bioRxiv - Synthetic Biology最新文献_第8页

Disentangling the regulatory response of Agrobacterium tumefaciens CHLDO to glyphosate for engineering whole-cell phosphonate biosensors 厘清农杆菌 CHLDO 对草甘膦的调控响应以设计全细胞膦酸盐生物传感器

bioRxiv - Synthetic Biology Pub Date : 2024-07-19 DOI: 10.1101/2024.07.19.604230

Fiorella Masotti, Nicolas Krink, Nicolas Lencina, Natalia Gottig, Jorgelina Ottado, Pablo Ivan Nikel

引用次数: 0

High-throughput discovery of regulatory effector domains in human RNA-binding proteins 高通量发现人类 RNA 结合蛋白的调控效应结构域

bioRxiv - Synthetic Biology Pub Date : 2024-07-19 DOI: 10.1101/2024.07.19.604317

Abby R. Thurm, Yaara Finkel, Cecelia Andrews, Xiangmeng S. Cai, Colette Benko, Lacramioara Bintu

引用次数: 0

Preventing escape and malfunction of recoded cells due to tRNA base changes 防止因 tRNA 碱基变化导致重编码细胞逃逸和功能失常

bioRxiv - Synthetic Biology Pub Date : 2024-07-19 DOI: 10.1101/2024.07.18.604179

Anush Chiappino-Pepe, Felix Radford, Bogdan Budnik, Huseyin Tas, Teresa L Augustin, Hana M Burgess, Michael Moret, Azim M Dharani, Qinmei Zheng, Weicheng Fan, Maksud M Afrikawala, Shova Thapa, Erkin Kuru, Kamesh Narasimhan, Jorge A Marchand, Ramiro Martin Perrotta, Jonathan M Stokes, Jeantine E Lunshof, John D Aach, Jenny M Tam, George M Church

{"title":"Preventing escape and malfunction of recoded cells due to tRNA base changes","authors":"Anush Chiappino-Pepe, Felix Radford, Bogdan Budnik, Huseyin Tas, Teresa L Augustin, Hana M Burgess, Michael Moret, Azim M Dharani, Qinmei Zheng, Weicheng Fan, Maksud M Afrikawala, Shova Thapa, Erkin Kuru, Kamesh Narasimhan, Jorge A Marchand, Ramiro Martin Perrotta, Jonathan M Stokes, Jeantine E Lunshof, John D Aach, Jenny M Tam, George M Church","doi":"10.1101/2024.07.18.604179","DOIUrl":"https://doi.org/10.1101/2024.07.18.604179","url":null,"abstract":"Engineering the genetic code restricts DNA transfer (cellular bioisolation) and enables new chemistries via non-standard amino acid incorporation. These distinct properties make recoded cells state-of-the-art safe technologies. However, evolutionary pressures may endanger the longevity of the recoding. Here, we reveal that recoded Escherichia coli lacking 18,214 serine codons and two tRNASer can express wild-type antibiotic resistance genes and escape up to seven orders of magnitude faster than expected. We show a two-step escape process whereby recoded cells mistranslate antibiotic resistance genes to survive until modified or mutated tRNAs reintroduce serine into unassigned codons. We developed genetic-code-sensitive kill switches that sense serine incorporation and prevent cellular escape while preserving encoding of three distinct non-standard amino acids. This work lays the foundation for the long-term controlled function of cells that incorporate new chemistries, with implications for the design, use, and biosafety of synthetic genomes in clinical and environmental applications where physical containment is insufficient.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"27 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141745253","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

A modular cell-free protein biosensor platform using split T7 RNA polymerase 使用分裂 T7 RNA 聚合酶的模块化无细胞蛋白质生物传感器平台

bioRxiv - Synthetic Biology Pub Date : 2024-07-19 DOI: 10.1101/2024.07.19.604303

Megan A. McSweeney, Alexandra T. Patterson, Kathryn Loeffler, Regina Cuellar Lelo de Larrea, Monica P. McNerney, Ravi S. Kane, Mark P. Styczynski

