Bio-protocol最新文献

{"title":"Quantifying Intracellular Distributions of HaloTag-Labeled Proteins With SDS-PAGE and Epifluorescence Microscopy.","authors":"Julia Shangguan, Ronald S Rock","doi":"10.21769/BioProtoc.5391","DOIUrl":"10.21769/BioProtoc.5391","url":null,"abstract":"<p><p>Counting protein molecules helps reveal the organization of components within cellular structures and the stoichiometries of protein complexes. Existing protein and peptide quantitation methods vary in their complexity. Here, we report a straightforward workflow to measure the absolute number of HaloTag-labeled myosin 10 (Myo10) molecules in U2OS cells. Myo10 is a motor protein that plays a prominent role in cellular protrusion formation. Various biochemical and biological properties of Myo10 are established, but it is not well-defined how many molecules of Myo10 pack into narrow cellular structures called filopodia. We present a workflow for using SDS-PAGE to calibrate Myo10 signal with a reference protein, segmenting epifluorescence microscopy images to map Myo10 intracellular distribution, and interpreting the results to derive biological and functional insights. Our protocol is simple to employ and not only applicable for Myo10 research but also easily adaptable for other biological systems that use HaloTag. Key features • Combining SDS-PAGE densitometry with epifluorescence microscopy to quantitate HaloTag-labeled proteins in cells with readily available equipment. • Details for quantifying protein signal intensity in cellular compartments with semi-automated image segmentation.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 14","pages":"e5391"},"PeriodicalIF":1.1,"publicationDate":"2025-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12304454/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144755411","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Isolation and Culture of Ferret Airway Stem Cells.","authors":"Ziying Yan, John F Engelhardt, Feng Yuan","doi":"10.21769/BioProtoc.5399","DOIUrl":"10.21769/BioProtoc.5399","url":null,"abstract":"<p><p>Well-differentiated airway epithelial cultures are commonly used to study airway stem cell lineages, ion and fluid transport, respiratory virus infection and replication, and disease mechanisms in vitro. This culture model involves the isolation and expansion of airway stem cells followed by their differentiation at an air-liquid interface (ALI), a process that has been previously documented in humans and mice. Domestic ferrets (<i>Mustela putorius furo</i>) have gained considerable importance in respiratory disease research due to their notable susceptibility to these conditions and their anatomical similarities to humans. Here, we present a comprehensive description of the isolation and culture of stem/progenitor cells from the ferret airway, along with a protocol for their differentiation at the ALI. Our findings have demonstrated that this ferret culture system not only supports the differentiation of the predominant airway epithelial cell types but also facilitates the generation of rare airway epithelial subpopulations, including pulmonary ionocytes, tuft cells, and pulmonary neuroendocrine cells. Additionally, we provide a detailed procedure for measuring transepithelial ion transport relevant to airway diseases, particularly cystic fibrosis. The ability to isolate and culture ferret airway stem cells, combined with ALI differentiation and functional assessment of transepithelial ion transport, offers a powerful platform for evaluating genetic and pharmacologic interventions related to cystic fibrosis. Key features • A protocol for isolating ferret airway basal cells and generating air-liquid interface (ALI) cultures for electrophysiologic research. • Detailed procedures for propagating ferret airway basal cells and culturing in vitro well-differentiated airway epithelium. • A protocol for measuring ion transport, conductance, and immunofluorescence to identify airway cell types.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 14","pages":"e5399"},"PeriodicalIF":1.1,"publicationDate":"2025-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12304463/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144755407","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"<i>Thermus thermophilus</i> CRISPR Cas6 Heterologous Expression and Purification.","authors":"Junwei Wei, Mohamed Motawaa, Yingjun Li","doi":"10.21769/BioProtoc.5382","DOIUrl":"10.21769/BioProtoc.5382","url":null,"abstract":"<p><p>The CRISPR-Cas system of <i>Thermus thermophilus</i> has emerged as a potent biotechnological tool, particularly its Cas6 endonuclease, which plays a crucial role in CRISPR RNA (crRNA) maturation. This protocol details a robust and reproducible method for the high-level expression and purification of recombinant <i>T. thermophilus</i> Cas6 proteins (Cas6-1 and Cas6-2) in <i>E. coli</i>. We describe a streamlined approach