitch identification using Ligase-Assisted Selection for the Enrichment of Responsive Ribozymes (LigASERR).","authors":"Matthew C Haines,&nbsp;Marko Storch,&nbsp;Diego A Oyarzún,&nbsp;Guy-Bart Stan,&nbsp;Geoff S Baldwin","doi":"10.1093/synbio/ysz019","DOIUrl":"https://doi.org/10.1093/synbio/ysz019","url":null,"abstract":"<p><p><i>In vitro</i> selection of ligand-responsive ribozymes can identify rare, functional sequences from large libraries. While powerful, key caveats of this approach include lengthy and demanding experimental workflows; unpredictable experimental outcomes and unknown functionality of enriched sequences <i>in vivo</i>. To address the first of these limitations, we developed Ligase-Assisted Selection for the Enrichment of Responsive Ribozymes (LigASERR). LigASERR is scalable, amenable to automation and requires less time to implement compared to alternative methods. To improve the predictability of experiments, we modeled the underlying selection process, predicting experimental outcomes based on sequence and population parameters. We applied this new methodology and model to the enrichment of a known, <i>in vitro</i>-selected sequence from a bespoke library. Prior to implementing selection, conditions were optimized and target sequence dynamics accurately predicted for the majority of the experiment. In addition to enriching the target sequence, we identified two new, theophylline-activated ribozymes. Notably, all three sequences yielded riboswitches functional in <i>Escherichia coli,</i> suggesting LigASERR and similar <i>in vitro</i> selection methods can be utilized for generating functional riboswitches in this organism.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"4 1","pages":"ysz019"},"PeriodicalIF":0.0,"publicationDate":"2019-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysz019","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38439148","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}