bioRxiv - Synthetic Biology最新文献_第10页

Energy Aware Technology Mapping of Genetic Logic Circuits 遗传逻辑电路的能量感知技术映射

bioRxiv - Synthetic Biology Pub Date : 2024-06-27 DOI: 10.1101/2024.06.27.601038

Erik Kubaczka, Maximilian Gehri, Jérémie J. M. Marlhens, Tobias Schwarz, Maik Molderings, Nicolai Engelmann, Christian Hochberger, Heinz Koeppl

{"title":"Energy Aware Technology Mapping of Genetic Logic Circuits","authors":"Erik Kubaczka, Maximilian Gehri, Jérémie J. M. Marlhens, Tobias Schwarz, Maik Molderings, Nicolai Engelmann, Christian Hochberger, Heinz Koeppl","doi":"10.1101/2024.06.27.601038","DOIUrl":"https://doi.org/10.1101/2024.06.27.601038","url":null,"abstract":"Energy and its dissipation are fundamental to all living systems, including cells. Insufficient abundance of energy carriers -as caused by the additional burden of artificial genetic circuits- shifts a cell's priority to survival, also harming the functionality of the genetic circuit. Moreover, recent works have shown the importance of energy expenditure in information transmission. Despite living organisms being non-equilibrium systems, non-equilibrium models capable of accounting for energy dissipation and non-equilibrium response curves are not yet employed in genetic design automation (GDA) software. To this end, we introduce Energy Aware Technology Mapping, the automated design of genetic logic circuits with respect to energy efficiency and functionality. The basis for this is an energy aware non-equilibrium steady state (NESS) model of gene expression, capturing characteristics like energy dissipation -which we link to the entropy production rate- and transcriptional bursting, relevant to eukaryotes as well as prokaryotes. Our evaluation shows that a genetic logic circuit's functional performance and energy efficiency are disjoint optimization goals. For our benchmark, energy efficiency improves by 37.2% on average when comparing to functionally optimized variants. We discover a linear increase in energy expenditure and overall protein expression with the circuit size, where Energy Aware Technology Mapping allows for designing genetic logic circuits with the energy efficiency of circuits that are one to two gates smaller. Structural variants improve this further, while results show the Pareto dominance among structures of a single Boolean function. By incorporating energy demand into the design, Energy Aware Technology Mapping enables energy efficiency by design. This extends current GDA tools and complements approaches coping with burden <em>in vivo</em>.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"341 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141503904","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

Identification of novel broad host-range promoter sequences functional in diverse Pseudomonadota by a promoter-trap approach 通过启动子捕获方法识别在不同假单胞菌中发挥作用的新型宽宿主范围启动子序列

bioRxiv - Synthetic Biology Pub Date : 2024-06-27 DOI: 10.1101/2024.06.26.600856

Diego M. Roldan, Vanesa Amarelle

{"title":"Identification of novel broad host-range promoter sequences functional in diverse Pseudomonadota by a promoter-trap approach","authors":"Diego M. Roldan, Vanesa Amarelle","doi":"10.1101/2024.06.26.600856","DOIUrl":"https://doi.org/10.1101/2024.06.26.600856","url":null,"abstract":"Finding novel promoter sequences is a cornerstone of synthetic biology. To contribute to the expanding catalog of biological parts, we employed a promoter-trap approach to identify novel sequences within an Antarctic microbial community that act as broad host-range promoters functional in diverse Pseudomonadota. Using Pseudomonas putida KT2440 as host, we generated a library comprising approximately 2,000 clones resulting in the identification of thirteen functional promoter sequences, thereby expanding the genetic toolkit available for this chassis. Some of the discovered promoter sequences prove to be broad host-range as they drove gene expression not only in P. putida KT2440 but also in Escherichia coli DH5α, Cupriavidus taiwanensis R1T, Paraburkholderia phymatum STM 815T, Ensifer meliloti 1021, and an indigenous Antarctic bacterium, Pseudomonas sp. UYIF39. Our findings enrich the existing catalog of biological parts, offering a repertoire of broad host-range promoter sequences that exhibit functionality across diverse members of the phylum Pseudomonadota, proving Antarctic microbial community as a valuable resource for prospecting new biological parts for synthetic biology.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"23 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141503905","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