encompassing plasmid construction using seamless assembly, optimized bacterial heterologous expression, and multi-step purification leveraging affinity and size-exclusion chromatography. The protocol outlines the generation of both His-tagged and GST-tagged Cas6 variants, enabling flexibility in downstream applications. Key steps, including primer design, PCR optimization, competent cell transformation, and chromatography strategies, are meticulously detailed with critical parameters and troubleshooting guidance to ensure experimental success and high yields of highly pure and active <i>T. thermophilus</i> Cas6 proteins. This protocol is useful for researchers requiring purified <i>T. thermophilus</i> Cas6 for structural studies, biochemical characterization, and the development of CRISPR-based biotechnological tools. Key features • Robust method for expressing and purifying <i>Thermus thermophilus</i> Cas6 proteins in <i>E. coli.</i> • Seamless assembly cloning and dual affinity tagging system: Offers options for both His-tag and GST-tag purification strategies for increased versatility. • Applicable for diverse heterologous expression and purification of well-folding thermostable proteins in mesophilic host chassis cells [<i>E. coli</i> BL21(DE3)].</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 14","pages":"e5382"},"PeriodicalIF":1.1,"publicationDate":"2025-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12304457/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144755389","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Multi-phase Training in Virus-Like Particle Synthesis to Foster Science Self-efficacy in Students With Minimal Laboratory Experience.","authors":"Macie A Proctor-Roser, Marinca Faimau, Julianne Peabody, Krystal R Charley, Bryce Chackerian, Naomi R Lee","doi":"10.21769/BioProtoc.5381","DOIUrl":"10.21769/BioProtoc.5381","url":null,"abstract":"<p><p>Science self-efficacy describes the confidence individuals have in their ability to accomplish specific scientific practices. Self-efficacy is one factor linked to success and persistence within STEM fields. The purpose of this protocol is to provide research laboratories with effective methods for teaching and mentoring new students in molecular biology, specifically in the synthesis of virus-like particles (VLPs) derived from bacteriophages. VLPs are multivalent nanoparticle structures that can be utilized in multiple biomedical applications, including platforms for vaccine and drug delivery. Production of bacteriophage VLPs using bacterial expression systems is feasible in most laboratory settings. However, synthesizing and characterizing VLPs can be challenging for new researchers, especially those with minimal laboratory experience or a lack of foundational knowledge in molecular biology. To address this, a multi-phase training protocol was implemented to train new students in VLP synthesis, purification, and characterization. This model was optimized for training numerous high school and undergraduate students. By implementing this multi-phase methodology, the students' confidence in their abilities to perform specific tasks increased and likely enhanced their persistence in STEM. Key features • Multi-phase training model for new students. • Training phases that build to increase science self-efficacy. • Successful peer-to-peer training.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 14","pages":"e5381"},"PeriodicalIF":1.1,"publicationDate":"2025-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12304482/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144755409","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Protocol for 3D Bioprinting a Co-culture Skin Model Using a Natural Fibrin-Based Bioink as an Infection Model.","authors":"Giselle Y Díaz, Madeleine A Perry, Laura S Cárdenas, Victor A Da Silva, Kali Scheck, Silken A Tschofen, Stephen W Tuffs, Stephanie M Willerth","doi":"10.21769/BioProtoc.5380","DOIUrl":"10.21769/BioProtoc.5380","url":null,"abstract":"<p><p>The skin microbiome, a diverse community of microorganisms, plays a crucial role in maintaining skin health and homeostasis. Traditional studies have relied on two-dimensional (2D) models, which fail to recreate the complex three-dimensional (3D) architecture and cellular interactions of in vivo human skin, and animal models, which have species-specific physiology and accompanying ethical concerns. Consequently, both types of models fall short in accurately replicating skin physiology and understanding its complex microbial interactions. Three-dimensional bioprinting, an advanced tissue engineering technology, addresses these limitations by creating custom-designed tissue scaffolds using biomaterial-based bioinks containing living cells. This approach provides a more physiologically relevant 3D structure and microenvironment, allowing the incorporation of microbial communities to better reflect in vivo