Pub Date : 2024-07-27DOI: 10.1101/2024.07.26.605347
Vivian T Hu, Shahrzad Ezzatpour, Ekaterina T Selivanovitch, Jordan Carter, Julie Sahler, Richard A Adeleke, Avery August, Hector Aguilar-Carreno, Susan Daniel, Neha P Kamat
Membrane proteins expressed on the surface of enveloped viruses are potent antigens in a vaccine, yet are difficult to produce and present due to their instability without a lipid scaffold. Current vaccination strategies that incorporate viral membrane proteins, such as live attenuated viruses, inactivated viruses, or extracellular vesicles, have limitations including lengthy production time, poor immunogenicity, extensive processing steps, and/or poor stability. Cell-free protein synthesis of viral membrane proteins offers a rapid, one-step method to assemble vaccine nanoparticles via cotranslational folding of membrane proteins into nanoscale liposomes. Here, we develop a vaccine candidate for the deadly Nipah virus (NiV), a highly lethal virus listed by the World Health Organization as a priority pathogen, by cell-free expressing two full-length Nipah virus membrane proteins. We demonstrate that both NiV fusion protein (NiV F) and NiV glycoprotein (NiV G) can be expressed and cotranslationally integrated into liposomes and that they fold into their native conformation. We find the removal of a signal peptide sequence and alteration of liposome lipid composition improves viral membrane protein incorporation. Furthermore, a lipid adjuvant, monophosphoryl lipid A (MPLA), can be readily added to liposomes without disrupting protein-vesicle loading or protein folding conformations. Finally, we demonstrate that our generated liposomal formulations lead to enhanced humoral responses in mice compared to empty and single-protein controls. This work establishes a platform to quickly assemble and present membrane antigens as multivalent vaccines that will enable a rapid response to the broad range of emerging pathogenic threats.
{"title":"Cell-free expression of Nipah virus transmembrane proteins for proteoliposome vaccine design","authors":"Vivian T Hu, Shahrzad Ezzatpour, Ekaterina T Selivanovitch, Jordan Carter, Julie Sahler, Richard A Adeleke, Avery August, Hector Aguilar-Carreno, Susan Daniel, Neha P Kamat","doi":"10.1101/2024.07.26.605347","DOIUrl":"https://doi.org/10.1101/2024.07.26.605347","url":null,"abstract":"Membrane proteins expressed on the surface of enveloped viruses are potent antigens in a vaccine, yet are difficult to produce and present due to their instability without a lipid scaffold. Current vaccination strategies that incorporate viral membrane proteins, such as live attenuated viruses, inactivated viruses, or extracellular vesicles, have limitations including lengthy production time, poor immunogenicity, extensive processing steps, and/or poor stability. Cell-free protein synthesis of viral membrane proteins offers a rapid, one-step method to assemble vaccine nanoparticles via cotranslational folding of membrane proteins into nanoscale liposomes. Here, we develop a vaccine candidate for the deadly Nipah virus (NiV), a highly lethal virus listed by the World Health Organization as a priority pathogen, by cell-free expressing two full-length Nipah virus membrane proteins. We demonstrate that both NiV fusion protein (NiV F) and NiV glycoprotein (NiV G) can be expressed and cotranslationally integrated into liposomes and that they fold into their native conformation. We find the removal of a signal peptide sequence and alteration of liposome lipid composition improves viral membrane protein incorporation. Furthermore, a lipid adjuvant, monophosphoryl lipid A (MPLA), can be readily added to liposomes without disrupting protein-vesicle loading or protein folding conformations. Finally, we demonstrate that our generated liposomal formulations lead to enhanced humoral responses in mice compared to empty and single-protein controls. This work establishes a platform to quickly assemble and present membrane antigens as multivalent vaccines that will enable a rapid response to the broad range of emerging pathogenic threats.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"72 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141783937","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 : 2024-07-26DOI: 10.1101/2024.07.25.605042
Christian S. Diercks, Philipp Sondermann, Cynthia Rong, David A. Dik, Thomas G. Gillis, Yahui Ban, Peter G. Schultz
Systems that perform continuous hypermutation of designated genes without compromising the integrity of the host genome can dramatically accelerate the evolution of new or enhanced protein functions. We describe an orthogonal DNA replication system in E. coli based on the controlled expression of the replisome of bacteriophage T7. The system replicates circular plasmids that enable high transformation efficiencies and seamless integration into standard molecular biology workflows. Engineering of T7 DNA polymerase yielded variant proteins with mutation rates of 1.7 x 10-5 substitutions per base in vivo - 100,000-fold above the genomic mutation rate. Continuous evolution using the mutagenic T7 replisome was demonstrated by expanding the substrate scope of TEM-1 β-lactamase and increase activity 1,000-fold against clinically relevant monobactam and cephalosporin antibiotics in less than one week.
