bioRxiv - Synthetic Biology最新文献

Cas9AEY (Cas9-facilitated Homologous Recombination Assembly of non-specific Escherichia coli yeast vector) method of constructing large-sized DNA. Cas9AEY（Cas9-facilitated Homologous Recombination Assembly of non-specific Escherichia coli yeast vector）构建大尺寸 DNA 的方法。

bioRxiv - Synthetic Biology Pub Date : 2024-09-18 DOI: 10.1101/2024.09.17.611575

xiaoshu ma, lei yang, hua ye

{"title":"Cas9AEY (Cas9-facilitated Homologous Recombination Assembly of non-specific Escherichia coli yeast vector) method of constructing large-sized DNA.","authors":"xiaoshu ma, lei yang, hua ye","doi":"10.1101/2024.09.17.611575","DOIUrl":"https://doi.org/10.1101/2024.09.17.611575","url":null,"abstract":"Saccharomyces cerevisiae is widely used in DNA assembly due to their efficient homologous recombination [1], but DNA assembly through yeast recombination in vivo usually requires the vector to have the ability to replicate in yeast. The CRISPR-Cas9 system can efficiently edit DNA [2,3], and the system can also be used for DNA editing of plasmids. In this paper, a yeast universal element is selected, which can be inserted into the vector, so that the vector can replicate in yeast cells, and then the intermediate plasmid containing yeast universal element can be obtained by recombination in yeast. At the same time, a pCas-SmR plasmid was designed in this paper. After Donor DNA is added, the CRISPR-Cas9 system can accurately and efficiently knock out the yeast universal element in the intermediate plasmid, remove the pCas-SmR plasmid through sucrose screening, and finally obtain a pure plasmid. Saccharomyces cerevisiae cells are widely used in DNA assembly due to their efficient homologous recombination [1], but DNA assembly through yeast recombination in vivo usually requires the vector to have the ability to replicate in yeast. The CRISPR-Cas9 system can efficiently edit DNA [2,3], and the system can also be used for DNA editing of plasmids. In this paper, a yeast universal element is selected, which can be inserted into the vector, so that the vector has the ability to replicate in yeast cells, and then the intermediate plasmid containing yeast universal element can be obtained by recombination in yeast. At the same time, a pCas-SmR plasmid was designed in this paper. After Donor DNA is added, the CRISPR-Cas9 system can accurately and efficiently knock out the yeast universal element in the intermediate plasmid, remove the pCas-SmR plasmid through sucrose screening, and finally obtain a pure knocked out plasmid.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142250791","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

DNA-templated spatially controlled proteolysis targeting chimeras for CyclinD1-CDK4/6 complex protein degradation DNA模板空间控制蛋白水解靶向嵌合体促进CyclinD1-CDK4/6复合物蛋白质降解

bioRxiv - Synthetic Biology Pub Date : 2024-09-18 DOI: 10.1101/2024.09.18.613743

Rong Zheng, Abhay Prasad, Deeksha Satyabola, Yang Xu, Hao Yan

{"title":"DNA-templated spatially controlled proteolysis targeting chimeras for CyclinD1-CDK4/6 complex protein degradation","authors":"Rong Zheng, Abhay Prasad, Deeksha Satyabola, Yang Xu, Hao Yan","doi":"10.1101/2024.09.18.613743","DOIUrl":"https://doi.org/10.1101/2024.09.18.613743","url":null,"abstract":"Constraining proximity-based drugs, such as proteolysis-targeting chimeras (PROTACs), into its bioactive conformation can significantly impact their selectivity and potency. However, traditional methods for achieving this often involve complex and time-consuming synthetic procedures. Here, we introduced an alternative approach by demonstrating DNA-templated spatially controlled PROTACs (DTACs), which leverage the programmability of nucleic-acid based self-assembly for efficient synthesis, providing precise control over inhibitors spacing and orientation. The resulting constructs revealed distance-and orientation-dependent selectivity and degradation potency for the CyclinD1-CDK4/6 protein complex in cancer cells. Notably, an optimal construct DTAC-V1 demonstrated the unprecedented synchronous degradation of entire CyclinD1-CDK4/6 complex. This resulted in the effective cell cycle arrest in G1 phase, and further therapeutic studies showed its potent anti-tumor effects compared to inhibitors alone. These findings present a novel framework for PROTACs design, offering critical insights that may inform the development of other proximity-induced therapeutic modalities.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142250790","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

