Pub Date : 2024-07-19DOI: 10.1101/2024.07.19.604230
Fiorella Masotti, Nicolas Krink, Nicolas Lencina, Natalia Gottig, Jorgelina Ottado, Pablo Ivan Nikel
Phosphonates (PHTs), organic compounds with a stable C-P bond, are widely distributed in nature. Glyphosate (GP), a synthetic PHT, is extensively used in agriculture and has been linked to various human health issues and environmental damage. Given the prevalence of GP, developing cost-effective, on-site methods for GP detection is key for assessing pollution and reducing exposure risks. We adopted Agrobacterium tumefaciens CHLDO, a natural GP degrader, as the source of genetic parts for constructing PHT biosensors. In this species, the phn gene cluster, encoding the C-P lyase pathway, is regulated by the PhnF transcriptional repressor and is part of the Pho regulon. We selected the phnG promoter, which displays a dose-dependent response to GP, to build a set of whole-cell biosensors. Through stepwise optimization of the transcriptional cascade, we created a biosensor capable of detecting GP in the 0.25-50 uM range in various samples, including soil and water.
膦酸盐（PHTs）是一种具有稳定 C-P 键的有机化合物，在自然界中广泛分布。草甘膦（GP）是一种人工合成的 PHT，广泛用于农业，与各种人类健康问题和环境破坏有关。鉴于 GP 的普遍存在，开发具有成本效益的现场 GP 检测方法是评估污染和降低暴露风险的关键。我们采用农杆菌 CHLDO（一种天然 GP 降解菌）作为构建 PHT 生物传感器的基因部件来源。在该物种中，编码 C-P 裂解酶途径的 phn 基因簇受 PhnF 转录抑制因子调控，是 Pho 调节子的一部分。我们选择了phnG启动子来构建全细胞生物传感器。通过逐步优化转录级联，我们创建了一种生物传感器，能够检测各种样品（包括土壤和水）中 0.25-50 uM 范围内的 GP。
{"title":"Disentangling the regulatory response of Agrobacterium tumefaciens CHLDO to glyphosate for engineering whole-cell phosphonate biosensors","authors":"Fiorella Masotti, Nicolas Krink, Nicolas Lencina, Natalia Gottig, Jorgelina Ottado, Pablo Ivan Nikel","doi":"10.1101/2024.07.19.604230","DOIUrl":"https://doi.org/10.1101/2024.07.19.604230","url":null,"abstract":"Phosphonates (PHTs), organic compounds with a stable C-P bond, are widely distributed in nature. Glyphosate (GP), a synthetic PHT, is extensively used in agriculture and has been linked to various human health issues and environmental damage. Given the prevalence of GP, developing cost-effective, on-site methods for GP detection is key for assessing pollution and reducing exposure risks. We adopted Agrobacterium tumefaciens CHLDO, a natural GP degrader, as the source of genetic parts for constructing PHT biosensors. In this species, the phn gene cluster, encoding the C-P lyase pathway, is regulated by the PhnF transcriptional repressor and is part of the Pho regulon. We selected the phnG promoter, which displays a dose-dependent response to GP, to build a set of whole-cell biosensors. Through stepwise optimization of the transcriptional cascade, we created a biosensor capable of detecting GP in the 0.25-50 uM range in various samples, including soil and water.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"39 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141745364","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 : 2024-07-19DOI: 10.1101/2024.07.19.604317
Abby R. Thurm, Yaara Finkel, Cecelia Andrews, Xiangmeng S. Cai, Colette Benko, Lacramioara Bintu
RNA regulation plays an integral role in tuning gene expression and is controlled by thousands of RNA-binding proteins (RBPs). We develop and use a high-throughput recruitment assay (HT-RNA-Recruit) to identify regulatory domains within human RBPs by recruiting over 30,000 protein tiles from 367 RBPs to a reporter mRNA. We discover over 100 unique RNA-regulatory effectors in 86 distinct RBPs, presenting evidence that RBPs contain functionally separable domains that dictate their post-transcriptional control of gene expression, and identify some with unique activity at 5' or 3'UTRs. We identify some domains that downregulate gene expression both when recruited to DNA and RNA, and dissect their mechanisms of regulation. Finally, we build a synthetic RNA regulator that can stably maintain gene expression at desired levels that are predictable by a mathematical model. This work serves as a resource for human RNA-regulatory effectors and expands the synthetic repertoire of RNA-based genetic control tools.