Tuning the performance of a TphR-based terephthalate biosensor with a design of experiments approach 用实验设计方法调整基于 TphR 的对苯二甲酸盐生物传感器的性能

bioRxiv - Synthetic Biology Pub Date : 2024-06-27 DOI: 10.1101/2024.06.26.600737

Guadalupe Alvarez Gonzalez, Micaela Chacon, Thomas Butterfield, Neil Dixon

{"title":"Tuning the performance of a TphR-based terephthalate biosensor with a design of experiments approach","authors":"Guadalupe Alvarez Gonzalez, Micaela Chacon, Thomas Butterfield, Neil Dixon","doi":"10.1101/2024.06.26.600737","DOIUrl":"https://doi.org/10.1101/2024.06.26.600737","url":null,"abstract":"Transcription factor-based biosensors are genetic tools that aim to predictability link the presence of a specific input stimuli to a tailored gene expression output. The performance characteristics of a biosensor fundamentally determines its potential applications. However, current methods to engineer and optimise tailored biosensor responses are highly nonintuitive, and struggle to investigate multidimensional sequence/design space efficiently. In this study we employ a design of experiments (DoE) approach to build a framework for efficiently engineering activator-based biosensors with tailored performances, and we apply the framework for the development of biosensors for the polyethylene terephthalate (PET) plastic degradation monomer terephthalate (TPA). We simultaneously engineer the core promoter and operator regions of the responsive promoter, and by employing a dual refactoring approach, we were able to explore an enhanced biosensor design space and assign their causative performance effects. The approach employed here serves as a foundational framework for engineering transcriptional biosensors and enabled development of tailored biosensors with enhanced dynamic range and diverse signal output, sensitivity, and steepness. We further demonstrate its applicability on the development of tailored biosensors for primary screening of PET hydrolases and enzyme condition screening, demonstrating the potential of statistical modelling in optimizing biosensors for tailored industrial and environmental applications.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"18 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141524889","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

Nuclear assembly in giant unilamellar vesicles encapsulating Xenopus egg extract 包裹爪蟾卵提取物的巨型单拉美拉尔囊泡中的核组装

bioRxiv - Synthetic Biology Pub Date : 2024-06-25 DOI: 10.1101/2024.06.25.600006

Sho Takamori, Hisatoshi Mimura, Toshihisa Osaki, Tomo Kondo, Miyuki Shintomi, Keishi Shintomi, Miho Ohsugi, Shoji Takeuchi

{"title":"Nuclear assembly in giant unilamellar vesicles encapsulating Xenopus egg extract","authors":"Sho Takamori, Hisatoshi Mimura, Toshihisa Osaki, Tomo Kondo, Miyuki Shintomi, Keishi Shintomi, Miho Ohsugi, Shoji Takeuchi","doi":"10.1101/2024.06.25.600006","DOIUrl":"https://doi.org/10.1101/2024.06.25.600006","url":null,"abstract":"The reconstitution of a cell nucleus in a lipid bilayer-enclosed synthetic cell makes great strides in bottom-up synthetic biology. In this study, we propose a method for assembling a nucleus in giant unilamellar vesicles (GUVs). To induce reconstitution of the nucleus, we utilise interphase egg extract of African clawed frogs Xenopus laevis, known as a biochemically controllable cell-free system capable of transforming an added sperm chromatin into a nucleus in vitro. We enhanced GUV-formation efficiency by the inverted emulsion method through incorporating prolonged waiting time and adding chloroform into lipid-dispersed oil, facilitating subsequent nuclear assembly reactions in the GUVs. Characterisation of nucleus-like structures formed in the GUVs revealed the presence of dense DNA and accumulated GFP-NLS in the structure, indicative of functional nuclear import. Immunostaining further validated the presence of nuclear pore complexes on the surfaces of these nucleus-like structures. Moreover, we observed a positive correlation between sizes of GUV and nucleus-like structure/nucleus. Our approach provides insights into nuclear assembly in lipid bilayer-enclosed cell-like confinement and becomes a platform for constructing artificial cellular systems that closely mimic eukaryotic cells.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"9 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141524999","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