在不损害宿主基因组完整性的情况下对指定基因进行连续超突变的系统可以大大加速新的或增强的蛋白质功能的进化。我们描述了一种基于噬菌体 T7 复制体受控表达的大肠杆菌正交 DNA 复制系统。该系统可复制环形质粒，从而实现高转化效率，并无缝集成到标准分子生物学工作流程中。通过对 T7 DNA 聚合酶进行工程改造，产生了变异蛋白质，其体内每个碱基的突变率为 1.7 x 10-5 次置换，比基因组突变率高出 10 万倍。通过扩大 TEM-1 β-内酰胺酶的底物范围，并在不到一周的时间内将其对临床相关单内酰胺类和头孢菌素类抗生素的活性提高 1000 倍，证明了诱变 T7 复制体的持续进化。
{"title":"An Orthogonal T7 Replisome for Continuous Hypermutation and Accelerated Evolution in E. coli","authors":"Christian S. Diercks, Philipp Sondermann, Cynthia Rong, David A. Dik, Thomas G. Gillis, Yahui Ban, Peter G. Schultz","doi":"10.1101/2024.07.25.605042","DOIUrl":"https://doi.org/10.1101/2024.07.25.605042","url":null,"abstract":"Systems that perform continuous hypermutation of designated genes without compromising the integrity of the host genome can dramatically accelerate the evolution of new or enhanced protein functions. We describe an orthogonal DNA replication system in E. coli based on the controlled expression of the replisome of bacteriophage T7. The system replicates circular plasmids that enable high transformation efficiencies and seamless integration into standard molecular biology workflows. Engineering of T7 DNA polymerase yielded variant proteins with mutation rates of 1.7 x 10-5 substitutions per base in vivo - 100,000-fold above the genomic mutation rate. Continuous evolution using the mutagenic T7 replisome was demonstrated by expanding the substrate scope of TEM-1 β-lactamase and increase activity 1,000-fold against clinically relevant monobactam and cephalosporin antibiotics in less than one week.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"95 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141783938","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 : 2024-07-24DOI: 10.1101/2024.07.23.604805
Elizabeth Vaisbourd, Anat Bren, Uri Alon, David Shaanan Glass
Plasmids are an essential tool for basic research and biotechnology applications. To optimize plasmid-based circuits, it is crucial to control plasmid integrity, including the formation of plasmid multimers. Multimers are tandem repeats of entire plasmids formed during replication by failed dimer resolution. Multimers can affect the behavior of synthetic circuits, especially ones that include DNA-editing enzymes. However, occurrence of multimers is not commonly assayed. Here we survey four commonly used plasmid backbones for occurrence of multimers in cloning (JM109) and wild-type (MG1655) strains. We find that multimers occur appreciably only in MG1655, with the fraction of plasmids existing as multimers increasing with both plasmid copy number and culture passaging. In contrast, introduction of multimers into JM109 can produce strains containing only multimers. We present an MG1655 ΔrecA single-locus knockout that avoids multimer production. These results can aid synthetic biologists in improving design and reliability of plasmid-based circuits.