{"title":"High-throughput discovery of regulatory effector domains in human RNA-binding proteins","authors":"Abby R. Thurm, Yaara Finkel, Cecelia Andrews, Xiangmeng S. Cai, Colette Benko, Lacramioara Bintu","doi":"10.1101/2024.07.19.604317","DOIUrl":"https://doi.org/10.1101/2024.07.19.604317","url":null,"abstract":"RNA regulation plays an integral role in tuning gene expression and is controlled by thousands of RNA-binding proteins (RBPs). We develop and use a high-throughput recruitment assay (HT-RNA-Recruit) to identify regulatory domains within human RBPs by recruiting over 30,000 protein tiles from 367 RBPs to a reporter mRNA. We discover over 100 unique RNA-regulatory effectors in 86 distinct RBPs, presenting evidence that RBPs contain functionally separable domains that dictate their post-transcriptional control of gene expression, and identify some with unique activity at 5' or 3'UTRs. We identify some domains that downregulate gene expression both when recruited to DNA and RNA, and dissect their mechanisms of regulation. Finally, we build a synthetic RNA regulator that can stably maintain gene expression at desired levels that are predictable by a mathematical model. This work serves as a resource for human RNA-regulatory effectors and expands the synthetic repertoire of RNA-based genetic control tools.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"77 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141745252","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 : 2024-07-19DOI: 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
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.
基因编码工程限制了 DNA 的转移（细胞生物分离），并通过非标准氨基酸的加入实现了新的化学反应。这些独特的特性使重新编码细胞成为最先进的安全技术。然而，进化压力可能会危及重编码的寿命。在这里，我们揭示了缺乏 18,214 个丝氨酸密码子和两个 tRNASer 的重编码大肠杆菌能够表达野生型抗生素耐药基因，而且逃逸速度比预期快七个数量级。我们展示了一个两步逃逸过程，在这一过程中，重新编码的细胞错误翻译抗生素抗性基因，直到修改或突变的 tRNA 将丝氨酸重新引入未指定的密码子中才得以存活。我们开发了对基因编码敏感的杀伤开关，它能感知丝氨酸的加入，防止细胞逃逸，同时保留三种不同的非标准氨基酸的编码。这项工作为长期控制加入新化学物质的细胞的功能奠定了基础，对临床和环境应用中物理封闭性不足的合成基因组的设计、使用和生物安全性具有重要意义。
{"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}
Pub Date : 2024-07-19DOI: 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
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.
{"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}
Pub Date : 2024-07-19DOI: 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
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.
{"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}
Pub Date : 2024-07-18DOI: 10.1101/2024.07.18.604150
Chelsea Y. Hu, John B. McManus, Fatemeh Aghlmand, Elin M Larsson, Azita Emami, Richard M Murray
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.
{"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}
Pub Date : 2024-07-12DOI: 10.1101/2024.07.12.603202
Ryota Tanaka, Kenji Tamao, Mana Ono, Seiya Yamayoshi, Yoshihiro Kawaoka, Masayuki Su'etsugu, Hiroyuki Noji, Kazuhito V. Tabata
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.
反向遗传学系统可从编码病毒基因组的质粒中产生流感病毒，是病毒感染机制基础研究和疫苗开发等应用研究的有力工具。然而，使用大肠杆菌（E. coli）克隆构建质粒的传统方法非常耗时，而且难以处理对大肠杆菌有毒的基因编码 DNA 或高度重复的序列。这些限制妨碍了病毒的快速合成。在这项研究中，我们建立了一种非常快速的基于体外单锅质粒构建（IVOC）的病毒合成方法。这种方法将用于病毒合成的基因组质粒构建时间从数天或更长时间大幅缩短至约 8 小时。此外，感染性病毒的合成产量与传统的基于大肠杆菌克隆的方法相近，且准确度高。从 IVOC 产物中生成携带报告基因的重组病毒也证明了这种方法的适用性。这种方法有望进一步加深人们对流感病毒的了解，并适用于其他 RNA 病毒。
{"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}
Pub Date : 2024-07-10DOI: 10.1101/2024.07.10.602858
Elif Gediz Kocaoglan, Andreas Andreou, Jessica Nirkko, Marisol Ochoa-Villarreal, Gary Loake, Naomi Nakayama
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.