Model-guided design of microRNA-based gene circuits supports precise dosage of transgenic cargoes into diverse primary cells 以模型为指导设计基于 microRNA 的基因回路，支持将转基因货物精确植入不同的原代细胞中

bioRxiv - Synthetic Biology Pub Date : 2024-06-25 DOI: 10.1101/2024.06.25.600629

Kasey S Love, Christopher P Johnstone, Emma L Peterman, Stephanie Gaglione, Kate E Galloway

{"title":"Model-guided design of microRNA-based gene circuits supports precise dosage of transgenic cargoes into diverse primary cells","authors":"Kasey S Love, Christopher P Johnstone, Emma L Peterman, Stephanie Gaglione, Kate E Galloway","doi":"10.1101/2024.06.25.600629","DOIUrl":"https://doi.org/10.1101/2024.06.25.600629","url":null,"abstract":"To realize the potential of engineered cells in therapeutic applications, transgenes must be expressed within the window of therapeutic efficacy. Differences in copy number and other sources of extrinsic noise generate variance in transgene expression and limit the performance of synthetic gene circuits. In a therapeutic context, supraphysiological expression of transgenes can compromise engineered phenotypes and lead to toxicity. To ensure a narrow range of transgene expression, we design and characterize Compact microRNA-Mediated Attenuator of Noise and Dosage (ComMAND), a single-transcript, microRNA-based incoherent feedforward loop. We tune the ComMAND output profile, and we model the system to explore additional tuning strategies. By comparing ComMAND to two-gene implementations, we highlight the precise control afforded by the single-transcript architecture, particularly at relatively low copy numbers. We show that ComMAND tightly regulates transgene expression from lentiviruses and precisely controls expression in primary human T cells, primary rat neurons, primary mouse embryonic fibroblasts, and human induced pluripotent stem cells. Finally, ComMAND effectively sets levels of the clinically relevant transgenes FMRP1 and FXN within a narrow window. Together, ComMAND is a compact tool well-suited to precisely specify expression of therapeutic cargoes.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"48 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141525001","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

Effect of Over-expression of GRXs on Thermo and Acetic Acid Stress Tolerance of Saccharomyces cerevisiae 过表达 GRXs 对酿酒酵母耐受热应激和醋酸应激的影响

bioRxiv - Synthetic Biology Pub Date : 2024-06-25 DOI: 10.1101/2024.06.24.600531

Lin Wang, Li-shuang Zhang, Mei-ling Zhang, Ya-xin He, Yuan Yu, Ke Xu

引用次数: 0

SubtiToolKit - a bioengineering kit for Bacillus subtilis and Gram-positive bacteria. SubtiToolKit - 用于枯草杆菌和革兰氏阳性菌的生物工程试剂盒。

bioRxiv - Synthetic Biology Pub Date : 2024-06-25 DOI: 10.1101/2024.06.24.600474

Joaquin Caro Astorga, Koray Malci, Tom Ellis, Paul James, Erika Debenedictis, Hia Ming, Mat Rogan

引用次数: 0

Engineered yeast multicellularity via synthetic cell-cell adhesion and direct-contact signalling 通过合成细胞-细胞粘附和直接接触信号设计酵母多细胞性

