bioRxiv - Synthetic Biology最新文献_第5页

Cargo quantification of functionalized DNA origami for therapeutic application 用于治疗的功能化 DNA 折纸的货物定量

bioRxiv - Synthetic Biology Pub Date : 2024-08-27 DOI: 10.1101/2024.08.27.609963

Olivia Young, Hawa Dembele, Anjali Rajwar, Ick Chan Kwon, Ju Hee Ryu, William Shih, Yang Zeng

引用次数: 0

Expanding the biotechnological scope of metabolic sensors through computation-aided designs 通过计算辅助设计扩大代谢传感器的生物技术范围

bioRxiv - Synthetic Biology Pub Date : 2024-08-23 DOI: 10.1101/2024.08.23.609350

Enrico Orsi, Helena Schulz-Mirbach, Charles A.R. Cotton, Ari Satanowski, Henrik Petri, Susanne L. Arnold, Natalia Grabarczyk, Rutger Verbakel, Karsten S. Jensen, Stefano Donati, Nicole Paczia, Timo Glatter, Andreas Markus Kueffner, Tanguy Chotel, Farah Schillmueller, Alberto De Maria, Hai He, Steffen N. Lindner, Elad Noor, Arren Bar-Even, Tobias J. Erb, Pablo Ivan Nikel

{"title":"Expanding the biotechnological scope of metabolic sensors through computation-aided designs","authors":"Enrico Orsi, Helena Schulz-Mirbach, Charles A.R. Cotton, Ari Satanowski, Henrik Petri, Susanne L. Arnold, Natalia Grabarczyk, Rutger Verbakel, Karsten S. Jensen, Stefano Donati, Nicole Paczia, Timo Glatter, Andreas Markus Kueffner, Tanguy Chotel, Farah Schillmueller, Alberto De Maria, Hai He, Steffen N. Lindner, Elad Noor, Arren Bar-Even, Tobias J. Erb, Pablo Ivan Nikel","doi":"10.1101/2024.08.23.609350","DOIUrl":"https://doi.org/10.1101/2024.08.23.609350","url":null,"abstract":"Metabolic sensors are microbial strains modified so that biomass formation correlates with the availability of specific metabolites. These sensors are essential for bioengineering (e.g. in growth-coupled designs) but creating them is often a time-consuming and low-throughput process that can potentially be streamlined by in silico analysis. Here, we present the systematic workflow of designing, implementing, and testing versatile Escherichia coli metabolic sensor strains. Glyoxylate, a key metabolite in (synthetic) CO2 fixation and carbon-conserving pathways, served as the test molecule. Through iterative screening of a compact metabolic model, we identified non-trivial growth-coupled designs that resulted in six metabolic sensors with a wide sensitivity range for glyoxylate, spanning three orders of magnitude in detected concentrations. We further adapted these E. coli strains for sensing glycolate and demonstrated their utility in both pathway engineering (testing a key metabolic module via glyoxylate) and applications in environmental monitoring (quantifying glycolate produced by photosynthetic microalgae). The versatility and ease of implementation of this workflow make it suitable for designing and building multiple metabolic sensors for diverse biotechnological applications.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"51 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206782","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Confinement and Catalysis Within De Novo Designed Peptide Barrels 新设计肽桶内的封闭和催化作用

bioRxiv - Synthetic Biology Pub Date : 2024-08-22 DOI: 10.1101/2024.08.22.609140

Rokas Petrenas, Olivia A. Hawkins, Jacob F. Jones, D. Arne Scott, Jordan M. Fletcher, Ulrike Obst, Lucia Lombardi, Fabio Pirro, Graham J. Leggett, Thomas A. A. Oliver, Derek N. Woolfson

{"title":"Confinement and Catalysis Within De Novo Designed Peptide Barrels","authors":"Rokas Petrenas, Olivia A. Hawkins, Jacob F. Jones, D. Arne Scott, Jordan M. Fletcher, Ulrike Obst, Lucia Lombardi, Fabio Pirro, Graham J. Leggett, Thomas A. A. Oliver, Derek N. Woolfson","doi":"10.1101/2024.08.22.609140","DOIUrl":"https://doi.org/10.1101/2024.08.22.609140","url":null,"abstract":"De novo protein design has advanced such that many peptide assemblies and protein structures can be generated predictably and quickly. The drive now is to bring functions to these structures, for example, small-molecule binding and catalysis. The formidable challenge of binding and orienting multiple small molecules to direct chemistry is particularly important for paving the way to new functionalities. To address this, here we describe the design, characterization, and application of small-molecule:peptide ternary complexes in aqueous solution. This uses alpha-helical barrel (alphaHB) peptide assemblies, which comprise 5 or more alpha-helices arranged around central channels. These channels are solvent accessible, and their internal dimensions and chemistries can be altered predictably. Thus, alphaHBs are analogous to Prime molecular flasks made in supramolecular, polymer, and materials chemistry. Using Forster resonance energy transfer as a readout, we demonstrate that specific alphaHBs can accept two different organic dyes, 1,6-diphenyl-1,3,5-hexatriene and Nile Red in close proximity. In addition, two anthracene molecules can be accommodated within an alphaHB to promote photocatalytic anthracene-dimer formation. However, not all ternary complexes are productive, either in energy transfer or photocatalysis, illustrating the control that can be exerted by judicious choice and design of the alphaHB.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"16 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206787","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Multi-Layer Autocatalytic Feedback Enables Integral Control Amidst Resource Competition and Across Scales 多层自催化反馈可在资源竞争中实现跨尺度综合控制