conditions. Here, we present a protocol for 3D bioprinting an in vitro skin infection model by co-culturing human keratinocytes and dermal fibroblasts in a high-viscosity, fibrin-based bioink to mimic the dermis and epidermis. The bioprinted skin tissue was co-infected with <i>Staphylococcus aureus</i> and <i>Staphylococcus epidermidis</i> to mimic bacterial skin disease. Bacterial survival was assessed through colony-forming unit enumeration. By incorporating bacteria, this protocol offers the potential to serve as a more representative in vivo 3D bioprinted skin infection model, providing a platform to study host-microbe interactions, immune responses, and the development of antimicrobial therapeutics. Key features • This protocol provides a detailed description of the cell culture process for both keratinocyte and fibroblast cells. • This protocol outlines step-by-step preparation of the high-viscosity fibrin bioink and chemical crosslinker. • The protocol uses an extrusion-based bioprinter, with an easy-to-follow methodology that clarifies the printing details, including the incorporation of skin cells into the bioink. • This protocol details how the bacteria are inoculated into the construct to achieve the co-infection 3D skin model.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 14","pages":"e5380"},"PeriodicalIF":1.1,"publicationDate":"2025-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12304462/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144755410","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Inducible HIV-1 Reservoir Reduction Assay (HIVRRA), a Fast and Sensitive Assay to Test Cytotoxicity and Potency of Cure Strategies to Reduce the Replication-Competent HIV-1 Reservoir in Ex Vivo PBMCs.","authors":"Jade Jansen, Teunis B H Geijtenbeek, Neeltje A Kootstra","doi":"10.21769/BioProtoc.5384","DOIUrl":"10.21769/BioProtoc.5384","url":null,"abstract":"<p><p>The HIV-1 reservoir, consisting of transcriptionally silent integrated HIV-1 proviruses, is a major barrier to a cure, as it persists during effective antiretroviral therapy (ART) and is the source of viral rebound upon treatment interruption. Some of the strategies explored for HIV cure focus on the identification of compounds to either reactivate and eliminate the HIV reservoir (\"shock and kill\") or to prevent HIV reservoir reactivation and induce deep proviral latency (\"block and lock\"). Paramount in developing these HIV-1 cure strategies is determining the effect of the compounds on the size of the inducible HIV-1 reservoir in blood from people living with HIV-1 (PWH). Traditionally, viral outgrowth assays have been the primary method to determine the inducible HIV-1 reservoir in CD4+ T cells from PWH. However, these assays are labor-intensive, time-consuming, and often have low sensitivity. We have recently developed the inducible HIV-1 reservoir reduction assay (HIVRRA), a rapid, cost-effective, and sensitive method to measure the impact of compounds on the inducible replication-competent HIV-1 reservoir in total peripheral blood mononuclear cells (PBMCs) from PWH ex vivo. The HIVRRA simultaneously evaluates the effect of test conditions on the size of the inducible replication-competent HIV-1 reservoir as well as the specificity and toxicity of the test strategy. Using total PBMCs instead of purified CD4+ T cells reduces processing time and resource requirements. This makes the HIVRRA a more practical, scalable tool for evaluating potential HIV-1 cure strategies. Key features • The HIVRRA builds on the TZM-BL cell-based assay to quantify the HIV-1 reservoir by Sanyal et al.'s [1] method. • The HIVRRA uses total PBMCs from PWH to determine infectious units per million cells. • The HIVRRA requires low PBMC input compared to other reservoir analysis methods. • The HIVRRA determines the toxicity of the compounds on HIV-1-infected and uninfected cells in the same assay.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 14","pages":"e5384"},"PeriodicalIF":1.1,"publicationDate":"2025-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12340236/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144839370","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Workflow for Fluorescence-Targeted Lamella Milling From Vitrified Cells With a Coincident Fluorescence, Electron, and Ion Beam Microscope.","authors":"Elise G Perton, Daan B Boltje, Arjen J Jakobi, Jacob P Hoogenboom","doi":"10.21769/BioProtoc.5390","DOIUrl":"10.21769/BioProtoc.5390","url":null,"abstract":"<p><p>Cryo-electron tomography (cryo-ET) is the main technique to image the structure of biological macromolecules inside their cellular environment. The samples for cryo-ET must be thinner than 200 nm, which is not compatible with micron-sized cells. A focused ion beam (FIB), in conjunction with a scanning electron microscope (SEM) to navigate the sample, can be used to ablate material from vitrified cells such that a