bioRxiv - Synthetic Biology最新文献_第7页

Cell-free expression of Nipah virus transmembrane proteins for proteoliposome vaccine design 无细胞表达尼帕病毒跨膜蛋白以设计蛋白脂质体疫苗

bioRxiv - Synthetic Biology Pub Date : 2024-07-27 DOI: 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

{"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}

引用次数: 0

An Orthogonal T7 Replisome for Continuous Hypermutation and Accelerated Evolution in E. coli 用于大肠杆菌连续超突变和加速进化的正交 T7 Replisome

bioRxiv - Synthetic Biology Pub Date : 2024-07-26 DOI: 10.1101/2024.07.25.605042

Christian S. Diercks, Philipp Sondermann, Cynthia Rong, David A. Dik, Thomas G. Gillis, Yahui Ban, Peter G. Schultz

引用次数: 0

Preventing plasmid multimer formation in commonly used synthetic biology plasmids 防止常用合成生物学质粒中质粒多聚体的形成

bioRxiv - Synthetic Biology Pub Date : 2024-07-24 DOI: 10.1101/2024.07.23.604805

Elizabeth Vaisbourd, Anat Bren, Uri Alon, David Shaanan Glass

引用次数: 0

Expression and characterization of the complete cyanophage genome PP in the heterologous host Synechococcus elongatus PCC 7942 完整蓝藻噬菌体基因组 PP 在异源宿主伸长 Synechococcus PCC 7942 中的表达和特征描述

bioRxiv - Synthetic Biology Pub Date : 2024-07-24 DOI: 10.1101/2024.07.23.604706

Guorui Li, Jia Feng, Xiaofei Zhu, Yujie Chai, Tao Sun, Jianlan Jiang

{"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}

引用次数: 0

Integrative multi-omics data elucidating the biosynthesis and regulatory mechanisms of furanocoumarins in Angelica dahurica 多组学整合数据阐明白芷中呋喃香豆素的生物合成和调控机制

bioRxiv - Synthetic Biology Pub Date : 2024-07-24 DOI: 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

{"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}

引用次数: 0

Super-resolution imaging of proteins inside live mammalian cells with mLIVE-PAINT 利用 mLIVE-PAINT 对哺乳动物活细胞内的蛋白质进行超分辨率成像

bioRxiv - Synthetic Biology Pub Date : 2024-07-22 DOI: 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

引用次数: 0

Fine Tuning Genetic Circuits via Host Context and RBS Modulation 通过宿主语境和 RBS 调节微调遗传回路

bioRxiv - Synthetic Biology Pub Date : 2024-07-21 DOI: 10.1101/2024.07.20.604438

Dennis Tin Chat Chan, Lena Winter, Johan Bjerg, Stina Krsmanovic, Geoff S. Baldwin, Hans C. Bernstein

{"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}

引用次数: 0

A scalable genetic tool for the functional analysis of the signal recognition particle. 用于信号识别粒子功能分析的可扩展遗传工具。

bioRxiv - Synthetic Biology Pub Date : 2024-07-20 DOI: 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

{"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}

引用次数: 0

A facile chemical strategy to synthesize precise AAV-protein conjugates for targeted gene delivery 合成用于靶向基因递送的精确 AAV 蛋白共轭物的简便化学策略

bioRxiv - Synthetic Biology Pub Date : 2024-07-20 DOI: 10.1101/2024.07.20.604406

Quan Pham, Jake Glicksman, Seyed Sadegh Shahraeini, Boyang Han, Delilah Jewel, Conor Loynd, Soumya Jyoti Singha Roy, Abhishek Chatterjee

{"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}

引用次数: 0

Prevention of ribozyme catalysis through cDNA synthesis enables accurate RT-qPCR measurements of context-dependent ribozyme activity 通过 cDNA 合成防止核糖酶催化，可对上下文相关的核糖酶活性进行精确的 RT-qPCR 测量

bioRxiv - Synthetic Biology Pub Date : 2024-07-19 DOI: 10.1101/2024.07.19.604288

Nina Y Alperovich, Olga B Vasilyeva, Samuel W Schaffter

{"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}

引用次数: 0