bioRxiv - Synthetic Biology最新文献_第4页

Engineering pathogen-inducible promoters for conferring disease resistance in tomato 改造病原体诱导启动子，赋予番茄抗病性

bioRxiv - Synthetic Biology Pub Date : 2024-08-31 DOI: 10.1101/2024.08.30.610566

Wei Wei, Doogie Kim, Naio Koehler, Ashley Bendl, Myeong-Je Cho, Ksenia Krasileva

{"title":"Engineering pathogen-inducible promoters for conferring disease resistance in tomato","authors":"Wei Wei, Doogie Kim, Naio Koehler, Ashley Bendl, Myeong-Je Cho, Ksenia Krasileva","doi":"10.1101/2024.08.30.610566","DOIUrl":"https://doi.org/10.1101/2024.08.30.610566","url":null,"abstract":"Plant diseases pose a significant threat to global crop production. Most disease resistance genes used in crop breeding programs encode nucleotide-binding leucine-rich repeat receptors (NLRs) that are limited in pathogen specificity and durability. In this study, we leveraged synthetic biology to develop an inducible broad-spectrum resistance in tomatoes. Constitutive expression of autoactive NLRs in plants leads to robust resistance against multiple pathogens but significantly stunts growth. We expressed autoactive NLRs under the control of pathogen-inducible (PI) promoters to mitigate the fitness costs. Taking advantage of extensive, new genomic and transcriptomic resources, we identified PI promoters that responded to multiple pathogens but not abiotic stress. We further validated functionality of predicted elements through a promoter luciferase assay. We generated significant resistance in transgenic tomatoes but we also encountered unwanted expression induction of the native promoter regions in flowers which led to lethal fruit development. Thus, we pursued promoter engineering for fine-tuning the induction. We identified cis-regulatory regions responsible for pathogen-inducibility through promoter bashing experiments and recombined the native promoter with the inducible part and the core promoter. Furthermore, we rationally created synthetic promoters showing a gradient of expression levels, which will allow for selection for transgenic tomatoes with the best performance. We found that the spacing between functional sequences, repeat number of inducible sequences, and core promoters all influence the outcome of engineering. Our study outlines a framework for developing broad-spectrum synthetic immune constructs with reduced fitness cost and provides examples of pathogen-inducible promoter engineering.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142226314","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

Effects of lipid membranes on RNA catalytic activity and stability 脂质膜对 RNA 催化活性和稳定性的影响

bioRxiv - Synthetic Biology Pub Date : 2024-08-31 DOI: 10.1101/2024.08.31.610601

Tomasz Czerniak, James Peter Saenz

{"title":"Effects of lipid membranes on RNA catalytic activity and stability","authors":"Tomasz Czerniak, James Peter Saenz","doi":"10.1101/2024.08.31.610601","DOIUrl":"https://doi.org/10.1101/2024.08.31.610601","url":null,"abstract":"RNA plays crucial roles in cellular organization and metabolism, and modulating its activity is essential for maintaining cellular functions. RNA activity, involving both catalytic (ribozymes) and translation processes, is controlled via myriad mechanisms involving different binding partners such as proteins and smaller polar solutes. We previously reported that lipid membranes can directly interact with the artificial R3C ribozyme, changing its activity; however, the effect of lipids on naturally occurring ribozymes remains unknown. Here, we report that both catalytic activity and RNA integrity can be controlled by the presence of different lipid membranes. Lipid gel membranes decreased the activity of hepatitis delta virus (HDV) and increased the hammerhead (HH) ribozyme reaction yield. The presence of lipid liquid membrane lattices triggered RNA degradation, with greater degradation occurring in the single-stranded regions of RNA. The interplay between RNA activity and stability in the presence of different lipid membranes introduces multiple possibilities, where different combinations of ribozyme and lipid membrane composition could produce different effects on activity. Taken together, these observations support the hypothesis that the activity of both natural and artificial RNAs can be modulated by lipid membranes, which, in turn, contribute to the development of novel riboswitch-like molecules and lipid membrane-based RNA-biosensors.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206735","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

