Microbiology

Genetic modification is essential for understanding parasite biology, yet it remains challenging in Plasmodium. This is partially due to the parasite’s low genetic tractability and reliance on homologous recombination, since the parasites lack the canonical non-homologous end-joining pathway. Existing approaches, such as the PlasmoGEM project, enable genome-wide knockouts but remain limited in coverage and flexibility. Here, we present the Plasmodium berghei high-throughput (PbHiT) system, a scalable CRISPR-Cas9 protocol for efficient genome editing in rodent malaria parasites. The PbHiT method uses a single cloning step to generate vectors in which a guide RNA (gRNA) is physically linked to short (100 bp) homology arms, enabling precise integration at the target locus upon transfection. The gRNA also serves as a unique barcode, allowing pooled vector transfections and identification of mutants by downstream gRNA sequencing. The PbHiT system reliably recapitulates known mutant growth phenotypes and supports both knockout and tagging strategies. This protocol provides a reproducible and scalable tool for genome editing in P. berghei, enabling both targeted functional studies and high-throughput genetic screens. Additionally, we provide an online resource covering the entire P. berghei protein-coding genome and describe a step-by-step pooled ligation approach for large-scale vector production.

Accurate profiling of soil and root-associated bacterial communities is essential for understanding ecosystem functions and improving sustainable agricultural practices. Here, a comprehensive, modular workflow is presented for the analysis of full-length 16S rRNA gene amplicons generated with Oxford Nanopore long-read sequencing. The protocol integrates four standardized steps: (i) quality assessment and filtering of raw reads with NanoPlot and NanoFilt, (ii) removal of plant organelle contamination using a curated Viridiplantae Kraken2 database, (iii) species-level taxonomic assignment with Emu, and (iv) downstream ecological analyses, including rarefaction, diversity metrics, and functional inference. Leveraging high-performance computing resources, the workflow enables parallel processing of large datasets, rigorous contamination control, and reproducible execution across environments. The pipeline’s efficiency is demonstrated on full-length 16S rRNA gene datasets from yellow pea rhizosphere and root samples, with high post-filter read retention and high-resolution community profiles. Automated SLURM scripts and detailed documentation are provided in a public GitHub repository (https://github.com/henrimdias/emu-microbiome-HPC; release v1.0.2, emu-pipeline-revised) and archived on Zenodo (DOI: 10.5281/zenodo.17764933).

Toxoplasma gondii is an apicomplexan parasite that infects a wide variety of eukaryotic hosts and causes toxoplasmosis. The cell cycle of T. gondii exhibits a distinct architecture and regulation that differ significantly from those observed in well-studied eukaryotic models. To better understand the tachyzoite cell cycle, we developed a fluorescent ubiquitination-based cell cycle indicator (FUCCI) system that enables real-time visualization and quantitative assessment of the different cell cycle phases via immunofluorescence microscopy. Quantitative immunofluorescence and live-cell imaging of the ToxoFUCCI^S probe with specific cell cycle markers revealed substantial overlap between cell cycle phases S, G₂, mitosis, and cytokinesis, further confirming the intricacy of the apicomplexan cell cycle.

During herpesvirus replication, capsids are assembled inside the nucleus and translocated into the cytosol by a non-canonical nucleocytoplasmic export process termed nuclear egress. Traditional methods of measuring nuclear egress rely on imaging-based technologies such as confocal and electron microscopy. These techniques are labor-intensive, limited by the number of cells that can be examined, and may not accurately represent the entire population, generating a potential bias during data interpretation. To overcome these problems, we have developed a flow cytometry–based method to measure HSV-1 nuclear egress that we termed FLARE (FLow cytometry–based Assay of nucleaR Egress). This assay uses a double fluorescent reporter system, utilizing HSV-1-tdTomato to identify infected cells and an Alexa Fluor-488-conjugated, capsid-specific antibody to detect cytosolic capsids, thereby distinguishing infected cells with nuclear egress from those without it. This assay provides more quantitative results than traditional methods, enables large-scale high throughput, and can be adapted for use with other herpesviruses.

Since its introduction, the CRISPR/Cas9 system has been used in many organisms for precise and rapid genome editing, as well as for editing multiple genes at once. This targeted mutagenesis makes it easy to analyze the function of a gene of interest (goi). The standard method for genetic manipulation of the model organism Neurospora crassa has been homologous recombination. It is well established and widely used to create knock-out or overexpression mutants. The recently developed CRISPR/Cas9 system is an addition to the toolkit for genetically manipulating N. crassa. For this protocol, a strain stably expressing the Cas9 endonuclease is required. After designing the gRNA with the online tool CHOP-CHOP, a synthetic gRNA is used to transform macroconidia via electroporation. Combining the goi-gRNA with a gRNA targeting the csr-1 gene as a selection marker allows for easy identification of colonies with mutations at the target site of the goi, since the obtained resistance to Cyclosporin A (CsA) allows for selecting editing events. The mutation type can be detected by PCR of the edited gene region followed by Sanger sequencing. This system is fast and easy to handle, offering an attractive alternative to homologous recombination, especially for targeting multiple genes simultaneously.