bioRxiv - Synthetic Biology Pub Date : 2024-06-24 DOI: 10.1101/2024.06.23.600301

Fankang Meng, William M Shaw, Yue Kei Keith Kam, Tom Ellis

{"title":"Engineered yeast multicellularity via synthetic cell-cell adhesion and direct-contact signalling","authors":"Fankang Meng, William M Shaw, Yue Kei Keith Kam, Tom Ellis","doi":"10.1101/2024.06.23.600301","DOIUrl":"https://doi.org/10.1101/2024.06.23.600301","url":null,"abstract":"Coordination of behaviour in multicellular systems is one the main ways that nature increases the complexity of biological function in organisms and communities. While Saccharomyces cerevisiae yeast is used extensively in research and biotechnology, it is a unicellular organism capable of only limited multicellular states. Here we expand the possibilities for engineering multicellular behaviours in yeast by developing modular toolkits for two key mechanisms seen in multicellularity, contact-dependent signalling and specific cell-to-cell adhesion. MARS (Mating-peptide Anchored Response System) is a toolkit based on surface-displayed fungal mating peptides and G protein-coupled receptor (GPCR) signalling which can mimic juxtacrine signalling between yeasts. SATURN (Saccharomyces Adhesion Toolkit for multicellUlar patteRNing) surface displays adhesion-proteins pairs on yeasts and facilitates the creation of cell aggregation patterns. Together they can be used to create multicellular logic circuits, equivalent to developmental programs that lead to cell differentiation based on the local population. Using MARS and SATURN, we further developed JUPITER (JUxtacrine sensor for Protein-protein InTERaction), a genetic sensor for assaying protein-protein interactions in culture, demonstrating this as a tool to select for high affinity binders among a population of mutated nanobodies. Collectively, MARS, SATURN, and JUPITER present valuable tools that facilitate the engineering of complex multicellularity with yeast and expand the scope of its biotechnological applications.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"2015 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141503906","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

In- & Out-Cloning: Plasmid toolboxes for scarless transcription unit and modular Golden Gate acceptor plasmid assembly 内克隆和外克隆：用于无痕转录单元和模块化金门接受质粒组装的质粒工具箱

bioRxiv - Synthetic Biology Pub Date : 2024-06-22 DOI: 10.1101/2024.06.22.600171

Stijn T. de Vries, Tania S. Koebel, Ahmet Sanal, Daniel Schindler

{"title":"In- & Out-Cloning: Plasmid toolboxes for scarless transcription unit and modular Golden Gate acceptor plasmid assembly","authors":"Stijn T. de Vries, Tania S. Koebel, Ahmet Sanal, Daniel Schindler","doi":"10.1101/2024.06.22.600171","DOIUrl":"https://doi.org/10.1101/2024.06.22.600171","url":null,"abstract":"Golden Gate cloning has become one of the most important DNA assembly strategies. The construction of standardized and reusable part libraries, their assembly into transcription units, and the subsequent assembly of multigene constructs is highly reliable and sustainable. Researchers can quickly construct derivatives of their assemblies or entire pathways, and importantly, the standardization of Golden Gate assemblies is compatible with laboratory automation. Most Golden Gate strategies rely on four nucleotide overhangs generated by commonly used Type IIS enzymes. However, reduction to three nucleotide overhangs allows the use of codons as fusion sites and reduces potential scar sequences. This is particularly important when studying biological functions, as additional nucleotides may alter the structure or stability of the transcribed RNA. To address this issue we use SapI, a Type IIS enzyme generating three nucleotide overhangs, for transcription unit assembly, allowing for codon-based fusion in coding sequences. We created a corresponding plasmid toolbox for basic part generation and transcription unit assembly, a workflow we term In-Cloning. In-Cloning is downstream compatible with the Modular Cloning standard developed by Sylvestre Marillonnet's group for standardized assembly of multigene constructs. However, the multigene construct plasmids may not be compatible for use with the model organism of choice. Therefore, we have developed a workflow called Out-Cloning to rapidly generate Golden Gate acceptor plasmids. Out-Cloning uses standardized plasmid parts that are assembled into Golden Gate acceptor plasmids using flexible linkers. This allows the systematic construction of acceptor plasmids needed to transfer assembled DNA into the organism of interest.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"8 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-06-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141503907","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

Optimization of plant-derived punicic acid synthesis in Saccharomyces cerevisiae by Ty retrotransposon-targeted random gene shuffling 通过 Ty 反转座子靶向随机基因洗牌优化酿酒酵母中植物来源的布匿酸合成

bioRxiv - Synthetic Biology Pub Date : 2024-06-21 DOI: 10.1101/2024.06.20.599979

Juli Wang, Guanqun Gavin Chen

引用次数: 0