{"title":"A modular cell-free protein biosensor platform using split T7 RNA polymerase","authors":"Megan A. McSweeney, Alexandra T. Patterson, Kathryn Loeffler, Regina Cuellar Lelo de Larrea, Monica P. McNerney, Ravi S. Kane, Mark P. Styczynski","doi":"10.1101/2024.07.19.604303","DOIUrl":"https://doi.org/10.1101/2024.07.19.604303","url":null,"abstract":"Conventional laboratory protein detection techniques are not suitable for point-of-care (POC) use because they require expensive equipment and laborious protocols, and existing POC assays suffer from long development timescales. Here, we describe a modular cell-free biosensing platform for generalizable protein detection that we call TLISA (T7 RNA polymerase-Linked ImmunoSensing Assay), designed for extreme flexibility and equipment-free use. TLISA uses a split T7 RNA polymerase fused to affinity domains against a protein. The target antigen drives polymerase reassembly, inducing reporter expression. We characterize the platform, then demonstrate its modularity by using 16 affinity domains against four different antigens with minimal protocol optimization. We show TLISA is suitable for POC use by sensing human biomarkers in serum and saliva with a colorimetric readout within one hour and by demonstrating functionality after lyophilization. Altogether, this technology could have potentially revolutionary impacts, enabling truly rapid, reconfigurable, equipment-free detection of virtually any protein.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"8 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141745251","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

Engineered endosymbionts that modulate primary macrophage function and attenuate tumor growth by shifting the tumor microenvironment 通过改变肿瘤微环境来调节原发性巨噬细胞功能并抑制肿瘤生长的工程内生菌

bioRxiv - Synthetic Biology Pub Date : 2024-07-19 DOI: 10.1101/2024.07.18.604190

Cody S Madsen, Ashley V Makela, Chima V Maduka, Emily M Greeson, Anthony Tundo, Evran Ural, Satyajit H Kulkarni, Ahmed Zarea, Matti Kiupel, Maryam Sayadi, Christopher H Contag

{"title":"Engineered endosymbionts that modulate primary macrophage function and attenuate tumor growth by shifting the tumor microenvironment","authors":"Cody S Madsen, Ashley V Makela, Chima V Maduka, Emily M Greeson, Anthony Tundo, Evran Ural, Satyajit H Kulkarni, Ahmed Zarea, Matti Kiupel, Maryam Sayadi, Christopher H Contag","doi":"10.1101/2024.07.18.604190","DOIUrl":"https://doi.org/10.1101/2024.07.18.604190","url":null,"abstract":"Modulating gene expression in macrophages can be used to improve tissue regeneration and to redirect tumor microenvironments (TME) toward positive therapeutic outcomes. We have developed Bacillus subtilis as an engineered endosymbiont (EES) capable of residing inside the eukaryotic host cell cytoplasm and controlling the fate of macrophages. Secretion of mammalian transcription factors (TFs) from B. subtilis that expresses listeriolysin O (LLO; allowing the EES to escape destruction by the macrophage) modulated expression of surface markers, cytokines and chemokines, indicating functional changes in a macrophage/monocyte cell line. The engineered B. subtilis LLO TF strains were evaluated in murine bone marrow-derived macrophages (BMDMs) by flow cytometry, chemokine/cytokine profiling, metabolic assays and RNA-Seq. Delivery of TFs by the EES shifted BMDM gene expression, production of cytokine and chemokines and metabolic patterns, indicating that the TF strains could guide primary macrophage function. Thereafter, the ability of the TF strains to alter the TME was characterized in vivo, in an orthotopic murine model of triple-negative breast cancer to assess therapeutic effects. The TF strains altered the TME by shifting immune cell composition and attenuating tumor growth. Additionally, multiple doses of the TF strains were well-tolerated by the mice. The use of B. subtilis LLO TF strains as EES showed promise as a unique cancer immunotherapy by directing immune function intracellularly. The uses of EES could be expanded to modulate other mammalian cells over a range of biomedical applications.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"15 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141745254","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

A Field-Deployable Arsenic Sensor Integrating Bacillus Megaterium with CMOS Technology 将巨型芽孢杆菌与 CMOS 技术相结合的可现场部署的砷传感器

bioRxiv - Synthetic Biology Pub Date : 2024-07-18 DOI: 10.1101/2024.07.18.604150

Chelsea Y. Hu, John B. McManus, Fatemeh Aghlmand, Elin M Larsson, Azita Emami, Richard M Murray