thin lamella remains. However, the preparation of lamellae with a FIB-SEM is blind to the location of specific cellular structures and biomolecules. Furthermore, the thickness and uniformity of lamella, while crucial for high-quality tomograms, cannot be established accurately with the FIB-SEM. These limitations strongly affect the success rate for cryo-ET on FIB-milled lamellae and thereby the total throughput of the workflow. To mitigate these problems, a coincident light, electron, and ion beam cryo-microscope was developed by retrofitting a fluorescence microscope, cryogenic microcooler, and piezo stage on a FIB-SEM. The fluorescence of molecules of interest can be monitored in real time while milling to ensure the final lamella contains the structure of interest. In addition, reflected light microscopy can be used for thickness and quality control of the lamella. In this protocol, we will describe how the coincident microscope can be used to prepare lamellae from vitrified cells. Key features • Step-by-step protocol for fluorescence-guided FIB-milling with a coincident three-beam cryogenic microscope as described in [1]. • Details about sample loading and unloading, as well as the lamella milling workflow with graphical explanations. • Quality control of lamella, including thickness, uniformity, and ice contamination.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 14","pages":"e5390"},"PeriodicalIF":1.1,"publicationDate":"2025-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12304472/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144755418","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluation of Translation Rate Through L-azidohomoalanine (AHA) Incorporation and Subsequent Alkyne Fluorophore-Mediated Click Chemistry in Yeast.","authors":"Mainak Pratim Jha, Koyeli Mapa","doi":"10.21769/BioProtoc.5379","DOIUrl":"10.21769/BioProtoc.5379","url":null,"abstract":"<p><p>Accurate measurement of protein translation rates is crucial for understanding cellular processes and disease mechanisms. However, existing methods for quantifying translation rates in yeast cells are limited. Here, we present a streamlined protocol for measuring protein translation rates in <i>Saccharomyces cerevisiae</i> using the methionine analog L-azidohomoalanine (AHA), which is the L isoform of this synthetic amino acid, and fluorophore-labeled alkyne dye-based Click chemistry. Our method involves incorporating AHA into newly synthesized proteins, followed by detection using confocal microscopy, flow cytometry, and SDS-PAGE. We validated our protocol by measuring translation rates under various stress conditions, including heat stress, endoplasmic reticulum (ER) stress induced by tunicamycin, and translation inhibition by cycloheximide. Confocal microscopy revealed differential AHA incorporation and fluorescence intensity across conditions. Flow cytometry quantitatively confirmed significant increases in translation rates under heat stress and decreases under ER stress compared to unstressed conditions at 6 and 24 h post-treatment. Imaging of gels under fluorescence detectors following SDS-PAGE further visualized newly synthesized proteins, with no detectable translation after cycloheximide treatment. Our protocol offers enhanced precision and selectivity compared to existing methods for mammalian cells and represents the first standardized approach for measuring translation rates in yeast. Despite limitations in required specialized equipment and expertise, this method holds promise for diverse applications in biotechnology and biomedical research, enabling investigations into protein synthesis regulation in yeast systems. Key features • This study presents the first standardized protocol for measuring protein translation in budding yeast using AHA and Click chemistry, addressing yeast-specific challenges effectively. • The study uses microscopy, flow cytometry, and fluorescence gel imaging to robustly validate yeast translation rates, ensuring reliable, reproducible results across cellular and biochemical levels. • The method detects translation changes under stress: increased with heat, decreased with ER stress, and halted by cycloheximide, highlighting its sensitivity for proteostasis research. • Despite requiring specialized equipment and expertise, the method offers valuable applications in biomedical research, metabolic engineering, and drug screening focused on protein homeostasis in yeast.