bioRxiv - Synthetic Biology Pub Date : 2024-08-22 DOI: 10.1101/2024.08.22.609155

Armin M. Zand, Stanislav Anastassov, Timothy Frei, Mustafa Khammash

{"title":"Multi-Layer Autocatalytic Feedback Enables Integral Control Amidst Resource Competition and Across Scales","authors":"Armin M. Zand, Stanislav Anastassov, Timothy Frei, Mustafa Khammash","doi":"10.1101/2024.08.22.609155","DOIUrl":"https://doi.org/10.1101/2024.08.22.609155","url":null,"abstract":"Integral feedback control strategies have proven effective in regulating protein expression in unpredictable cellular environments. These strategies, grounded in model-based designs and control theory, have advanced synthetic biology applications. Autocatalytic integral feedback controllers, utilizing positive autoregulation for integral action, are particularly promising due to their similarity to natural behaviors like self-replication and positive feedback seen across biological scales. However, their effectiveness is often hindered by resource competition and context-dependent couplings. This study addresses these challenges with a multi-layer feedback strategy, enabling population-level integral feedback and multicellular integrators. We provide a generalized mathematical framework for modeling resource competition in complex genetic networks, supporting the design of intracellular control circuits. Our controller motif demonstrated precise regulation in tasks ranging from gene expression control to population growth in multi-strain communities. We also explore a variant capable of ratiometric control, proving its effectiveness in managing gene ratios and co-culture compositions in engineered microbial ecosystems. These findings offer a versatile approach to achieving robust adaptation and homeostasis from subcellular to multicellular scales.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"22 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206789","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Magnetic Activation of Spherical Nucleic Acids for the Remote Control of Synthetic Cells 磁激活球形核酸以远程控制合成细胞

bioRxiv - Synthetic Biology Pub Date : 2024-08-21 DOI: 10.1101/2024.08.21.608917

Ellen Parkes, Assala Al Samad, Giacomo Mazzotti, Charlie Newell, Brian Ng, Amy Radford, Michael J Booth

引用次数: 0

Covalently linked adenovirus-AAV complexes as a novel platform technology for gene therapy 共价连接的腺病毒-AAV 复合物作为基因治疗的新型平台技术

bioRxiv - Synthetic Biology Pub Date : 2024-08-21 DOI: 10.1101/2024.08.21.609008

Logan Thrasher Collins, Wandy Beatty, Buhle Moyo, Michele Alves-Bezerra, Ayrea Hurley, William Lagor, Gang Bao, Zhi Hong Lu, David T Curiel

{"title":"Covalently linked adenovirus-AAV complexes as a novel platform technology for gene therapy","authors":"Logan Thrasher Collins, Wandy Beatty, Buhle Moyo, Michele Alves-Bezerra, Ayrea Hurley, William Lagor, Gang Bao, Zhi Hong Lu, David T Curiel","doi":"10.1101/2024.08.21.609008","DOIUrl":"https://doi.org/10.1101/2024.08.21.609008","url":null,"abstract":"Adeno-associated virus (AAV) has found immense success as a delivery system for gene therapy, yet the small 4.7 kb packaging capacity of the AAV sharply limits the scope of its application. In addition, high doses of AAV are frequently required to facilitate therapeutic effects, leading to acute toxicity issues. While dual and triple AAV approaches have been developed to mitigate the packaging capacity problem, these necessitate even higher doses to ensure that co-infection occurs at sufficient frequency. To address these challenges, we herein describe a novel delivery system consisting of adenovirus (Ad) covalently linked to multiple adeno-associated virus (AAV) capsids as a new way of more efficiently co-infecting cells with lower overall amounts of AAVs. We utilize the DogTag-DogCatcher (DgT-DgC) molecular glue system to construct our AdAAVs and we demonstrate that these hybrid virus complexes achieve enhanced co-transduction of cultured cells. This technology may eventually broaden the utility of AAV gene delivery by providing an alternative to dual or triple AAV which can be employed at lower dose while reaching higher co-transduction efficiency.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"13 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206784","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Genetic bioaugmentation-mediated bioremediation of terephthalate in soil microcosms using an engineered environmental plasmid 利用工程化环境质粒在土壤微生态系统中对对苯二甲酸盐进行基因生物增强介导的生物修复