Analysis and control of untemplated DNA polymerase activity for guided synthesis of kilobase-scale DNA sequences 分析和控制非模板 DNA 聚合酶活性，引导合成千碱基级 DNA 序列

bioRxiv - Synthetic Biology Pub Date : 2024-08-30 DOI: 10.1101/2024.08.29.610422

Simeon D. Castle, Adrian Woolfson, Gregory Linshiz, Blake T. Riley, Ifor D.W. Samuel, Philipp Holliger, Lauren Oldfield, Andrew Hessel, Thomas E. Gorochowski

{"title":"Analysis and control of untemplated DNA polymerase activity for guided synthesis of kilobase-scale DNA sequences","authors":"Simeon D. Castle, Adrian Woolfson, Gregory Linshiz, Blake T. Riley, Ifor D.W. Samuel, Philipp Holliger, Lauren Oldfield, Andrew Hessel, Thomas E. Gorochowski","doi":"10.1101/2024.08.29.610422","DOIUrl":"https://doi.org/10.1101/2024.08.29.610422","url":null,"abstract":"DNA polymerases are complex molecular machines able to replicate genetic material using a template-driven process. While the copying function of these enzymes is well established, their ability to perform untemplated DNA synthesis is less well characterized. Here, we explore the ability of DNA polymerases to synthesize DNA fragments in the absence of template. We use long-read nanopore sequencing and real-time PCR to observe the synthesis of pools of DNA products derived from a diverse set of natural and engineered DNA polymerases across varying temperatures and buffer compositions. We detail the features of the DNA fragments generated, enrichment of select sequence motifs, and demonstrate that the sequence composition of the synthesized DNA may be altered by modifying environmental conditions. This work provides an extensive data set to better discern the process of untemplated DNA polymerase activity and may support its potential repurposing as a technology for the guided synthesis of DNA sequences on the kilobase-scale and beyond.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206733","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

Genome-wide A to G and C to T Mutations Induced by Functional TadA Variants in Escherichia coli 大肠杆菌中功能性 TadA 变体诱导的全基因组 A 到 G 和 C 到 T 突变

bioRxiv - Synthetic Biology Pub Date : 2024-08-30 DOI: 10.1101/2024.08.29.610230

Hao Wang, Zhengxin Dong, Jingyi Shi, Lei Chen, Tao Sun, Weiwen Zhang

{"title":"Genome-wide A to G and C to T Mutations Induced by Functional TadA Variants in Escherichia coli","authors":"Hao Wang, Zhengxin Dong, Jingyi Shi, Lei Chen, Tao Sun, Weiwen Zhang","doi":"10.1101/2024.08.29.610230","DOIUrl":"https://doi.org/10.1101/2024.08.29.610230","url":null,"abstract":"The fusion expression of DNA replication-related proteins with nucleotide deaminase enzymes promotes random mutations in bacterial genomes, thereby increasing genetic diversity. However, most previous studies have focused on cytosine deaminase, which only produces C to T mutations, significantly limiting the variety of mutation types. In this study, we developed a fusion expression system in Escherichia coli by combining DnaG (RNA primase) with adenine deaminase TadA-8e (DnaG-TadA), which rapidly introduced A to G mutations into the E. coli genome, resulting in a 664-fold increase in mutation rate. Additionally, we engineered a dual-functional TadA variant, TadAD, and fused it with DnaG. This construct introduced both C to T and A to G mutations into the genome, with the mutation rate further increased by 370-fold upon co-expression with an uracil glycosylase inhibitor (DnaG-TadAD-UGI). We applied DnaG-TadA and DnaG-TadAD-UGI to adaptive laboratory evolution for Cd2+ and kanamycin resistance, achieving an 8.0 mM Cd2+ and 200 ug/mL kanamycin tolerance within just 17 days and 132 hours, respectively. Compared to standard evolution methods, the final tolerance levels increased by 320% and 266%, respectively. Our work offers a novel strategy for random mutagenesis in E. coli and potentially other species.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206732","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

Design of diverse, functional mitochondrial targeting sequences across eukaryotic organisms using variational autoencoder 利用变异自动编码器设计真核生物中多样化的功能性线粒体靶向序列

bioRxiv - Synthetic Biology Pub Date : 2024-08-29 DOI: 10.1101/2024.08.28.610205

Aashutosh Girish Boob, Shih-I Tan, Airah Zaidi, Nilmani Singh, Xueyi Xue, Shuaizhen Zhou, Teresa A Martin, Li-Qing Chen, Huimin Zhao