High-efficiency transformation and gene expression in Picosynechococcus sp. PCC 7002 Picosynechococcus sp. PCC 7002 的高效转化和基因表达

bioRxiv - Synthetic Biology Pub Date : 2024-09-17 DOI: 10.1101/2024.09.17.613521

Andrew P Hren, Joshua P Abraham, Melissa P. Tumen-Velasquez, Michael Melesse Vergara, Adam M Guss, William G Alexander, Brian F Pfleger, Jerome M Fox, Carrie A Eckert

引用次数: 0

A modular system for programming multistep activation of endogenous genes in stem cells 干细胞内源基因多步激活编程模块系统

bioRxiv - Synthetic Biology Pub Date : 2024-09-17 DOI: 10.1101/2024.09.17.613466

Anupama K Puppala, Andrew C Nielsen, Maureen R Regan, Georgina E Mancinelli, Renee F DePooter, Stephen Arnovitz, Caspian Harding, Michaele McGregor, Nikolas G Balanis, Ryan Clarke, Brad J Merrill

引用次数: 0

Metabolite-responsive Control of Transcription by Phase Separation-based Synthetic Organelles 基于相分离的合成细胞器对转录的代谢物响应控制

bioRxiv - Synthetic Biology Pub Date : 2024-09-17 DOI: 10.1101/2024.09.17.613456

Carolina Jerez-Longres, Wilfried Weber

{"title":"Metabolite-responsive Control of Transcription by Phase Separation-based Synthetic Organelles","authors":"Carolina Jerez-Longres, Wilfried Weber","doi":"10.1101/2024.09.17.613456","DOIUrl":"https://doi.org/10.1101/2024.09.17.613456","url":null,"abstract":"Living natural materials have remarkable sensing abilities that translate external cues into functional changes of the material. The reconstruction of such sensing materials in bottom-up synthetic biology provides the opportunity to develop synthetic materials with life-like sensing and adaptation ability. Key to such functions are material modules that translate specific input signals into a biomolecular response. Here, we engineer a synthetic organelle based on liquid-liquid phase separation that translates a metabolic signal into the regulation of gene transcription. To this aim, we engineer the pyruvate-dependent repressor PdhR to undergo liquid-liquid phase separation in vitro by fusion to intrinsically disordered regions. We demonstrate that the resulting coacervates bind DNA harbouring PdhR-responsive operator sites in a pyruvate dose-dependent and reversible manner. We observed that the activity of transcription units on the DNA was strongly attenuated following recruitment to the coacervates. However, the addition of pyruvate resulted in a reversible and dose-dependent reconstitution of transcriptional activity. The coacervate-based synthetic organelles linking metabolic cues to transcriptional signals represent a materials approach to confer stimulus-responsiveness to minimal bottom-up synthetic biological systems and open opportunities in materials for sensor applications.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142250793","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

Mutual dependence between membrane phase separation and bacterial division protein dynamics in synthetic cell models 合成细胞模型中膜相分离与细菌分裂蛋白动态之间的相互依存关系

