Synthetic biology (Oxford, England)最新文献

{"title":"pBLAM1-x: standardized transposon tools for high-throughput screening.","authors":"Lorea Alejaldre, Ana-Mariya Anhel, Ángel Goñi-Moreno","doi":"10.1093/synbio/ysad012","DOIUrl":"10.1093/synbio/ysad012","url":null,"abstract":"<p><p>The engineering of pre-defined functions in living cells requires increasingly accurate tools as synthetic biology efforts become more ambitious. Moreover, the characterization of the phenotypic performance of genetic constructs demands meticulous measurements and extensive data acquisition for the sake of feeding mathematical models and matching predictions along the design-build-test lifecycle. Here, we developed a genetic tool that eases high-throughput transposon insertion sequencing (TnSeq): the pBLAM1-x plasmid vectors carrying the Himar1 Mariner transposase system. These plasmids were derived from the mini-Tn5 transposon vector pBAMD1-2 and built following modular criteria of the Standard European Vector Architecture (SEVA) format. To showcase their function, we analyzed sequencing results of 60 clones of the soil bacterium <i>Pseudomonas putida</i> KT2440. The new pBLAM1-x tool has already been included in the latest SEVA database release, and here we describe its performance using laboratory automation workflows. <b>Graphical Abstract</b>.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"8 1","pages":"ysad012"},"PeriodicalIF":0.0,"publicationDate":"2023-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10306358/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9730140","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":"Biosynthesis of cannabigerol and cannabigerolic acid: the gateways to further cannabinoid production.","authors":"Lewis J Kearsey, Cunyu Yan, Nicole Prandi, Helen S Toogood, Eriko Takano, Nigel S Scrutton","doi":"10.1093/synbio/ysad010","DOIUrl":"10.1093/synbio/ysad010","url":null,"abstract":"<p><p>Cannabinoids are a therapeutically valuable class of secondary metabolites with a vast number of substituents. The native cannabinoid biosynthetic pathway of <i>Cannabis sativa</i> generates cannabigerolic acid (CBGA), the common substrate to multiple cannabinoid synthases. The bioactive decarboxylated analog of this compound, cannabigerol (CBG), represents an alternate gateway into the cannabinoid space as a substrate either to non-canonical cannabinoid synthase homologs or to synthetic chemical reactions. Herein, we describe the identification and repurposing of aromatic prenyltransferase (AtaPT), which when coupled with native enzymes of <i>C. sativa</i> can form an <i>Escherichia coli</i> production system for CBGA in cell lysates and CBG in whole cells. Engineering of AtaPT, guided by structural analysis, was performed to enhance its kinetics toward CBGA production for subsequent use in a proof-of-concept lysate system. For the first time, we show a synthetic biology platform for CBG biosynthesis in <i>E. coli</i> cells by employing AtaPT under an optimized microbial system. Our results have therefore set the foundation for sustainable production of well-researched and rarer cannabinoids in an <i>E. coli</i> chassis. <b>Graphical Abstract</b>.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"8 1","pages":"ysad010"},"PeriodicalIF":0.0,"publicationDate":"2023-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10263468/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9655827","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":"Rewiring cell-free metabolic flux in <i>E. coli</i> lysates using a block-push-pull approach.","authors":"Jaime Lorenzo N Dinglasan,&nbsp;Mitchel J Doktycz","doi":"10.1093/synbio/ysad007","DOIUrl":"https://doi.org/10.1093/synbio/ysad007","url":null,"abstract":"<p><p>Cell-free systems can expedite the design and implementation of biomanufacturing processes by bypassing troublesome requirements associated with the use of live cells. In particular, the lack of survival objectives and the open nature of cell-free reactions afford engineering approaches that allow purposeful direction of metabolic flux. The use of lysate-based systems to produce desired small molecules can result in competitive titers and productivities when compared to their cell-based counterparts. However, pathway crosstalk within endogenous lysate metabolism can compromise conversion yields by diverting carbon flow away from desired products. Here, the 'block-push-pull' concept of conventional cell-based metabolic engineering was adapted to develop a cell-free approach that efficiently directs carbon flow in lysates from glucose and toward endogenous ethanol synthesis. The approach is readily adaptable, is relatively rapid and allows for the manipulation of central metabolism in cell extracts. In implementing this approach, a block strategy is first optimized, enabling selective enzyme removal from the lysate to the point of eliminating by-product-forming activity while channeling flux through the target pathway. This is complemented with cell-free metabolic engineering methods that manipulate the lysate proteome and reaction environment to push through bottlenecks and pull flux toward ethanol. The approach incorporating these block, push and pull strategies maximized the glucose-to-ethanol conversion in an <i>Escherichia coli</i> lysate that initially had low ethanologenic potential. A 10-fold improvement in the percent yield is demonstrated. To our knowledge, this is the first report of successfully rewiring lysate carbon flux without source strain optimization and completely transforming the consumed input substrate to a desired output product in a lysate-based, cell-free system.