Methods in molecular biology最新文献_第7页

Targeted Forward Genetics: Saturating Mutational Analyses of Specific Target Loci Within the Genome. 目标前向遗传学：基因组内特定目标位点的饱和突变分析。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4168-2_16

Reine U Protacio, Wayne P Wahls

引用次数: 0

Combining Oligo Pools and Golden Gate Cloning to Create Protein Variant Libraries or Guide RNA Libraries for CRISPR Applications. 结合寡核苷酸库和金门克隆技术，创建用于 CRISPR 应用的蛋白质变体库或引导 RNA 库。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4220-7_15

Alicia Maciá Valero, Rianne C Prins, Thijs de Vroet, Sonja Billerbeck

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Golden Gate-Assisted Gene Doctoring for Streamlined and Efficient Recombineering in Bacteria. 黄金闸门辅助基因诊断，实现简化高效的细菌重组。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4220-7_19

Nicholas M Thomson

{"title":"Golden Gate-Assisted Gene Doctoring for Streamlined and Efficient Recombineering in Bacteria.","authors":"Nicholas M Thomson","doi":"10.1007/978-1-0716-4220-7_19","DOIUrl":"10.1007/978-1-0716-4220-7_19","url":null,"abstract":"Gene Doctoring is a genetic modification technique for E. coli and related bacteria, in which the Red-recombinase from bacteriophage λ mediates chromosomal integration of a fragment of DNA by homologous recombination (known as recombineering). In contrast to the traditional recombineering method, the integrated fragment for Gene Doctoring is supplied on a donor plasmid rather than as a linear DNA. This protects the DNA from degradation, facilitates transformation, and ensures multiple copies are present per cell, increasing the efficiency and making the technique particularly suitable for strains that are difficult to modify. Production of the donor plasmid has, until recently, relied on traditional cloning techniques that are inflexible, tedious, and inefficient. This protocol describes a procedure for Gene Doctoring combined with Golden Gate assembly of a donor plasmid, using a custom-designed plasmid backbone, for rapid and simple production of complex, multi-part assemblies. Insertion of a gene for superfolder green fluorescent protein, with selection by tetracycline resistance, into E. coli strain MG1655 is used as an example but in principle the method can be tailored for virtually any modification in a wide range of bacteria.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2850 ","pages":"345-363"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142372292","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 DNA Construct Design for High-Throughput Golden Gate Assembly. 用于高通量金门组装的模块化 DNA 构建设计

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4220-7_4

Peter Vegh, Elliott Chapman, Craig Gilmour, Rennos Fragkoudis

引用次数: 0

Standardized Golden Gate Assembly Metadata Representation Using SBOL. 使用 SBOL 的标准化金门组件元数据表示。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4220-7_6

Gonzalo Vidal, Carolus Vitalis, Johan Guillén

引用次数: 0

Use of Bio-Layer Interferometry (BLI) to Measure Binding Affinities of SNAREs and Phosphoinositides. 利用生物层干涉法（BLI）测量SNAREs和磷酸肌苷的结合亲和力。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4314-3_7

Jorge D Calderin, Chi Zhang, Timothy J C Tan, Nicholas C Wu, Rutilio Fratti

{"title":"Use of Bio-Layer Interferometry (BLI) to Measure Binding Affinities of SNAREs and Phosphoinositides.","authors":"Jorge D Calderin, Chi Zhang, Timothy J C Tan, Nicholas C Wu, Rutilio Fratti","doi":"10.1007/978-1-0716-4314-3_7","DOIUrl":"https://doi.org/10.1007/978-1-0716-4314-3_7","url":null,"abstract":"Bio-Layer Interferometry (BLI) is a technique that uses optical biosensing to analyze interactions between molecules. The analysis of molecular interactions is measured in real-time and does not require fluorescent tags. BLI uses disposable biosensors that come in a variety of formats to bind different ligands including biotin, hexahistidine, GST, and the Fc portion of antibodies. Unlike surface plasmon resonance (SPR), BLI is an open system that does not require microfluidics, which eliminates issues that result from clogging and changes in viscosity. Importantly, BLI readings can be completed in minutes and can be formatted for high throughput screening. Here we use biotinylated short chain phosphoinositides and phosphatidic acid bound to streptavidin BLI biosensors to measure the binding of the soluble Qc SNARE Vam7 from Saccharomyces cerevisiae. Unlike most SNAREs, Vam7 lacks a transmembrane domain or lipid anchor to associate with membranes. Instead Vam7 associates to yeast vacuolar membranes using its N-terminal PX domain that binds to phosphatidylinositol 3-phosphate (PI3P) and phosphatidic acid (PA). Using full length Vam7, Vam7Y42A, and PX domain alone, we determined and compared the dissociation constants (KD) of each to biotinylated PI3P and PA biosensors.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2887 ","pages":"103-117"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142979108","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

Bio- and Chemoinformatic Approaches for Metabolomics Data Analysis. 代谢组学数据分析的生物和化学信息学方法。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4334-1_4

Michael Witting, Johannes Rainer

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Investigating the Functions of Hox Genes Using Planarian Asexual Reproduction. 利用涡虫无性生殖研究Hox基因的功能。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4322-8_7

Rachel B Gandee, Susanna M Reigner, Christopher P Arnold

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Bayesian Phylogenetic Lineage Reconstruction with Loss of Heterozygosity Mutations Derived from Single-Cell RNA Sequencing. 基于单细胞RNA测序的杂合性突变缺失的贝叶斯系统发育谱系重建。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4310-5_1

Donovan J Anderson, Marshall S Horwitz

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GEMLI: Gene Expression Memory-Based Lineage Inference from Single-Cell RNA-Sequencing Datasets. GEMLI：单细胞rna测序数据集的基因表达记忆谱系推断。

Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4310-5_19

A S Eisele, D M Suter

{"title":"GEMLI: Gene Expression Memory-Based Lineage Inference from Single-Cell RNA-Sequencing Datasets.","authors":"A S Eisele, D M Suter","doi":"10.1007/978-1-0716-4310-5_19","DOIUrl":"https://doi.org/10.1007/978-1-0716-4310-5_19","url":null,"abstract":"Gene expression memory-based lineage inference (GEMLI) is a computational tool allowing to predict cell lineages solely from single-cell RNA-sequencing (scRNA-seq) datasets and is publicly available as an R package on GitHub. GEMLI is based on the occurrence of gene expression memory, i.e., the gene-specific maintenance of expression levels through cell divisions. This represents a shift away from experimental lineage tracing techniques based on genetic marks or physical cell lineage separation and greatly eases and expands lineage annotation. GEMLI allows to study cell lineages during differentiation in development, homeostasis, and regeneration, as well as disease onset and progression in various physiological and pathological contexts. This makes it possible to dissect cell type-specific gene expression memory, to discriminate symmetric and asymmetric cell fate decisions, and to reconstruct individual multicellular structures from pooled scRNA-seq datasets. GEMLI is particularly promising for its ability to identify small lineages in human samples, a context in which no other lineage tracing methods are applicable. In this chapter, we provide a detailed protocol of the GEMLI R package usage on gene expression matrices derived from standard scRNA-seq on various platforms. We cover the use of the main function to predict cell lineages and how to adjust its parameters to different tasks. We also show how lineage information is extracted, visualized, and fine-tuned. Finally, we describe the use of the package's functions for the detailed analysis of the predicted cell lineages. This includes the analysis of gene expression memory, cell type composition of individual large lineages, and identification of lineages at the transition point between two cell types.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2886 ","pages":"375-400"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142915347","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