{"title":"Design of diverse, functional mitochondrial targeting sequences across eukaryotic organisms using variational autoencoder","authors":"Aashutosh Girish Boob, Shih-I Tan, Airah Zaidi, Nilmani Singh, Xueyi Xue, Shuaizhen Zhou, Teresa A Martin, Li-Qing Chen, Huimin Zhao","doi":"10.1101/2024.08.28.610205","DOIUrl":"https://doi.org/10.1101/2024.08.28.610205","url":null,"abstract":"Mitochondria play a key role in energy production and cellular metabolism, making them a promising target for metabolic engineering and disease treatment. However, despite the known influence of passenger proteins on localization efficiency, only a few protein-localization tags have been characterized for mitochondrial targeting. To address this limitation, we exploited Variational Autoencoder (VAE), an unsupervised deep learning framework, to design novel mitochondrial targeting sequences (MTSs). In silico analysis revealed that a high fraction of generated peptides are functional and possess features important for mitochondrial targeting. Additionally, we devised a sampling scheme to indirectly address biases arising from the differences in mitochondrial protein import machinery and characterized artificial MTSs in four eukaryotic organisms. These sequences displayed significant diversity, sharing less than 60% sequence identity with MTSs in the UniProt database. Moreover, we trained a separate VAE and employed latent space interpolation to design dual targeting sequences capable of targeting both mitochondria and chloroplasts, shedding light on their evolutionary origins. As a proof-of-concept, we demonstrate the application of these artificial MTSs in increasing titers of 3-hydroxypropionic acid through pathway compartmentalization and improving 5-aminolevulinate synthase delivery by 1.62-fold and 4.76-fold, respectively. Overall, our work not only demonstrates the potential of generative artificial intelligence in designing novel, functional mitochondrial targeting sequences but also highlights their utility in engineering mitochondria for both fundamental research and practical applications in biology.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206736","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

Genomically recoded Escherichia coli with optimized functional phenotypes 具有优化功能表型的基因组重编码大肠杆菌

bioRxiv - Synthetic Biology Pub Date : 2024-08-29 DOI: 10.1101/2024.08.29.610322

Farren Isaacs, Colin Hemez, Kyle Mohler, Felix Radford, Jack Moen, Jesse Rinehart

{"title":"Genomically recoded Escherichia coli with optimized functional phenotypes","authors":"Farren Isaacs, Colin Hemez, Kyle Mohler, Felix Radford, Jack Moen, Jesse Rinehart","doi":"10.1101/2024.08.29.610322","DOIUrl":"https://doi.org/10.1101/2024.08.29.610322","url":null,"abstract":"Genomically recoded organisms hold promise for many biotechnological applications, but they may exhibit substantial fitness defects relative to their non-recoded counterparts. We used targeted metabolic screens, genetic analysis, and proteomics to identify the origins of fitness impairment in a model recoded organism, Escherichia coli C321.∆A. We found that defects in isoleucine biosynthesis and release factor activity, caused by mutations extant in all K-12 lineage strains, elicited profound fitness impairments in C321.∆A, suggesting that genome recoding exacerbates suboptimal traits present in precursor strains. By correcting these and other C321.∆A-specific mutations, we engineered C321.∆A strains with doubling time reductions of 17% and 42% in rich and minimal medium, respectively, compared to ancestral C321. Strains with improved growth kinetics also demonstrated enhanced ribosomal non-standard amino acid incorporation capabilities. Proteomic analysis indicated that C321.∆A lacks the ability to regulate essential amino acid and nucleotide biosynthesis pathways, and that targeted mutation reversion restored regulatory capabilities. Our work outlines a strategy for the rapid and precise phenotypic optimization of genomically recoded organisms and other engineered microbes.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206737","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