bioRxiv - Synthetic Biology Pub Date : 2024-09-17 DOI: 10.1101/2024.09.17.613417

Nishu Kanwa, Shunshi Kohyama, Leonard Fröhlich, Amogh Desai, Petra Schwille

{"title":"Mutual dependence between membrane phase separation and bacterial division protein dynamics in synthetic cell models","authors":"Nishu Kanwa, Shunshi Kohyama, Leonard Fröhlich, Amogh Desai, Petra Schwille","doi":"10.1101/2024.09.17.613417","DOIUrl":"https://doi.org/10.1101/2024.09.17.613417","url":null,"abstract":"Cell membranes in bacteria are laterally polarized to produce specific environments for membrane proteins, e.g., proteins involved in cell division which accumulate at mid-cell or the cell poles. An interesting result of such membrane-lipid interplay is the reorganization of lipid domains together with membrane-bound proteins at the onset of cell division, suggesting a functional significance of membrane compartments in the cell cycle. Here, by adopting the key bacterial division proteins MinCDE and FtsZ as an archetypal spatial patterning system, we present a simple vesicle-based in vitro model to explore the mutual dependence of protein pattern formation and membrane heterogeneity. Like many other peripheral membrane proteins, MinDE exhibit preferential binding and macro-scale pattern formation at Ld domains, which leads to altered oscillation mode selection in phase-separated membrane compartments (GUVs). Moreover, incorporating bacterial division proteins within phase-separated GUVs leads to blebbing-like membrane deformations followed by the reorganization of Lo domains aligning at the neck region of the bleb, which agrees well with the domain rearrangement in bacterial membranes immediately preceding the radial constriction process. Overall, the presented in vitro model system showcases a basic framework to better comprehend the cellular division mechanism in consideration of complex cellular lipid environments.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142250795","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

Modular Nano-Scaffold Biocatalysis for Superior PET Depolymerization and Valorization 模块化纳米支架生物催化技术实现卓越的 PET 解聚和增值

bioRxiv - Synthetic Biology Pub Date : 2024-09-16 DOI: 10.1101/2024.09.16.613172

Yujia Zhang, Chongsen Li, Ehsan Hashemi, Enting Xu, Xuemei Yang, Yanbing Lin, Hui Gao, Zhuobin Liang

{"title":"Modular Nano-Scaffold Biocatalysis for Superior PET Depolymerization and Valorization","authors":"Yujia Zhang, Chongsen Li, Ehsan Hashemi, Enting Xu, Xuemei Yang, Yanbing Lin, Hui Gao, Zhuobin Liang","doi":"10.1101/2024.09.16.613172","DOIUrl":"https://doi.org/10.1101/2024.09.16.613172","url":null,"abstract":"The global crisis of polyethylene terephthalate (PET) waste demands innovative solutions for sustainable management. Current approaches are often inefficient, energy-intensive, and result in incomplete depolymerization. Here, we introduce SPEED (Scaffold-enabled PET Enzyme Ensemble-augmented Degradation), a transformative biocatalytic platform engineered for the superior degradation across diverse PET substrates. Through the strategic combination of complementary PET hydrolases on a tailored protein nano-scaffold and extensive optimization, SPEED achieves near-complete depolymerization of PET into its constituent monomers, exceeding existing biocatalytic systems' efficiency by up to two orders of magnitude. The platform's versatility and industrial relevance are further demonstrated through successful integration with metal-organic frameworks (MOFs) for enhanced stability and reusability, enabling PET upcycling into valuable products, and its compatibility with a yeast-based live cell system for surface display. SPEED's high efficiency, adaptability, and cost-effectiveness position it as a powerful technology to accelerate sustainable plastic waste management and drive a circular PET economy.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142250796","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

De novo RNA base editing in plant organelles with engineered synthetic P-type PPR editing factors 利用人工合成的 P 型 PPR 编辑因子在植物细胞器中进行新的 RNA 碱基编辑

bioRxiv - Synthetic Biology Pub Date : 2024-09-16 DOI: 10.1101/2024.09.13.612905

Sebastien Mathieu, Elena Lesch, Shahinez Garcia, Stefanie Graindorge, Mareike Schallenberg-Rudinger, Kamel Hammani