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"8 1","pages":"ysad007"},"PeriodicalIF":0.0,"publicationDate":"2023-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10615139/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"71430011","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":"Functional Synthetic Biology.","authors":"Ibrahim Aldulijan, Jacob Beal, Sonja Billerbeck, Jeff Bouffard, Gaël Chambonnier, Nikolaos Ntelkis, Isaac Guerreiro, Martin Holub, Paul Ross, Vinoo Selvarajah, Noah Sprent, Gonzalo Vidal, Alejandro Vignoni","doi":"10.1093/synbio/ysad006","DOIUrl":"10.1093/synbio/ysad006","url":null,"abstract":"<p><p>Synthetic biologists have made great progress over the past decade in developing methods for modular assembly of genetic sequences and in engineering biological systems with a wide variety of functions in various contexts and organisms. However, current paradigms in the field entangle sequence and functionality in a manner that makes abstraction difficult, reduces engineering flexibility and impairs predictability and design reuse. Functional Synthetic Biology aims to overcome these impediments by focusing the design of biological systems on function, rather than on sequence. This reorientation will decouple the engineering of biological devices from the specifics of how those devices are put to use, requiring both conceptual and organizational change, as well as supporting software tooling. Realizing this vision of Functional Synthetic Biology will allow more flexibility in how devices are used, more opportunity for reuse of devices and data, improvements in predictability and reductions in technical risk and cost.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"8 1","pages":"ysad006"},"PeriodicalIF":2.6,"publicationDate":"2023-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10105873/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9736864","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":"Construction of a mini-RNA replicon in <i>Escherichia coli</i>.","authors":"Akiko Kashiwagi,&nbsp;Tetsuya Yomo","doi":"10.1093/synbio/ysad004","DOIUrl":"https://doi.org/10.1093/synbio/ysad004","url":null,"abstract":"<p><p>How the ribonucleic acid (RNA) world transited to the deoxyribonucleic acid (DNA) world has remained controversial in evolutionary biology. At a certain time point in the transition from the RNA world to the DNA world, 'RNA replicons', in which RNAs produce proteins to replicate their coding RNA, and 'DNA replicons', in which DNAs produce RNA to synthesize proteins that replicate their coding DNA, can be assumed to coexist. The coexistent state of RNA replicons and DNA replicons is desired for experimental approaches to determine how the DNA world overtook the RNA world. We constructed a mini-RNA replicon in <i>Escherichia coli</i>. This mini-RNA replicon encoded the β subunit, one of the subunits of the Qβ replicase derived from the positive-sense single-stranded Qβ RNA phage and is replicated by the replicase in <i>E. coli</i>. To maintain the mini-RNA replicon persistently in <i>E. coli</i> cells, we employed a system of α complementation of LacZ that was dependent on the Qβ replicase, allowing the cells carrying the RNA replicon to grow in the lactose minimal medium selectively. The coexistent state of the mini-RNA replicon and DNA replicon (<i>E. coli</i> genome) was successively synthesized. The coexistent state can be used as a starting system to experimentally demonstrate the transition from the RNA-protein world to the DNA world, which will contribute to progress in the research field of the origin of life.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"8 1","pages":"ysad004"},"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/71/70/ysad004.PMC10013734.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9125769","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 biology regulation in Europe: containment, release and beyond.","authors":"Lalitha S Sundaram,&nbsp;James W Ajioka,&nbsp;Jennifer C Molloy","doi":"10.1093/synbio/ysad009","DOIUrl":"https://doi.org/10.1093/synbio/ysad009","url":null,"abstract":"<p><p>While synthetic biology is hoped to hold promise and potential to address pressing global challenges, the issue of regulation is an under-appreciated challenge. Particularly in Europe, the regulatory frameworks involved are rooted in historical concepts based on containment and release. Through a series of case studies including a field-use biosensor intended to detect arsenic in well water in Nepal and Bangladesh, and insects engineered for sterility, we explore the implications that this regulatory and conceptual divide has had on the deployment of synthetic biology projects in different national contexts. We then consider some of the broader impacts that regulation can have on the development of synthetic biology as a field, not only in Europe but also globally, with a particular emphasis on low- and middle-income countries. We propose that future regulatory adaptability would be increased by moving away from a containment and release dichotomy and toward a more comprehensive assessment that accounts for the possibility of varying degrees of 'contained release'. <b>Graphical Abstract</b>.