Most viruses extensively remodel their host cells to establish productive infection. Visualization of virus-induced cellular remodeling by electron microscopy (EM) has been revolutionized in recent years by advances in cryo-focused ion beam (cryo-FIB) milling paired with cryo-electron tomography (cryo-ET). As cryo-FIB/ET becomes more widely available, there is a need for beginner-friendly guides to optimize the preparation of virus-infected mammalian cells on EM grids. Here, we provide an in-house protocol for new users for preparing samples of cells infected with herpes simplex virus 1 (HSV-1) for cryo-FIB/ET. This protocol guides users in how to seed infected cells onto grids, blot, and plunge-freeze grids using basic, manual equipment. It also provides tips on how to screen and prioritize grids for efficient milling and data collection.

Optogenetic stimulation of peripheral motor nerves is a promising technique for modulating neural activity via illumination of light-sensitive ion channels known as opsins. Stimulating muscle activity through this method offers many advantages, such as a physiological recruitment order of motor units, reduced fatigue, and target-specific stimulation, which make it a favorable option for use in many neuroscience and motor rehabilitation applications. To enable such optical stimulation, opsin expression in peripheral nerves can be achieved either with transgenic animal models or through injection of viral vectors. In this protocol, we describe a method for driving peripheral nerve opsin expression via intramuscular adeno-associated virus (AAV) injection with the goal of enhancing virus uptake by targeting injections to neuromuscular junctions with electrical stimulation. We also describe procedures for non-invasively assessing functional opsin expression over time with transdermal optical stimulation of opsin-labeled nerves and electromyography (EMG) recordings. The presence of time-locked EMG spikes 4–8 ms after each stimulation pulse demonstrates that functional opsin expression is present at a given assessment time point. Onset of functional optical sensitivity generally occurs 2–4 weeks following virus injection, and sensitivity generally peaks or plateaus between 6–10 weeks. Stimulation sequences such as light intensity, stimulation pulse width, and frequency sweeps provide further information on functional opsin expression at the testing timepoint. The methods presented here can be used for driving functional opsin expression with a standard AAV6 vector commonly used in similar experiments or as a protocol for assessing peripheral nerve opsin expression with novel viral vectors.

This protocol presents a modified version of the Filterprep method originally reported in New Biotechnology, adding an optional step to reduce endotoxin levels. Filterprep is a simple, rapid, and cost-effective approach to plasmid DNA purification that couples ethanol precipitation with a single spin-column filtration step, eliminating chaotropic salts and silica binding. The formulations and parameters are fully transparent and do not rely on proprietary buffers, using only standard laboratory reagents and widely available miniprep columns. Under matched conditions, the method recovers high-purity plasmid DNA with yields up to fivefold higher than those obtained with representative commercial midiprep kits. The workflow is readily adoptable in most molecular biology laboratories and, under routine conditions, can be completed in approximately 40 min. The resulting DNA is suitable for molecular cloning, PCR, sequencing, and other downstream biochemical applications. Endotoxin is a lipopolysaccharide (LPS) found in the outer membrane of Gram-negative bacteria and may carry over during plasmid preparation. For experiments requiring lower endotoxin input, an optional modification resuspends the DNA pellet in a Triton X-114 wash buffer before column loading to decrease lipopolysaccharide carryover. The method is modular and extensible, allowing adjustment of precipitation and wash conditions, variation in the number of washes, selection of alternative column formats, and integration of endotoxin-reduction modules without altering the core principle. These features facilitate troubleshooting and quality control, enable scaling from routine batches to larger culture volumes and higher throughput, and allow seamless integration with existing workflows.

The exploration of microbial genomes through next-generation sequencing (NGS) and genome mining has transformed the discovery of natural products, revealing an immense reservoir of previously untapped chemical diversity. Bacteria remain a prolific source of specialized metabolites with potential applications in medicine and biotechnology. Here, we present a protocol to access novel biosynthetic gene clusters (BGCs) that encode natural products from soil bacteria. The protocol uses a combination of Oxford Nanopore Technology (ONT) sequencing, de novo genome assembly, antiSMASH for BGC identification, and transformation-associated recombination (TAR) for cloning the BGCs. We used this protocol to allow the detection of large BGCs at a relatively fast and low-cost DNA sequencing. The protocol can be applied to diverse bacteria, provided that sufficient high-molecular-weight DNA can be obtained for long-read sequencing. Moreover, this protocol enables subsequent cloning of uncharacterized BGCs into a genome engineering-ready vector, illustrating the capabilities of this powerful and cost-effective strategy.

The tissue explant culture (histoculture) is a method that involves maintaining small pieces taken from an organ ex vivo or post mortem in a controlled laboratory setting. Such a technique has a number of advantages: unlike the 2D, organoid, or on-chip cultures, tissue explants preserve the whole complexity of the original tissue in vivo, its structure, extracellular matrix, and the diverse cell populations, including resident immune cells. The explant culture method can be applied to human tissue specimens obtained from biopsies or autopsies, provided that proper ethical protocols are followed. This avoids the difficulties that may arise in translating results obtained on animal models into biomedical research for humans. This advantage makes histocultures especially desirable for studying human pathogenesis in the course of infectious diseases. The disadvantage of the method is the limited lifespan of the cultured tissues; however, a number of approaches allow extending tissue viability to a period sufficient for observing the infection onset and development. Here, we provide a protocol for lung explant maintenance that allows tracing the local effects of infection with SARS-CoV-2 in humans. Further applications of the lung tissues cultured according to this protocol include, but are not limited to, histochemical and immunohistochemical studies and microscopy, FACS, qPCR, and ELISA-based analysis of the conditioned culture media.