质粒是基础研究和生物技术应用的重要工具。要优化基于质粒的电路，控制质粒的完整性至关重要，包括质粒多聚体的形成。多聚体是整个质粒在复制过程中由于二聚体解析失败而形成的串联重复。多聚体会影响合成电路的行为，尤其是包含 DNA 编辑酶的电路。然而，多聚体的出现并不常见。在这里，我们调查了四种常用质粒骨架在克隆（JM109）和野生型（MG1655）菌株中出现多聚体的情况。我们发现，只有在 MG1655 中才会出现明显的多聚体，质粒多聚体的比例随着质粒拷贝数和培养传代的增加而增加。相比之下，将多聚体引入 JM109 能产生只含有多聚体的菌株。我们介绍了一种避免产生多聚体的 MG1655 ΔrecA 单基因敲除方法。这些结果有助于合成生物学家改进基于质粒的电路的设计和可靠性。
{"title":"Preventing plasmid multimer formation in commonly used synthetic biology plasmids","authors":"Elizabeth Vaisbourd, Anat Bren, Uri Alon, David Shaanan Glass","doi":"10.1101/2024.07.23.604805","DOIUrl":"https://doi.org/10.1101/2024.07.23.604805","url":null,"abstract":"Plasmids are an essential tool for basic research and biotechnology applications. To optimize plasmid-based circuits, it is crucial to control plasmid integrity, including the formation of plasmid multimers. Multimers are tandem repeats of entire plasmids formed during replication by failed dimer resolution. Multimers can affect the behavior of synthetic circuits, especially ones that include DNA-editing enzymes. However, occurrence of multimers is not commonly assayed. Here we survey four commonly used plasmid backbones for occurrence of multimers in cloning (JM109) and wild-type (MG1655) strains. We find that multimers occur appreciably only in MG1655, with the fraction of plasmids existing as multimers increasing with both plasmid copy number and culture passaging. In contrast, introduction of multimers into JM109 can produce strains containing only multimers. We present an MG1655 ΔrecA single-locus knockout that avoids multimer production. These results can aid synthetic biologists in improving design and reliability of plasmid-based circuits.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"23 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141783942","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}
Cyanophages are considered a promising biological management option for treating cyanobacterial blooms. Broadening the host range of cyanophages and/or shortening the lysis cycle by designing and synthesizing artificial cyanophages are potential strategies to enhance their effectiveness and efficiency. However, the rescue of artificial cyanophage genomes remains unexplored. In this study, we achieved the integration of a full-length cyanophage genome, PP, which originally infects Plectonema boryanum FACHB-240, into the model cyanobacterium Synechococcus elongatus PCC 7942. Since the integration of these large fragments (~42 kb) into cyanobacteria depended on conjugation via Escherichia coli, the toxic open reading frames (ORFs) of PP to E. coli were first identified, leading to the identification of toxic ORF6, ORF11, and ORF22. The original PP genome was then rearranged, and the three toxic ORFs were controlled using a tandem induction switch. The full length of the PP genome was integrated into the genome of S. elongatus PCC 7942 via two rounds of homologous recombination. Interestingly, compared to the control strain, the integration of the PP genome decreased photosynthesis and carbon fixation in S. elongatus PCC 7942, exhibiting cyanophage-like behavior. Transcriptomic analysis revealed that 32 of the 41 ORFs of the PP genome were transcribed in S. elongatus PCC 7942, significantly altering the energy metabolism and carbon fixation pathways. These influences were further demonstrated using metabolomics. This study provides a comprehensive approach for the artificial design and integration of cyanophage genomes in cyanobacteria, laying the foundation for their real rescue in the future.