bioRxiv - Synthetic Biology Pub Date : 2024-08-19 DOI: 10.1101/2024.08.19.608593

Alejandro Marquiegui Alvaro, Anastasia Kottara, MICAELA CHACON, Michael Brockhurst, Neil Dixon

{"title":"Genetic bioaugmentation-mediated bioremediation of terephthalate in soil microcosms using an engineered environmental plasmid","authors":"Alejandro Marquiegui Alvaro, Anastasia Kottara, MICAELA CHACON, Michael Brockhurst, Neil Dixon","doi":"10.1101/2024.08.19.608593","DOIUrl":"https://doi.org/10.1101/2024.08.19.608593","url":null,"abstract":"Harnessing in situ microbial communities to clean-up polluted natural environments is a potentially efficient means of bioremediation, but often the necessary genes to breakdown pollutants are missing. Genetic bioaugmentation, whereby the required genes are delivered to resident bacteria via horizonal gene transfer, offers a promising solution to this problem. Here we engineered a conjugative plasmid previously isolated from soil, pQBR57, to carry a synthetic set of genes allowing bacteria to consume terephthalate, a chemical component of plastics commonly released during their manufacture and breakdown. Our engineered plasmid caused a low fitness cost and was stably maintained in terephthalate contaminated soil by the bacterium P. putida. Plasmid carriers efficiently bioremediated contaminated soil, achieving complete breakdown of 3.2 mg/g of terephthalate within 8 days. The engineered plasmid horizontally transferred the synthetic operon to P. fluorescens in situ, and the resulting transconjugants degraded 10 mM terephthalate during a 180-hour incubation. Our findings show that environmental plasmids carrying synthetic catabolic operons can be useful tools for in situ engineering of microbial communities to perform clean-up even of complex environments like soil.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"25 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206783","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

APEX: Automated Protein EXpression in Escherichia coli APEX：在大肠杆菌中自动表达蛋白质

bioRxiv - Synthetic Biology Pub Date : 2024-08-13 DOI: 10.1101/2024.08.13.607171

Martyna Kasprzyk, Michael A Herrera, Giovanni Stracquadanio

{"title":"APEX: Automated Protein EXpression in Escherichia coli","authors":"Martyna Kasprzyk, Michael A Herrera, Giovanni Stracquadanio","doi":"10.1101/2024.08.13.607171","DOIUrl":"https://doi.org/10.1101/2024.08.13.607171","url":null,"abstract":"Heterologous protein expression is an indispensable strategy to generate significant amounts of recombinant proteins. To this end, Escherichia coli is one the most used microbial host for recombinant protein production due to its rapid\u0000growth, well-characterised genetics, and ability to produce recombinant proteins in high yields using modern recombinant DNA technology. However, while there is a plethora of robust protein expression protocols for E.coli, these methods are often unsuitable for high-throughput screening due to their\u0000significant resource and time consumption; these protocols are also susceptible to operator error and inconsistency.\u0000To address these challenges, we have developed APEX, a robust and automated protocol for recombinant protein production in E. coli. APEX leverages the accessible, open-source Opentrons OT-2 platform to automate microbial handling and protein expression with high precision and reproducibility. APEX can be configured to perform heat shock transformation, colony selection, colony sampling, inoculation, subculturing and\u0000protein expression using a low-cost, minimal OT-2 hardware setup. We further demonstrate the efficacy of our automated transformation workflows using a variety of plasmids (2.7-17.7 kb), and exemplify the automated hetrologous expression of a diverse array of proteins (27-222 kDa). Designed with\u0000customization, modularity and user-friendliness in mind, APEX can be easily adapted to the operator's needs without requiring any coding expertise.\u0000APEX is available at https://github.com/stracquadaniolab/apex-nf under the AGPL3 license.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"10 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206786","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Discovery of a single-subunit oligosaccharyltransferase that enables glycosylation of full-length IgG antibodies in Escherichia coli 发现一种单亚基寡糖基转移酶，它能使大肠杆菌中的全长 IgG 抗体糖基化

bioRxiv - Synthetic Biology Pub Date : 2024-08-12 DOI: 10.1101/2024.08.12.607630

Belen Sotomayor, Thomas Connor Donahue, Sai Pooja Mahajan, May N Taw, Sophia W Hulbert, Erik J Bidstrup, D. Natasha Owitipana, Alexandra Pang, Xu Yang, Souvik Ghosal, Christopher A Alabi, Parastoo Azadi, Jeffrey J. Gray, Michael C Jewett, Lai-Xi Wang, Matthew P DeLisa