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"8 1","pages":"ysad009"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10173542/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9523184","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 artificial protein translation language makes bacteria resistant to viruses.","authors":"David M Truong","doi":"10.1093/synbio/ysad011","DOIUrl":"https://doi.org/10.1093/synbio/ysad011","url":null,"abstract":"© The Author(s) 2023. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. American statesman Benjamin Franklin once stated, ‘... in this world nothing can be said to be certain, except death and taxes’. Had he been a microbiologist, he would have known about another near certainty: that viruses and the organisms they infect, will continue waging cycles of genetic conflict where the host builds immunity against a virus and the virus finds a way around it. But what if we had a way to break that cycle? In a recent paper by Nyerges et al. from George Church’s lab at Harvard Medical School in the USA, the authors engineered the bacterium Escherichia coli to be resistant to bacteriophages (bacterial viruses) by using the phage’s reliance on host machinery against them—viruses rely almost entirely on their host’s protein machinery to reproduce, as they cannot replicate on their own (1). To achieve this, they generated a new artificial genetic ‘language’ by which messenger RNAs (mRNAs) are read and translated into proteins. Importantly, the E. coli host neither needs nor uses this artificial language to make proteins, but crucially, the bacteriophage does. The system might not only prohibit phage infections but also enable ‘biocontainment’ of the engineered bacteria to prevent escape into the wild. The genetic code transcribed into mRNAs, which code for proteins, is divided into a three-nucleotide language called codons, which is universal to life as well as viruses. Codons are interpreted by molecules called transfer RNAs (tRNAs), which are bound to specific amino acids. There is redundancy in this language, as there are only 20 amino acids, but 64 possible codons. Because some amino acids have multiple codon/tRNA options, the field of synthetic genomics has long sought to repurpose excess codons for new designer functions, such as inputting non-natural amino acids into proteins (2). Synthetic genomics practitioners can generate organisms with new designer genomes using advanced DNA synthesis and genetic engineering, and it was a long-standing question whether phage infections could be prevented by recoding entire genomes. Indeed, prior work by the laboratory of Jason Chin at the Medical Research Council Laboratory of Molecular Biology in Cambridge, UK, fully recoded the E. coli genome to make the strain Syn61Δ3, in which they genome-wide repurposed two codons specifying serine and one stop sequence (3). In doing so, invading viruses now improperly make proteins, and the same team later concluded that Syn61Δ3 was resistant to viruses (4). The current study by Nyerges et al. found instead that Syn61Δ3 was only resistant to some bacteriophages but was susceptible to many other types. It turns out ","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"8 1","pages":"ysad011"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10256184/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9976933","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":"AssemblyTron: flexible automation of DNA assembly with Opentrons OT-2 lab robots.","authors":"John A Bryant,&nbsp;Mason Kellinger,&nbsp;Cameron Longmire,&nbsp;Ryan Miller,&nbsp;R Clay Wright","doi":"10.1093/synbio/ysac032","DOIUrl":"https://doi.org/10.1093/synbio/ysac032","url":null,"abstract":"<p><p>As one of the newest fields of engineering, synthetic biology relies upon a trial-and-error Design-Build-Test-Learn (DBTL) approach to simultaneously learn how a function is encoded in biology and attempt to engineer it. Many software and hardware platforms have been developed to automate, optimize and algorithmically perform each step of the DBTL cycle. However, there are many fewer options for automating the build step. Build typically involves deoxyribonucleic acid (DNA) assembly, which remains manual, low throughput and unreliable in most cases and limits our ability to advance the science and engineering of biology. Here, we present AssemblyTron, an open-source Python package to integrate j5 DNA assembly design software outputs with build implementation in Opentrons liquid handling robotics with minimal human intervention. We demonstrate the versatility of AssemblyTron through several scarless, multipart