{"title":"Expression and characterization of the complete cyanophage genome PP in the heterologous host Synechococcus elongatus PCC 7942","authors":"Guorui Li, Jia Feng, Xiaofei Zhu, Yujie Chai, Tao Sun, Jianlan Jiang","doi":"10.1101/2024.07.23.604706","DOIUrl":"https://doi.org/10.1101/2024.07.23.604706","url":null,"abstract":"Cyanophages are considered a promising biological management option for treating cyanobacterial blooms. Broadening the host range of cyanophages and/or shortening the lysis cycle by designing and synthesizing artificial cyanophages are potential strategies to enhance their effectiveness and efficiency. However, the rescue of artificial cyanophage genomes remains unexplored. In this study, we achieved the integration of a full-length cyanophage genome, PP, which originally infects Plectonema boryanum FACHB-240, into the model cyanobacterium Synechococcus elongatus PCC 7942. Since the integration of these large fragments (~42 kb) into cyanobacteria depended on conjugation via Escherichia coli, the toxic open reading frames (ORFs) of PP to E. coli were first identified, leading to the identification of toxic ORF6, ORF11, and ORF22. The original PP genome was then rearranged, and the three toxic ORFs were controlled using a tandem induction switch. The full length of the PP genome was integrated into the genome of S. elongatus PCC 7942 via two rounds of homologous recombination. Interestingly, compared to the control strain, the integration of the PP genome decreased photosynthesis and carbon fixation in S. elongatus PCC 7942, exhibiting cyanophage-like behavior. Transcriptomic analysis revealed that 32 of the 41 ORFs of the PP genome were transcribed in S. elongatus PCC 7942, significantly altering the energy metabolism and carbon fixation pathways. These influences were further demonstrated using metabolomics. This study provides a comprehensive approach for the artificial design and integration of cyanophage genomes in cyanobacteria, laying the foundation for their real rescue in the future.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141783946","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 : 2024-07-24DOI: 10.1101/2024.07.23.604792
jiaojiao ji, xiaoxu han, lanlan zang, yushan li, Liqun Lin, donghua hu, shichao sun, yonglin ren, Garth Maker, zefu lu, Li Wang
Furocoumarins (FCs) are crucial natural products playing a dual role as plant defense molecules and pharmacologically active substances. Angelica dahurica is a renowned herb with diverse and abundant FCs. However, the accumulation pattern over developmental stages, biosynthesis pathway and regulatory mechanisms of FCs in A. dahurica remain elusive, hindering the production of FCs via synthetic biology approaches. Here, we constructed a chromosome-level reference genome for A. dahurica and quantified the content dynamics of 17 coumarins across six developmental stages of its medicinal organ, root. It showed a gradual decrease in FC concentration with root enlargement. The combined analyses of transcriptomic and metabolomic data, together with in vivo enzymatic assay, confirmed that CYP71AZ18 was involved in the biosynthesis of bergaptol, whereas CYP71AZ19 and CYP83F95 contributed to the biosynthesis of xanthotoxol. Notably, CYP71AZ19 originated from a proximal duplication event of CYP71AZ18, specific to A. dahurica, subsequently undergoing neofunctionalization. Accessible chromatin regions (ACRs), especially proximal ACRs, are correlated with higher gene expression levels, including the three validated genes involved in FC biosynthesis, showing potential to regulate metabolite biosynthesis. Our findings provide new insights into the biosynthetic pathway of FCs and the epigenetic regulation of metabolite biosynthesis.
呋喃香豆素（FCs）是一种重要的天然产物，具有植物防御分子和药理活性物质的双重作用。白芷是一种著名的草本植物，含有多种丰富的 FCs。然而，白芷中FCs在不同发育阶段的积累模式、生物合成途径和调控机制仍然难以捉摸，阻碍了通过合成生物学方法生产FCs。在此，我们构建了白芷染色体级参考基因组，并量化了其药用器官根部六个发育阶段中 17 种香豆素的含量动态。结果表明，随着根的增大，FC的浓度逐渐降低。通过对转录组和代谢组数据的综合分析以及体内酶学检测，证实 CYP71AZ18 参与了香柑醇的生物合成，而 CYP71AZ19 和 CYP83F95 则参与了黄腐醇的生物合成。值得注意的是，CYP71AZ19 起源于 CYP71AZ18 的近端复制事件，为 A. dahurica 所特有，随后经历了新功能化。可进入的染色质区域（ACRs），尤其是近端 ACRs，与较高的基因表达水平相关，包括参与 FC 生物合成的三个验证基因，显示出调控代谢物生物合成的潜力。我们的研究结果为FCs的生物合成途径以及代谢物生物合成的表观遗传调控提供了新的见解。
{"title":"Integrative multi-omics data elucidating the biosynthesis and regulatory mechanisms of furanocoumarins in Angelica dahurica","authors":"jiaojiao ji, xiaoxu han, lanlan zang, yushan li, Liqun Lin, donghua hu, shichao sun, yonglin ren, Garth Maker, zefu lu, Li Wang","doi":"10.1101/2024.07.23.604792","DOIUrl":"https://doi.org/10.1101/2024.07.23.604792","url":null,"abstract":"Furocoumarins (FCs) are crucial natural products playing a dual role as plant defense molecules and pharmacologically active substances. Angelica dahurica is a renowned herb with diverse and abundant FCs. However, the accumulation pattern over developmental stages, biosynthesis pathway and regulatory mechanisms of FCs in A. dahurica remain elusive, hindering the production of FCs via