{"title":"Discovery of a single-subunit oligosaccharyltransferase that enables glycosylation of full-length IgG antibodies in Escherichia coli","authors":"Belen Sotomayor, Thomas Connor Donahue, Sai Pooja Mahajan, May N Taw, Sophia W Hulbert, Erik J Bidstrup, D. Natasha Owitipana, Alexandra Pang, Xu Yang, Souvik Ghosal, Christopher A Alabi, Parastoo Azadi, Jeffrey J. Gray, Michael C Jewett, Lai-Xi Wang, Matthew P DeLisa","doi":"10.1101/2024.08.12.607630","DOIUrl":"https://doi.org/10.1101/2024.08.12.607630","url":null,"abstract":"Human immunoglobulin G (IgG) antibodies are one of the most important classes of biotherapeutic agents and undergo glycosylation at the conserved N297 site in the CH2 domain, which is critical for IgG Fc effector functions and anti-inflammatory activity. Hence, technologies for producing authentically glycosylated IgGs are in high demand. While attempts to engineer Escherichia coli for this purpose have been described, they have met limited success due in part to the lack of available oligosaccharyltransferase (OST) enzymes that can install N-linked glycans within the QYNST sequon of the IgG CH2 domain. Here, we identified a previously uncharacterized single-subunit OST (ssOST) from the bacterium Desulfovibrio marinus that exhibited greatly relaxed substrate specificity and, as a result, was able to catalyze glycosylation of native CH2 domains in the context of both a hinge-Fc fragment and a full-length IgG. Although the attached glycans were bacterial in origin, conversion to a homogeneous, asialo complex-type G2 N-glycan at the QYNST sequon of the E. coli-derived hinge-Fc was achieved via chemoenzymatic glycan remodeling. Importantly, the resulting G2-hinge-Fc exhibited strong binding to human FcγRIIIa (CD16a), one of the most potent receptors for eliciting antibody-dependent cellular cytotoxicity (ADCC). Taken together, the discovery of DmPglB provides previously unavailable biocatalytic capabilities to the bacterial glycoprotein engineering toolbox and opens the door to using E. coli for the production and glycoengineering of human IgGs and fragments derived thereof.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"9 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206785","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

MMINT: a Metabolic Model Interactive Network Tool for the exploration and comparative visualisation of metabolic networks MMINT：用于探索和比较可视化代谢网络的代谢模型互动网络工具

bioRxiv - Synthetic Biology Pub Date : 2024-08-07 DOI: 10.1101/2024.08.06.606923

Juan P. Molina Ortiz, Matthew J. Morgan, Amy M. Paten, Andrew C. Warden, Philip Kilby

{"title":"MMINT: a Metabolic Model Interactive Network Tool for the exploration and comparative visualisation of metabolic networks","authors":"Juan P. Molina Ortiz, Matthew J. Morgan, Amy M. Paten, Andrew C. Warden, Philip Kilby","doi":"10.1101/2024.08.06.606923","DOIUrl":"https://doi.org/10.1101/2024.08.06.606923","url":null,"abstract":"Genome-scale metabolic models (GEMs) are essential tools in systems and synthetic biology, enabling the mathematical simulation of metabolic pathways encoded in genomes to predict phenotypes. The complexity of GEMs, however, can often limit the interpretation and comparison of their outputs. Here, we present MMINT (Metabolic Modelling Interactive Network Tool), designed to facilitate the exploration and comparison of metabolic networks. MMINT employs GEM networks and flux solutions derived from Constraint Based Analysis (e.g. Flux Balance Analysis) to create interactive visualizations. This tool allows for seamless toggling of source and target metabolites, network decluttering, enabling exploration and comparison of flux solutions by highlighting similarities and differences between metabolic states, which enhances the identification of mechanistic drivers of phenotypes. We demonstrate MMINT’s capabilities using the <em>Pyrococcus furiosus</em> GEM, showcasing its application in distinguishing the metabolic drivers of acetate- and ethanol-producing phenotypes. By providing an intuitive and responsive model-exploration experience, MMINT addresses the need for a tool that simplifies the interpretation of GEM outputs and supports the discovery of novel metabolic engineering strategies. MMINT is available at https://doi.org/10.6084/m9.figshare.26409328","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"30 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141969770","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0