Pub Date : 2024-08-27DOI: 10.1101/2024.08.27.609963
Olivia Young, Hawa Dembele, Anjali Rajwar, Ick Chan Kwon, Ju Hee Ryu, William Shih, Yang Zeng
In recent years, notable advances in nanotechnology-based drug delivery have emerged. A particularly promising platform in this field is DNA origami-based nanoparticles, which offer highly programmable surfaces, providing precise control over the nanoscale spacing and stoichiometry of various cargo. These versatile particles are finding diverse applications ranging from basic molecular biology to diagnostics and therapeutics. This growing interest creates the need for effective methods to quantify cargo on DNA origami nanoparticles. Our study consolidates several previously validated methods focusing on gel-based and fluorescence-based techniques, including multiplexed quantification of protein, peptide, and nucleic acid cargo on these nanoparticles. This work may serve as a valuable resource for groups researchers keen on utilizing DNA origami-based nanoparticles in therapeutic applications.
近年来，基于纳米技术的药物输送技术取得了显著进展。DNA 折纸纳米粒子是这一领域中一个特别有前途的平台，它具有高度可编程的表面，可精确控制各种货物的纳米级间距和化学计量。这些用途广泛的微粒正被广泛应用于从基础分子生物学到诊断和治疗的各个领域。这种日益增长的兴趣需要有效的方法来量化 DNA 折纸纳米粒子上的货物。我们的研究整合了之前几种经过验证的方法，重点是基于凝胶和荧光的技术，包括对这些纳米颗粒上的蛋白质、肽和核酸货物进行多重定量。这项工作可为热衷于利用基于 DNA 折纸的纳米粒子进行治疗的研究小组提供宝贵的资源。
{"title":"Cargo quantification of functionalized DNA origami for therapeutic application","authors":"Olivia Young, Hawa Dembele, Anjali Rajwar, Ick Chan Kwon, Ju Hee Ryu, William Shih, Yang Zeng","doi":"10.1101/2024.08.27.609963","DOIUrl":"https://doi.org/10.1101/2024.08.27.609963","url":null,"abstract":"In recent years, notable advances in nanotechnology-based drug delivery have emerged. A particularly promising platform in this field is DNA origami-based nanoparticles, which offer highly programmable surfaces, providing precise control over the nanoscale spacing and stoichiometry of various cargo. These versatile particles are finding diverse applications ranging from basic molecular biology to diagnostics and therapeutics. This growing interest creates the need for effective methods to quantify cargo on DNA origami nanoparticles. Our study consolidates several previously validated methods focusing on gel-based and fluorescence-based techniques, including multiplexed quantification of protein, peptide, and nucleic acid cargo on these nanoparticles. This work may serve as a valuable resource for groups researchers keen on utilizing DNA origami-based nanoparticles in therapeutic applications.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"62 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206781","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}
Pub Date : 2024-08-23DOI: 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
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.
{"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}
Pub Date : 2024-08-22DOI: 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
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.
{"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}
Pub Date : 2024-08-22DOI: 10.1101/2024.08.22.609155
Armin M. Zand, Stanislav Anastassov, Timothy Frei, Mustafa Khammash
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.
{"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}
Pub Date : 2024-08-21DOI: 10.1101/2024.08.21.608917
Ellen Parkes, Assala Al Samad, Giacomo Mazzotti, Charlie Newell, Brian Ng, Amy Radford, Michael J Booth
The advancement of synthetic cells as drug delivery devices hinges on the development of targeting strategies, in particular the controlled synthesis of biomolecules in-situ using a deeply penetrative stimulus. To address this, we have designed spherical nucleic acids comprising DNA promoter sequences decorating magnetic nanoparticle cores. By harnessing the heat dissipated from magnetic hyperthermia (a clinically-approved anticancer therapy) we tightly controlled cell-free protein synthesis. We then deployed a tissue phantom that is impenetrable by current activation methods to demonstrate the potential of this technology for the remote control of synthetic cells using deeply tissue-penetrating magnetic fields. This paves the way for targeting and controlling the in-situ synthesis of biomolecules deep within the body.
合成细胞作为药物输送设备的发展取决于靶向策略的开发，特别是利用深度穿透性刺激在原位控制合成生物分子。为此，我们设计了由 DNA 启动子序列组成的球形核酸，并以磁性纳米粒子为核心进行装饰。通过利用磁热效应（一种临床批准的抗癌疗法）产生的热量，我们严格控制了无细胞蛋白质合成。然后，我们部署了一个目前的活化方法无法穿透的组织模型，展示了这项技术利用深度穿透组织的磁场远程控制合成细胞的潜力。这为瞄准和控制体内深层生物分子的原位合成铺平了道路。
{"title":"Magnetic Activation of Spherical Nucleic Acids for the Remote Control of Synthetic Cells","authors":"Ellen Parkes, Assala Al Samad, Giacomo Mazzotti, Charlie Newell, Brian Ng, Amy Radford, Michael J Booth","doi":"10.1101/2024.08.21.608917","DOIUrl":"https://doi.org/10.1101/2024.08.21.608917","url":null,"abstract":"The advancement of synthetic cells as drug delivery devices hinges on the development of targeting strategies, in particular the controlled synthesis of biomolecules in-situ using a deeply penetrative stimulus. To address this, we have designed spherical nucleic acids comprising DNA promoter sequences decorating magnetic nanoparticle cores. By harnessing the heat dissipated from magnetic hyperthermia (a clinically-approved anticancer therapy) we tightly controlled cell-free protein synthesis. We then deployed a tissue phantom that is impenetrable by current activation methods to demonstrate the potential of this technology for the remote control of synthetic cells using deeply tissue-penetrating magnetic fields. This paves the way for targeting and controlling the in-situ synthesis of biomolecules deep within the body.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"11 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142226315","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}
Pub Date : 2024-08-21DOI: 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
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.
{"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}
Pub Date : 2024-08-19DOI: 10.1101/2024.08.19.608593
Alejandro Marquiegui Alvaro, Anastasia Kottara, MICAELA CHACON, Michael Brockhurst, Neil Dixon
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.
{"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}
Pub Date : 2024-08-13DOI: 10.1101/2024.08.13.607171
Martyna Kasprzyk, Michael A Herrera, Giovanni Stracquadanio
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 growth, 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 significant resource and time consumption; these protocols are also susceptible to operator error and inconsistency. To 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 protein 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 customization, modularity and user-friendliness in mind, APEX can be easily adapted to the operator's needs without requiring any coding expertise. APEX is available at https://github.com/stracquadaniolab/apex-nf under the AGPL3 license.
{"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}
Pub Date : 2024-08-12DOI: 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
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.
{"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}
Pub Date : 2024-08-07DOI: 10.1101/2024.08.06.606923
Juan P. Molina Ortiz, Matthew J. Morgan, Amy M. Paten, Andrew C. Warden, Philip Kilby
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 Pyrococcus furiosus 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
{"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}
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