{"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}
Pub Date : 2024-07-09DOI: 10.1101/2024.07.09.602704
Samir Hamadache, Yu K Huang, Adam Shedeed, Aqil Syed, Bogumil J Karas
Haemophilus influenzae is a bacterial species of interest for its medical relevance and utility as a model system. Despite its role in several landmark molecular and synthetic biology studies, H. influenzae remains underexplored as a potential chassis organism. The limited availability of reliable and convenient transformation methods and genetic tools for H. influenzae are obstacles to this end. However, a strain of H. influenzae Rd KW20 lacking the type II restriction endonucleases HindII and HindIII has previously been developed. Here, we show that this strain is more readily transformable by electroporation than wild-type Rd KW20. We also developed a series of multi-host plasmids carrying antibiotic selection and fluorescent visual markers based on the pSU20 vector. The availability of H. influenzae ΔHindII/III, paired with the electroporation method and plasmids presented here, will promote the exploration of H. influenzae as a host organism for synthetic biology applications.
流感嗜血杆菌（Haemophilus influenzae）是一种具有医学意义和模型系统用途的细菌。尽管流感嗜血杆菌在一些具有里程碑意义的分子和合成生物学研究中发挥了重要作用，但作为一种潜在的底盘生物，流感嗜血杆菌仍未得到充分开发。用于流感嗜血杆菌的可靠、方便的转化方法和遗传工具有限，是实现这一目标的障碍。不过，此前已经开发出了一株缺乏 II 型限制性内切酶 HindII 和 HindIII 的流感杆菌 Rd KW20。在这里，我们发现这种菌株比野生型 Rd KW20 更容易通过电穿孔进行转化。我们还在 pSU20 载体的基础上开发了一系列携带抗生素选择和荧光可视标记的多宿主质粒。H. influenzae ΔHindII/III的可用性，加上本文介绍的电穿孔方法和质粒，将促进将 H. influenzae 作为宿主生物用于合成生物学应用的探索。
{"title":"Deletion of HindIIR and HindIIIR improves DNA transfer via electroporation to Haemophilus influenzae Rd","authors":"Samir Hamadache, Yu K Huang, Adam Shedeed, Aqil Syed, Bogumil J Karas","doi":"10.1101/2024.07.09.602704","DOIUrl":"https://doi.org/10.1101/2024.07.09.602704","url":null,"abstract":"Haemophilus influenzae is a bacterial species of interest for its medical relevance and utility as a model system. Despite its role in several landmark molecular and synthetic biology studies, H. influenzae remains underexplored as a potential chassis organism. The limited availability of reliable and convenient transformation methods and genetic tools for H. influenzae are obstacles to this end. However, a strain of H. influenzae Rd KW20 lacking the type II restriction endonucleases HindII and HindIII has previously been developed. Here, we show that this strain is more readily transformable by electroporation than wild-type Rd KW20. We also developed a series of multi-host plasmids carrying antibiotic selection and fluorescent visual markers based on the pSU20 vector. The availability of H. influenzae ΔHindII/III, paired with the electroporation method and plasmids presented here, will promote the exploration of H. influenzae as a host organism for synthetic biology applications.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"42 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141569782","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 : 2024-07-08DOI: 10.1101/2024.07.07.602432
Wei Li Ong, Zhi Li, Kian Hong Ng, Kang Zhou
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 in a significant 5.4-fold improvement in mannanase activity under the nprE promoter. The nprE promoter 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. Lastly, 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 mannanase expression in B. subtilis and its potential application in the biotransformation of PKM and other mannan-rich bioresources for improved feed utilization.
{"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}
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