引用次数: 0

Decoding Specificity of Cyanobacterial MysDs in Mycosporine-Like Amino Acid Biosynthesis through Heterologous Expression in Saccharomyces cerevisiae 通过在酿酒酵母中异源表达解码蓝藻 MysDs 在类霉菌素氨基酸合成过程中的特异性

bioRxiv - Synthetic Biology Pub Date : 2024-09-15 DOI: 10.1101/2024.09.14.613006

Xiaoyou Zheng, Peifeng Xie, Andrew Chen Cai, Yuze Jiang, Sirui Huang, Xiaochong Ma, Honghao Su, Boxiang Wang

{"title":"Decoding Specificity of Cyanobacterial MysDs in Mycosporine-Like Amino Acid Biosynthesis through Heterologous Expression in Saccharomyces cerevisiae","authors":"Xiaoyou Zheng, Peifeng Xie, Andrew Chen Cai, Yuze Jiang, Sirui Huang, Xiaochong Ma, Honghao Su, Boxiang Wang","doi":"10.1101/2024.09.14.613006","DOIUrl":"https://doi.org/10.1101/2024.09.14.613006","url":null,"abstract":"Mycosporine-like amino acids (MAAs) are potent natural UV-protectants, but their industrial production is hindered by efficiency and sustainability issues of large-scale extraction of their native hosts. Heterologous expression of MAA biosynthesis pathway genes in chassis organisms provides a promising alternative route, though the substrate promiscuity of the ATP-grasp ligase MysD complicates the biosynthesis of specific MAAs. In this study, we developed a <em>Saccharomyces cerevisiae</em> strain with enhanced capacity of producing mycosporine-glycine (MG), through genomic expression of biosynthesis pathway genes and knockout of competing pathway genes. This strain serves as an efficient MysD expression platform, which converts MG into shinorine and porphyra-334. Through structural modelling, site-directed mutagenesis and mutant characterization, we identified two residues on the omega-loop of MysD involved in determining product specificity. We further characterized the product specificity of 20 MysDs from diverse cyanobacterial lineages and confirmed the residue pattern-product specificity correlation. Our findings provide guidance for screening, selecting, and designing novel MysDs for industrial-scale MAA production through heterologous expression.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142250797","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

Light-triggered protease-mediated release of actin-bound cargo from synthetic cells 光诱导蛋白酶介导合成细胞释放肌动蛋白结合的货物

bioRxiv - Synthetic Biology Pub Date : 2024-09-15 DOI: 10.1101/2024.09.15.613133

Mousumi Akter, Hossein Moghimianavval, Gary D Luker, Allen P Liu

{"title":"Light-triggered protease-mediated release of actin-bound cargo from synthetic cells","authors":"Mousumi Akter, Hossein Moghimianavval, Gary D Luker, Allen P Liu","doi":"10.1101/2024.09.15.613133","DOIUrl":"https://doi.org/10.1101/2024.09.15.613133","url":null,"abstract":"Synthetic cells offer a versatile platform for addressing biomedical and environmental challenges, due to their modular design and capability to mimic cellular processes such as biosensing, intercellular communication, and metabolism. Constructing synthetic cells capable of stimuli-responsive secretion is vital for applications in targeted drug delivery and biosensor development. Previous attempts at engineering secretion for synthetic cells have been confined to non-specific cargo release via membrane pores, limiting the spatiotemporal precision and specificity necessary for selective secretion. Here, we designed and constructed a protein-based platform termed TEV Protease-mediated Releasable Actin-binding protein (TRAP) for selective, rapid, and triggerable secretion in synthetic cells. TRAP is designed to bind tightly to reconstituted actin networks and is proteolytically released from bound actin, followed by secretion via cell-penetrating peptide membrane translocation. We demonstrated TRAP's efficacy in facilitating light-activated secretion of both fluorescent and luminescent proteins. By equipping synthetic cells with a controlled secretion mechanism, TRAP paves the way for the development of stimuli-responsive biomaterials, versatile synthetic cell-based biosensing systems, and therapeutic applications through the integration of synthetic cells with living cells for targeted delivery of protein therapeutics.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142250832","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