DNA assemblies, beginning from fragment amplification. We show that AssemblyTron can perform polymerase chain reactions across a range of fragment lengths and annealing temperatures by using an optimal annealing temperature gradient calculation algorithm. We then demonstrate that AssemblyTron can perform Golden Gate and homology-dependent <i>in vivo</i> assemblies (IVAs) with comparable fidelity to manual assemblies by simultaneously building four four-fragment assemblies of chromoprotein reporter expression plasmids. Finally, we used AssemblyTron to perform site-directed mutagenesis reactions via homology-dependent IVA also achieving comparable fidelity to manual assemblies as assessed by sequencing. AssemblyTron can reduce the time, training, costs and wastes associated with synthetic biology, which, along with open-source and affordable automation, will further foster the accessibility of synthetic biology and accelerate biological research and engineering.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"8 1","pages":"ysac032"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9832943/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10536129","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":"Automated cell segmentation for reproducibility in bioimage analysis.","authors":"Michael C Robitaille,&nbsp;Jeff M Byers,&nbsp;Joseph A Christodoulides,&nbsp;Marc P Raphael","doi":"10.1093/synbio/ysad001","DOIUrl":"https://doi.org/10.1093/synbio/ysad001","url":null,"abstract":"<p><p>Live-cell imaging is extremely common in synthetic biology research, but its ability to be applied reproducibly across laboratories can be hindered by a lack of standardized image analysis. Here, we introduce a novel cell segmentation method developed as part of a broader Independent Verification & Validation (IV&V) program aimed at characterizing engineered <i>Dictyostelium</i> cells. Standardizing image analysis was found to be highly challenging: the amount of human judgment required for parameter optimization, algorithm tweaking, training and data pre-processing steps forms serious challenges for reproducibility. To bring automation and help remove bias from live-cell image analysis, we developed a self-supervised learning (SSL) method that recursively trains itself directly from motion in live-cell microscopy images without any end-user input, thus providing objective cell segmentation. Here, we highlight this SSL method applied to characterizing the engineered <i>Dictyostelium</i> cells of the original IV&V program. This approach is highly generalizable, accepting images from any cell type or optical modality without the need for manual training or parameter optimization. This method represents an important step toward automated bioimage analysis software and reflects broader efforts to design accessible measurement technologies to enhance reproducibility in synthetic biology research.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"8 1","pages":"ysad001"},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9933842/pdf/ysad001.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10774161","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":"Periplasmic stress contributes to a trade-off between protein secretion and cell growth in <i>Escherichia coli</i> Nissle 1917.","authors":"Sivaram Subaya Emani,&nbsp;Anton Kan,&nbsp;Timothy Storms,&nbsp;Shanna Bonanno,&nbsp;Jade Law,&nbsp;Sanhita Ray,&nbsp;Neel S Joshi","doi":"10.1093/synbio/ysad013","DOIUrl":"https://doi.org/10.1093/synbio/ysad013","url":null,"abstract":"<p><p>Maximizing protein secretion is an important target in the design of engineered living systems. In this paper, we characterize a trade-off between cell growth and per-cell protein secretion in the curli biofilm secretion system of <i>Escherichia coli</i> Nissle 1917. Initial characterization using 24-h continuous growth and protein production monitoring confirms decreased growth rates at high induction, leading to a local maximum in total protein production at intermediate induction. Propidium iodide (PI) staining at the endpoint indicates that cellular death is a dominant cause of growth reduction. Assaying variants with combinatorial constructs of inner and outer membrane secretion tags, we find that diminished growth at high production is specific to secretory variants associated with periplasmic stress mediated by outer membrane secretion and periplasmic accumulation of protein containing the outer membrane transport tag. RNA sequencing experiments indicate upregulation of known periplasmic stress response genes in the highly secreting variant, further implicating periplasmic stress in the growth-secretion trade-off. Overall, these results motivate additional strategies for optimizing total protein production and longevity of secretory engineered living systems <b>Graphical Abstract</b>.</p>","PeriodicalId":74902,"journal":{"name":"Synthetic biology (Oxford, England)","volume":"8 1","pages":"ysad013"},"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/c2/ae/ysad013.PMC10439730.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10199999","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}