Synthetic biology (Oxford, England)最新文献

{"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}

{"title":"Analysis of primitive genetic interactions for the design of a genetic signal differentiator.","authors":"Wolfgang Halter, Richard M Murray, Frank Allgöwer","doi":"10.1093/synbio/ysz015","DOIUrl":"10.1093/synbio/ysz015","url":null,"abstract":"Abstract We study the dynamic and static input–output behavior of several primitive genetic interactions and their effect on the performance of a genetic signal differentiator. In a simplified design, several requirements for the linearity and time-scales of processes like transcription, translation and competitive promoter binding were introduced. By experimentally probing simple genetic constructs in a cell-free experimental environment and fitting semi-mechanistic models to these data, we show that some of these requirements can be verified, while others are only met with reservations in certain operational regimes. Analyzing the linearized model of the resulting genetic network, we conclude that it approximates a differentiator with relative degree one. Taking also the discovered nonlinearities into account and using a describing function approach, we further determine the particular frequency and amplitude ranges where the genetic differentiator can be expected to behave as such.","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"4 1","pages":"ysz015"},"PeriodicalIF":0.0,"publicationDate":"2019-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysz015","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38439146","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":"Life simplified: recompiling a bacterial genome for synonymous codon compression.","authors":"Joshua T Atkinson","doi":"10.1093/synbio/ysz017","DOIUrl":"https://doi.org/10.1093/synbio/ysz017","url":null,"abstract":"Researchers in the UK recently reported a strain of Escherichia coli with a completely synthetic 4-million-base-pair genome (1). This achievement sets a new world record in synthetic genomics by yielding a genome that is four times larger than the pioneering synthesis of the 1-million-base-pair Mycoplasma mycoides genome (2). Synthetic genomics is enabling the simplification of recoded organisms, the previous study minimized the total number of genes and this new study simplified the way those genes are encoded. Fredens and co-workers constructed an E. coli strain, dubbed Syn61, that utilizes just 61 codons for protein synthesis. Cells typically use 64 codons including 3 that encode termination of protein translation (stop codons). Eighteen of the 20 amino acids are encoded by 2–6 synonymous codons. Nature leverages these redundant codons to regulate the transfer of information from DNA to RNA to protein in a variety of ways (3). The extent to which these degenerate codons are needed for cell fitness is not known. To assess this question systematically, the team performed ‘synonymous codon compression’ on the E. coli genome, recoding 2 of the 6 codons encoding serine (TCG and TCA) and the amber stop codon (TAG) with synonymous codons. This study recoded an astonishing 18 214 codons, exceeding past recoding efforts by &gt;50-fold (4). To accomplish this tour de force, the authors used homologous recombination in Saccharomyces cerevisiae to assemble 37 bacterial artificial chromosomes ( 100 kilobase long) from 409 smaller synthetic DNA ( 10 kilobase). Using a method called ‘replicon excision for enhanced genome engineering through programmed recombination’, or REXER (3), they iteratively replaced segments of the E. coli genome with the synthetic DNA fragments. REXER uses a double selection strategy that leverages unique pairs of positive and negative selection markers embedded in both the genome and the synthetic DNA fragment and CRISPR/Cas9 DNA excision to increase the efficiency of lambda red recombination for large DNA fragments (3). The authors first performed REXER in parallel targeting eight different genomic regions to generate a library of partially recoded strains. Then, to assemble the full synthetic genome, they merged the engineered DNA in their strains using conjugative transfer and recombination. Relative to the parental strain, Syn61 displayed only minor growth defects with slightly elongated cells and enabled the deletion of a previously essential tRNA. This strain also showed increased viability when expressing tRNAs charged with a noncanonical amino acid (ncAA) that targets one of the removed codons. The application of synthetic genomics to the laboratory workhorse E. coli represents an important step towards enabling a future where synthetic biologists can readily design and write tailor-made genomes to generate synthetic organisms with user-specified functions. Codon compression leads to decreased infection by bacteriophage, as pha","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"4 1","pages":"ysz017"},"PeriodicalIF":0.0,"publicationDate":"2019-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysz017","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38439147","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":"PartCrafter: find, generate and analyze BioParts.","authors":"Emily Scher,&nbsp;Shay B Cohen,&nbsp;Guido Sanguinetti","doi":"10.1093/synbio/ysz014","DOIUrl":"https://doi.org/10.1093/synbio/ysz014","url":null,"abstract":"<p><p>The field of Synthetic Biology is both practically and philosophically reliant on the idea of BioParts-concrete DNA sequences meant to represent discrete functionalities. While there are a number of software tools which allow users to design complex DNA sequences by stitching together BioParts or genetic features into genetic devices, there is a lack of tools assisting Synthetic Biologists in finding BioParts and in generating new ones. In practice, researchers often find BioParts in an <i>ad hoc</i> way. We present PartCrafter, a tool which extracts and aggregates genomic feature data in order to facilitate the search for new BioParts with specific functionalities. PartCrafter can also turn a genomic feature into a BioPart by packaging it according to any manufacturing standard, codon optimizing it for a new host, and removing forbidden sites. PartCrafter is available at partcrafter.com.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"4 1","pages":"ysz014"},"PeriodicalIF":0.0,"publicationDate":"2019-06-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysz014","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38439145","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":"Broad range shuttle vector construction and promoter evaluation for the use of <i>Lactobacillus plantarum</i> WCFS1 as a microbial engineering platform.","authors":"Joseph R Spangler,&nbsp;Julie C Caruana,&nbsp;Daniel A Phillips,&nbsp;Scott A Walper","doi":"10.1093/synbio/ysz012","DOIUrl":"https://doi.org/10.1093/synbio/ysz012","url":null,"abstract":"<p><p>As the field of synthetic biology grows, efforts to deploy complex genetic circuits in nonlaboratory strains of bacteria will continue to be a focus of research laboratories. Members of the <i>Lactobacillus</i> genus are good targets for synthetic biology research as several species are already used in many foods and as probiotics. Additionally, <i>Lactobacilli</i> offer a relatively safe vehicle for microbiological treatment of various health issues considering these commensals are often minor constituents of the gut microbial community and maintain allochthonous behavior. In order to generate a foundation for engineering, we developed a shuttle vector for subcloning in <i>Escherichia coli</i> and used it to characterize the transcriptional and translational activities of a number of promoters native to <i>Lactobacillus plantarum</i> WCFS1. Additionally, we demonstrated the use of this vector system in multiple <i>Lactobacillus</i> species, and provided examples of non-native promoter recognition by both <i>L. plantarum</i> and <i>E. coli</i> strains that might allow a shortcut assessment of circuit outputs. A variety of promoter activities were observed covering a range of protein expression levels peaking at various times throughout growth, and subsequent directed mutations were demonstrated and suggested to further increase the degree of output tuning. We believe these data show the potential for <i>L. plantarum</i> WCFS1 to be used as a nontraditional synthetic biology chassis and provide evidence that our system can be transitioned to other probiotic <i>Lactobacillus</i> species as well.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"4 1","pages":"ysz012"},"PeriodicalIF":0.0,"publicationDate":"2019-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/synbio/ysz012","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38439144","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}