synthetic biology approaches. Here, we constructed a chromosome-level reference genome for A. dahurica and quantified the content dynamics of 17 coumarins across six developmental stages of its medicinal organ, root. It showed a gradual decrease in FC concentration with root enlargement. The combined analyses of transcriptomic and metabolomic data, together with in vivo enzymatic assay, confirmed that CYP71AZ18 was involved in the biosynthesis of bergaptol, whereas CYP71AZ19 and CYP83F95 contributed to the biosynthesis of xanthotoxol. Notably, CYP71AZ19 originated from a proximal duplication event of CYP71AZ18, specific to A. dahurica, subsequently undergoing neofunctionalization. Accessible chromatin regions (ACRs), especially proximal ACRs, are correlated with higher gene expression levels, including the three validated genes involved in FC biosynthesis, showing potential to regulate metabolite biosynthesis. Our findings provide new insights into the biosynthetic pathway of FCs and the epigenetic regulation of metabolite biosynthesis.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"43 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141783940","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 : 2024-07-22DOI: 10.1101/2024.07.22.604574
Haresh Bhaskar, Zoe Gidden, Gurvir Virdi, Dirk-Jan Kleinjan, Susan J. Rosser, Sonia Gandhi, Lynne Regan, Mathew H. Horrocks
Super-resolution microscopy has revolutionized biological imaging, enabling the visualization of structures at the nanometer length scale. Its application in live cells, however, has remained challenging. To address this, we adapted LIVE-PAINT, an approach we established in yeast, for application in live mammalian cells. Using the 101A/101B coiled-coil peptide pair as a peptide-based targeting system, we successfully demonstrate the super-resolution imaging of two distinct proteins in mammalian cells, one localized in the nucleus, and the second in the cytoplasm. This study highlights the versatility of LIVE-PAINT, suggesting its potential for live-cell super-resolution imaging across a range of protein targets in mammalian cells. We name the mammalian cell version of our original method mLIVE-PAINT.
{"title":"Super-resolution imaging of proteins inside live mammalian cells with mLIVE-PAINT","authors":"Haresh Bhaskar, Zoe Gidden, Gurvir Virdi, Dirk-Jan Kleinjan, Susan J. Rosser, Sonia Gandhi, Lynne Regan, Mathew H. Horrocks","doi":"10.1101/2024.07.22.604574","DOIUrl":"https://doi.org/10.1101/2024.07.22.604574","url":null,"abstract":"Super-resolution microscopy has revolutionized biological imaging, enabling the visualization of structures at the nanometer length scale. Its application in live cells, however, has remained challenging. To address this, we adapted LIVE-PAINT, an approach we established in yeast, for application in live mammalian cells. Using the 101A/101B coiled-coil peptide pair as a peptide-based targeting system, we successfully demonstrate the super-resolution imaging of two distinct proteins in mammalian cells, one localized in the nucleus, and the second in the cytoplasm. This study highlights the versatility of LIVE-PAINT, suggesting its potential for live-cell super-resolution imaging across a range of protein targets in mammalian cells. We name the mammalian cell version of our original method mLIVE-PAINT.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"44 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141783939","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 : 2024-07-21DOI: 10.1101/2024.07.20.604438
Dennis Tin Chat Chan, Lena Winter, Johan Bjerg, Stina Krsmanovic, Geoff S. Baldwin, Hans C. Bernstein
The choice of organism to host a genetic circuit, the chassis, is often defaulted to model organisms due to their amenability. The chassis-design space has therefore remained underexplored as an engineering variable. In this work, we explored the design space of a genetic toggle switch through variations in nine ribosome binding sites compositions and three host contexts, creating 27 circuit variants. Characterization of performance metrics in terms of toggle switch output and host growth dynamics unveils a spectrum of performance profiles from our circuit library. We find that changes in host-context causes large shifts in overall performance, while modulating ribosome binding sites leads to more incremental changes. We find that a combined ribosome binding site and host-context modulation approach can be used to fine tune the properties of a toggle switch according to user-defined specifications, such as towards greater signaling strength, inducer sensitivity or both. Other auxiliary properties, such as inducer tolerance, are also exclusively accessed through changes in host-context. We demonstrate here that exploration of the chassis-design space can offer significant value, reconceptualizing the chassis-organism as an important part in the synthetic biology toolbox with important implications for the field of synthetic biology.