{"title":"A Field-Deployable Arsenic Sensor Integrating Bacillus Megaterium with CMOS Technology","authors":"Chelsea Y. Hu, John B. McManus, Fatemeh Aghlmand, Elin M Larsson, Azita Emami, Richard M Murray","doi":"10.1101/2024.07.18.604150","DOIUrl":"https://doi.org/10.1101/2024.07.18.604150","url":null,"abstract":"Bacteria innately monitor their environment by dynamically regulating gene expression to respond to fluctuating conditions. Through synthetic biology, we can harness this natural capability to design cell-based sensors. Bacillus megaterium, a soil bacterium, stands out due to its remarkable heavy metal tolerance and sporulation ability, making it an ideal candidate for heavy metal detection with low transportation costs. However, challenges persist: the synthetic biology toolkit for this strain is underdeveloped and conventional whole-cell sensors necessitate specialized laboratory equipment to read the output. In our study, we genetically modified B. megaterium for arsenic detection, establishing a detection threshold below the EPA recommendation of 10 ppb for drinking water in both vegetative cell form and spore form. Additionally, we integrated both engineered B. megaterium living cells and spores with CMOS chip for field-deployable arsenic detection. We show that the limit of detection of our integrated sensor is applicable in soil and air arsenic contamination testing. As a proof of concept, this work paves the way for deploying our sensor in resource-limited settings, ensuring real-time arsenic detection in challenging environments.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"20 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141745255","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 vitro one-pot construction of influenza viral genomes for virus particle synthesis based on reverse genetics system 基于反向遗传学系统的用于病毒颗粒合成的流感病毒基因组体外一次性构建技术

bioRxiv - Synthetic Biology Pub Date : 2024-07-12 DOI: 10.1101/2024.07.12.603202

Ryota Tanaka, Kenji Tamao, Mana Ono, Seiya Yamayoshi, Yoshihiro Kawaoka, Masayuki Su'etsugu, Hiroyuki Noji, Kazuhito V. Tabata

{"title":"In vitro one-pot construction of influenza viral genomes for virus particle synthesis based on reverse genetics system","authors":"Ryota Tanaka, Kenji Tamao, Mana Ono, Seiya Yamayoshi, Yoshihiro Kawaoka, Masayuki Su'etsugu, Hiroyuki Noji, Kazuhito V. Tabata","doi":"10.1101/2024.07.12.603202","DOIUrl":"https://doi.org/10.1101/2024.07.12.603202","url":null,"abstract":"The reverse genetics system, which allows the generation of influenza viruses from plasmids encoding viral genome, is a powerful tool for basic research on viral infection mechanisms and application research such as vaccine development. However, conventional plasmid construction using Escherichia coli (E. coli) cloning is time-consuming and has difficulties handling DNA encoding genes toxic for E. coli or highly repeated sequences. These limitations hamper rapid virus synthesis. In this study, we establish a very rapid in vitro one-pot plasmid construction (IVOC) based virus synthesis. This method dramatically reduced the time for genome plasmid construction, which was used for virus synthesis, from several days or more to about 8 hours. Moreover, infectious viruses could be synthesized with a similar yield to the conventional E. coli cloning-based method with high accuracy. The applicability of this method was also demonstrated by the generation of recombinant viruses carrying reporter genes from the IVOC products. This method is expected to potentially advance further understanding of influenza viruses and apply to other RNA viruses.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"32 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141608414","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

Mobius Assembly for Plant Systems uncovers combinatorial interactions among promoters, coding sequences, and terminators in gene regulation 植物系统的莫比乌斯组装揭示了基因调控中启动子、编码序列和终止子之间的组合相互作用

bioRxiv - Synthetic Biology Pub Date : 2024-07-10 DOI: 10.1101/2024.07.10.602858

Elif Gediz Kocaoglan, Andreas Andreou, Jessica Nirkko, Marisol Ochoa-Villarreal, Gary Loake, Naomi Nakayama