Optimizing Plant Biofactories: Enhancing Recombinant Protein Production in Nicotiana benthamiana through Phytoplasma Effectors 优化植物生物工厂：通过植物原生质体效应器提高烟草植物重组蛋白的产量

bioRxiv - Synthetic Biology Pub Date : 2024-08-29 DOI: 10.1101/2024.08.29.610350

Md Saifur Rahman

引用次数: 0

Reusing excess staple oligonucleotides for economical production of DNA origami 重复使用多余的主食寡核苷酸，经济地制作 DNA 折纸

bioRxiv - Synthetic Biology Pub Date : 2024-08-28 DOI: 10.1101/2024.08.27.609958

Giorgia Isinelli, Christopher Wintersinger, Pascal Lill, Olivia Young, Jie Deng, William Shih, Yang Zeng

{"title":"Reusing excess staple oligonucleotides for economical production of DNA origami","authors":"Giorgia Isinelli, Christopher Wintersinger, Pascal Lill, Olivia Young, Jie Deng, William Shih, Yang Zeng","doi":"10.1101/2024.08.27.609958","DOIUrl":"https://doi.org/10.1101/2024.08.27.609958","url":null,"abstract":"DNA origami has enabled the development of responsive drug-delivery vehicles with precision features that were previously not attainable in bionanotechnology. To reduce the costs of creating therapeutics-scale amounts of DNA origami that need to bear costly modifications with high occupancy, we reused the excess staple oligonucleotides that are leftover from the folding process to fold additional origami. We determined that a DNA origami can be successfully folded with up to 80% cost savings by cyclic recovery and reuse of excess staple strands. We found evidence that higher quality staple strands are preferentially incorporated into origami, consistent with past reports, and therefore are preferentially depleted from the free-strand pool. The folding of DNA origami with staple strands that were reused up to eleven times was indistinguishable by our panel of assays versus a control folded with new strands, so long as the reused oligonucleotides were replenished each cycle with a small excess of fresh strands. We also observed a high degree of incorporation of guests on the DNA origami. By recovering, reusing, and replenishing excess staple oligonucleotides, it is possible to significantly lessen production costs to create well-formed origami, which is useful to allow more therapeutic designs to be tested.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206780","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

Phase Separation-based Antiviral Decoy Particles as Basis for Programmable Broad-spectrum Therapeutics 基于相分离的抗病毒诱饵粒子是可编程广谱疗法的基础

bioRxiv - Synthetic Biology Pub Date : 2024-08-28 DOI: 10.1101/2024.08.28.610020

Or Willinger, Naor Granik, Sarah Goldberg, Roee Amit

引用次数: 0

Engineering Intercellular Communication using M13 Phagemid and CRISPR-based Gene Regulation for Multicellular Computing in Escherichia coli 利用 M13 Phagemid 和基于 CRISPR 的基因调控技术进行细胞间通信，在大肠杆菌中实现多细胞计算

bioRxiv - Synthetic Biology Pub Date : 2024-08-28 DOI: 10.1101/2024.08.28.610043

Hadiastri Kusumawardhani, Florian Zoppi, Roberto Avendaño, Yolanda Schaerli

{"title":"Engineering Intercellular Communication using M13 Phagemid and CRISPR-based Gene Regulation for Multicellular Computing in Escherichia coli","authors":"Hadiastri Kusumawardhani, Florian Zoppi, Roberto Avendaño, Yolanda Schaerli","doi":"10.1101/2024.08.28.610043","DOIUrl":"https://doi.org/10.1101/2024.08.28.610043","url":null,"abstract":"Engineering multicellular consortia, where information processing is distributed across specialized cell types, offers a promising strategy for implementing sophisticated biocomputing systems. However, a major challenge remains in establishing orthogonal intercellular communication, or \"wires,\" within synthetic bacterial consortia. In this study, we address this bottleneck by integrating phagemid-mediated intercellular communication with CRISPR-based gene regulation for multicellular computing in synthetic <em>E. coli</em> consortia. We achieve intercellular communication by regulating the transfer of single guide RNAs (sgRNAs) encoded on M13 phagemids from sender to receiver cells. Once inside the receiver cells, the transferred sgRNAs mediate gene regulation via CRISPR interference. Leveraging this approach, we successfully constructed one-, two-, and four-input logic gates. Our work expands the toolkit for intercellular communication and paves the way for complex information processing in synthetic microbial consortia, with diverse potential applications, including biocomputing, biosensing, and biomanufacturing.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142206740","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