{"title":"Fine Tuning Genetic Circuits via Host Context and RBS Modulation","authors":"Dennis Tin Chat Chan, Lena Winter, Johan Bjerg, Stina Krsmanovic, Geoff S. Baldwin, Hans C. Bernstein","doi":"10.1101/2024.07.20.604438","DOIUrl":"https://doi.org/10.1101/2024.07.20.604438","url":null,"abstract":"The choice of organism to host a genetic circuit, the chassis, is often defaulted to model organisms due to their amenability. The chassis-design space has therefore remained underexplored as an engineering variable. In this work, we explored the design space of a genetic toggle switch through variations in nine ribosome binding sites compositions and three host contexts, creating 27 circuit variants. Characterization of performance metrics in terms of toggle switch output and host growth dynamics unveils a spectrum of performance profiles from our circuit library. We find that changes in host-context causes large shifts in overall performance, while modulating ribosome binding sites leads to more incremental changes. We find that a combined ribosome binding site and host-context modulation approach can be used to fine tune the properties of a toggle switch according to user-defined specifications, such as towards greater signaling strength, inducer sensitivity or both. Other auxiliary properties, such as inducer tolerance, are also exclusively accessed through changes in host-context. We demonstrate here that exploration of the chassis-design space can offer significant value, reconceptualizing the chassis-organism as an important part in the synthetic biology toolbox with important implications for the field of synthetic biology.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"41 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141745360","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 : 2024-07-20DOI: 10.1101/2024.07.20.602779
Lawton F Long, Shivani Biskunda, Ming"Peter" Yang, George C. Wu, Cassidy F Simas, Steven D Bruner, Carl A Denard
Mutations in the SRP54 gene are linked to the pathophysiology of severe congenital neutropenia (SCN). SRP54 is a key protein comprising one of the six protein subunits of the signal recognition particle responsible for co-translational targeting of proteins to the ER; mutations in SRP54 disrupt this process. Crystal structures and biochemical characterization of a few SRP54 mutants provide insights into how SRP54 mutations affect its function. However, to date, no scalable, flexible platform exists to study the sequence-structure-function relationships of SRP54 mutations and perform functional genomics and genome-wide association studies. In this work, we established a haploid model in Saccharomyces cerevisiae based on inducible gene expression that allows these relationships to be studied. We employed this model to test the function of orthologous clinical mutations to demonstrate the model's suitability for studying SCN. Lastly, we demonstrate the suspected dominant-negative phenotypes associated with SRP54 mutants. In doing so, we discovered for the first time that the most common yeast orthologous clinical mutation, S125del (T117del human orthologue) displayed the least severe growth defect while the less common G234E mutant (G226E human orthologue) displayed the most severe growth defect. The ability of this haploid model to recapitulate these phenotypes while remaining amenable to high-throughput screening approaches makes it a powerful tool for studying SRP54. Furthermore, the methodology used to create this model may also be used to study other human diseases involving essential and quasi-essential genes.