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 14","pages":"e5379"},"PeriodicalIF":1.1,"publicationDate":"2025-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12304456/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144755394","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Fluorescence Polarization-Based High-Throughput Screening Assay for Inhibitors Targeting Cathepsin L.","authors":"Keyu Guo, Baoqing You, Wenwen Zhou, Yan Li, Zhen Wang, Jing Zhang, Shuyi Si","doi":"10.21769/BioProtoc.5400","DOIUrl":"10.21769/BioProtoc.5400","url":null,"abstract":"<p><p>Cathepsin L (CTSL), a lysosomal cysteine protease belonging to the papain-like protease family, is primarily involved in intracellular protein degradation, antigen processing, and extracellular matrix remodeling. It plays critical roles in pathological conditions, including cancer metastasis, neurodegenerative disorders, and viral infection, due to dysregulated activity or overexpression. Thus, inhibitors targeting CTSL are under investigation for therapeutic applications. Current approaches for identifying CTSL inhibitors predominantly rely on fluorescence-labeled substrates, fluorescence resonance energy transfer (FRET), and cell-based screening assays. Here, we applied the principle of fluorescence polarization (FP) to the detection of substrate cleavage activity by CTSL through changes in millipolarization unit (mp) values and established a cost-effective, quantitative, reagent- and time-saving inhibitor high-throughput screening (HTS) assay. We also provide detailed steps for the expression and purification of highly active CTSL from eukaryotic cells, which lays a solid foundation for the FP-based assay. A key advantage of this assay lies in its reduced susceptibility to fluorescence interference, as the fluorescein isothiocyanate (FITC) fluorophore exhibits high quantum efficiency with an emission peak at 535 nm-a wavelength range distinct from most naturally occurring fluorescent molecules. The assay's adaptability to reaction time, temperature, and dimethyl sulfoxide (DMSO) concentration minimizes false-positive or false-negative results caused by minor experimental inconsistencies, streamlining the screening process. Furthermore, the protocol requires fewer operational steps, reduced incubation time, and lower quantities of CTSL and substrates compared to conventional methods. This rapid, cost-effective, and scalable approach aligns well with the demands of HTS platforms. Key features • Detailed procedures for eukaryotic expression, purification, and identification of active recombinant CTSL and determination of its biological activity. • Full description for application of fluorescence polarization (FP)-based high-throughput screening (HTS) assay targeting CTSL. • Elaboration for the application of the FP-based assay in CTSL activity evaluation or drug discovery. • Protocol is readily adaptable to other proteases with a similar catalytic mechanism.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 14","pages":"e5400"},"PeriodicalIF":1.1,"publicationDate":"2025-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12304460/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144755406","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Comprehensive Mapping of EZ-Tn5 Transposon Insertion Sites in <i>Pseudomonas argentinensis</i> SA190 Using RATE-PCR.","authors":"Büsra Elkatmis, Baoda Han, Sabiha Parween, Stanislav Kopriva, Heribert Hirt, Maged M Saad","doi":"10.21769/BioProtoc.5389","DOIUrl":"10.21769/BioProtoc.5389","url":null,"abstract":"<p><p>Transposon mutagenesis is a powerful tool for investigating gene function in bacteria, particularly in newly discovered species. In this study, we applied the hyperactive EZ-Tn5 transposase system to <i>Pseudomonas argentinensis</i> SA190, an endophytic bacterium known for enhancing plant resilience under drought stress. By leveraging the random amplification of transposon ends (RATE)-PCR method, we successfully mapped the insertion sites of the transposon within the SA190 genome. This approach enabled the precise identification of disrupted genes, offering insights into their roles in bacterial function and interaction with host plants. Our comprehensive protocol, including competent cell preparation, transformation, and insertion site mapping, provides a reliable framework for future studies aiming to explore gene function through mutagenesis. Key features • The use of the hyperactive EZ-Tn5 transposase system ensures efficient and detectable random mutagenesis across the <i>Pseudomonas argentinensis</i> SA190 genome, facilitating comprehensive gene disruption studies. • The technique is employed to identify and map the transposon insertion sites, allowing for precise determination of gene function and its impact on bacterial phenotypes. • This method enables the exploration of a broad range of gene functions within SA190, particularly those involved in plant growth promotion and stress tolerance. • This method can be readily adapted to generate mutant libraries in other bacterial species, emphasizing its transferability.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 14","pages":"e5389"},"PeriodicalIF":1.1,"publicationDate":"2025-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12304473/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144755392","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}