{"title":"Mobius Assembly for Plant Systems uncovers combinatorial interactions among promoters, coding sequences, and terminators in gene regulation","authors":"Elif Gediz Kocaoglan, Andreas Andreou, Jessica Nirkko, Marisol Ochoa-Villarreal, Gary Loake, Naomi Nakayama","doi":"10.1101/2024.07.10.602858","DOIUrl":"https://doi.org/10.1101/2024.07.10.602858","url":null,"abstract":"Plants are the primary biological platforms for producing food, energy, and materials in agriculture; however, they are largely untouched by synthetic biology-driven transformation in bioproduction technologies. Molecular tools for complex, multigene engineering are as yet limited, with development underway to enhance stability and predictivity. Here, we present a new standardized and streamlined toolkit for plant synthetic biology, Mobius Assembly for Plant Systems (MAPS). It is based on small plant binary vectors (pMAPs) that contain a fusion origin of replication that enhances plasmid yield in both E. coli and Agrobacterium. MAPS includes a new library of promoters and terminators with different activity levels; the parts were made small in size to improve construct stability and transformation efficiency. These promoters and terminators were characterized using a high-throughput protoplast expression assay. Our findings show a significant influence of terminators on gene expression, as the strength of a promoter can change more than 7 folds with the different terminators. Additionally, we have observed that changing the coding sequence changes the relative strength of promoter and terminator pairs, uncovering combinatorial interactions among all parts of a transcriptional unit. We further gained insights into the mechanisms of such interactions by analyzing RNA folding. These results contribute to improving stability, predictability, and orthogonality in synthetic biology of plant systems and beyond.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"324 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141587317","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

Deletion of HindIIR and HindIIIR improves DNA transfer via electroporation to Haemophilus influenzae Rd 删除 HindIIR 和 HindIIIR 可改善通过电穿孔向流感嗜血杆菌 Rd 转移 DNA 的效果

bioRxiv - Synthetic Biology Pub Date : 2024-07-09 DOI: 10.1101/2024.07.09.602704

Samir Hamadache, Yu K Huang, Adam Shedeed, Aqil Syed, Bogumil J Karas

引用次数: 0

Improving Mannanase Production in Bacillus subtilis for Fibre Hydrolysis during Solid-State Fermentation of Palm Kernel Meal 在棕榈仁粕固态发酵过程中提高枯草芽孢杆菌的甘露聚糖酶产量以水解纤维

bioRxiv - Synthetic Biology Pub Date : 2024-07-08 DOI: 10.1101/2024.07.07.602432

Wei Li Ong, Zhi Li, Kian Hong Ng, Kang Zhou

{"title":"Improving Mannanase Production in Bacillus subtilis for Fibre Hydrolysis during Solid-State Fermentation of Palm Kernel Meal","authors":"Wei Li Ong, Zhi Li, Kian Hong Ng, Kang Zhou","doi":"10.1101/2024.07.07.602432","DOIUrl":"https://doi.org/10.1101/2024.07.07.602432","url":null,"abstract":"The primary challenge in utilizing palm kernel meal (PKM, an agricultural by-product) as non-ruminant livestock feed is its high fibre content, predominantly in the form of mannan. Microbial fermentation offers an economically favourable alternative to enzyme supplementation for breaking down fibre in lignocellulosic biomass. In a recent study, we have isolated and characterized an undomesticated strain (Bacillus subtilis F6) that is able to secrete mannanase. In this work, the mannanase production was substantially improved by optimizing multiple regulatory elements controlling the mannanase expression. Mannanase GmuG, sourced from B. subtilis F6 and verified for its hydrolytic activity on PKM fibre, was expressed using a replicative plasmid (pBE-S). The recombinant strain of B. subtilis F6 exhibited 1.9-fold increase in the mannanase activity during solid-state fermentation. Optimization of signal peptide and ribosome binding site further enhanced mannanase activity by 3.1-fold. Subsequently, promoter screening based on highly transcribed genes in B. subtilis F6 resulted\u0000in a significant 5.4-fold improvement in mannanase activity under the nprE promoter. The nprE\u0000promoter was further refined by eliminating specific transcription factor binding sites, enhancing the mannanase activity further by 1.8-fold. Notably, a substantial 35-40% reduction in PKM fibre content was observed after 30 h of fermentation using the recombinant strains.\u0000Lastly, the highest mannanase-producing strain was examined for scaled-up fermentation. The impacts of fermentation on fibre and protein contents, as well as the surface morphology of PKM, were analysed. The outcomes of this study offer an efficient method for robust\u0000mannanase expression in B. subtilis and its potential application in the biotransformation of PKM and other mannan-rich bioresources for improved feed utilization.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"15 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141569784","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