{"title":"A scalable genetic tool for the functional analysis of the signal recognition particle.","authors":"Lawton F Long, Shivani Biskunda, Ming\"Peter\" Yang, George C. Wu, Cassidy F Simas, Steven D Bruner, Carl A Denard","doi":"10.1101/2024.07.20.602779","DOIUrl":"https://doi.org/10.1101/2024.07.20.602779","url":null,"abstract":"Mutations in the SRP54 gene are linked to the pathophysiology of severe congenital neutropenia (SCN). SRP54 is a key protein comprising one of the six protein subunits of the signal recognition particle responsible for co-translational targeting of proteins to the ER; mutations in SRP54 disrupt this process. Crystal structures and biochemical characterization of a few SRP54 mutants provide insights into how SRP54 mutations affect its function. However, to date, no scalable, flexible platform exists to study the sequence-structure-function relationships of SRP54 mutations and perform functional genomics and genome-wide association studies. In this work, we established a haploid model in Saccharomyces cerevisiae based on inducible gene expression that allows these relationships to be studied. We employed this model to test the function of orthologous clinical mutations to demonstrate the model's suitability for studying SCN. Lastly, we demonstrate the suspected dominant-negative phenotypes associated with SRP54 mutants. In doing so, we discovered for the first time that the most common yeast orthologous clinical mutation, S125del (T117del human orthologue) displayed the least severe growth defect while the less common G234E mutant (G226E human orthologue) displayed the most severe growth defect. The ability of this haploid model to recapitulate these phenotypes while remaining amenable to high-throughput screening approaches makes it a powerful tool for studying SRP54. Furthermore, the methodology used to create this model may also be used to study other human diseases involving essential and quasi-essential genes.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"17 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141745249","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}
The efficacy of current gene therapy approaches using adeno associated virus (AAV) vectors is limited by the poor control over their tissue tropism. Untargeted AAV vectors require high doses to achieve therapeutic efficacy, which is associated with toxic off-target impacts and increased therapeutic costs. The ability to reprogram existing AAV vectors to selectively transduce target tissues is essential to develop next-generation human gene therapies that are safer, more efficacious, and less expensive. Using selective and high-affinity antibodies and antibody-like proteins to retarget existing AAV vectors to bind novel cell-surface receptors offers an attractive and modular approach to reprogram their tropism. However, attaching these proteins onto the complex and delicate AAV capsids remains challenging. Here, we report a versatile chemical strategy to covalently attach recombinant proteins onto the capsid of AAV, using a combination of genetic code expansion and bioorthogonal conjugation chemistry. This method is efficient, and allows precise control over the site and stoichiometry of protein attachment onto the AAV capsid, enabling systematic optimization of the resulting conjugate. Using this approach, we generated conjugates of AAV2 with an anti-HER2 nanobody and a full-length anti-HER2 IgG, which show highly efficient and selective gene delivery into HER2+ cancer cells. Remarkably, the optimized AAV2-nanobody conjugate facilitated efficient transduction of HER2+ tumor xenograft in mice with little off-target gene expression, including in the liver. Programmable synthesis of AAV-protein conjugates using this method offers a promising new strategy to rationally engineer next-generation gene therapy vectors.
{"title":"A facile chemical strategy to synthesize precise AAV-protein conjugates for targeted gene delivery","authors":"Quan Pham, Jake Glicksman, Seyed Sadegh Shahraeini, Boyang Han, Delilah Jewel, Conor Loynd, Soumya Jyoti Singha Roy, Abhishek Chatterjee","doi":"10.1101/2024.07.20.604406","DOIUrl":"https://doi.org/10.1101/2024.07.20.604406","url":null,"abstract":"The efficacy of current gene therapy approaches using adeno associated virus (AAV) vectors is limited by the poor control over their tissue tropism. Untargeted AAV vectors require high doses to achieve therapeutic efficacy, which is associated with toxic off-target impacts and increased therapeutic costs. The ability to reprogram existing AAV vectors to selectively transduce target tissues is essential to develop next-generation human gene therapies that are safer, more efficacious, and less expensive. Using selective and high-affinity antibodies and antibody-like proteins to retarget existing AAV vectors to bind novel cell-surface receptors offers an attractive and modular approach to reprogram their tropism. However, attaching these proteins onto the complex and delicate AAV capsids remains challenging. Here, we report a versatile chemical strategy to covalently attach recombinant proteins onto the capsid of AAV, using a combination of genetic code expansion and bioorthogonal conjugation chemistry. This method is efficient, and allows precise control over the site and stoichiometry of protein attachment onto the AAV capsid, enabling systematic optimization of the resulting conjugate. Using this approach, we generated conjugates of AAV2 with an anti-HER2 nanobody and a full-length anti-HER2 IgG, which show highly efficient and selective gene delivery into HER2+ cancer cells. Remarkably, the optimized AAV2-nanobody conjugate facilitated efficient transduction of HER2+ tumor xenograft in mice with little off-target gene expression, including in the liver. Programmable synthesis of AAV-protein conjugates using this method offers a promising new strategy to rationally engineer next-generation gene therapy vectors.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"22 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141745361","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: 10.1101/2024.07.19.604288
Nina Y Alperovich, Olga B Vasilyeva, Samuel W Schaffter
Self-cleaving ribozymes are important tools in synthetic biology, biomanufacturing, and nucleic acid therapeutics. These broad applications deploy ribozymes in many genetic and environmental contexts, which can influence activity. Thus, accurate measurements of ribozyme activity across diverse contexts are crucial for validating new ribozyme sequences and ribozyme-based biotechnologies. Ribozyme activity measurements that rely on RNA extraction, such as RNA sequencing or reverse transcription-quantitative polymerase chain reaction (RT-qPCR), are generalizable to most applications and have high sensitivity. However, the activity measurement is indirect, taking place after RNA is isolated from the environment of interest and copied to DNA. So these measurements may not accurately reflect the activity in the original context. Here we develop and validate an RT-qPCR method for measuring context-dependent ribozyme activity using a set of self-cleaving RNAs for which context-dependent ribozyme cleavage is known in vitro. We find that RNA extraction and reverse transcription conditions can induce substantial ribozyme cleavage resulting in incorrect activity measurements with RT-qPCR. To restore the accuracy of the RT-qPCR measurements, we introduce an oligonucleotide into the sample preparation workflow that inhibits ribozyme activity. We then apply our method to measure ribozyme cleavage of RNAs produced in Escherichia coli (E. coli). These results have broad implications for many ribozyme measurements and technologies.
{"title":"Prevention of ribozyme catalysis through cDNA synthesis enables accurate RT-qPCR measurements of context-dependent ribozyme activity","authors":"Nina Y Alperovich, Olga B Vasilyeva, Samuel W Schaffter","doi":"10.1101/2024.07.19.604288","DOIUrl":"https://doi.org/10.1101/2024.07.19.604288","url":null,"abstract":"Self-cleaving ribozymes are important tools in synthetic biology, biomanufacturing, and nucleic acid therapeutics. These broad applications deploy ribozymes in many genetic and environmental contexts, which can influence activity. Thus, accurate measurements of ribozyme activity across diverse contexts are crucial for validating new ribozyme sequences and ribozyme-based biotechnologies. Ribozyme activity measurements that rely on RNA extraction, such as RNA sequencing or reverse transcription-quantitative polymerase chain reaction (RT-qPCR), are generalizable to most applications and have high sensitivity. However, the activity measurement is indirect, taking place after RNA is isolated from the environment of interest and copied to DNA. So these measurements may not accurately reflect the activity in the original context. Here we develop and validate an RT-qPCR method for measuring context-dependent ribozyme activity using a set of self-cleaving RNAs for which context-dependent ribozyme cleavage is known in vitro. We find that RNA extraction and reverse transcription conditions can induce substantial ribozyme cleavage resulting in incorrect activity measurements with RT-qPCR. To restore the accuracy of the RT-qPCR measurements, we introduce an oligonucleotide into the sample preparation workflow that inhibits ribozyme activity. We then apply our method to measure ribozyme cleavage of RNAs produced in Escherichia coli (E. coli). These results have broad implications for many ribozyme measurements and technologies.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"20 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141745362","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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