Microbiology

The Sox (SRY-related HMG-box) protein family plays a crucial role in cellular differentiation, development, and gene regulation, with the HMG (high-mobility group) domain responsible for DNA binding and transcriptional regulation. Proteins in the SOX gene family contain an HMG domain that shares 50% homology with the HMG domain of the sex-determining factor SRY gene. The SOX gene family comprises 30 proteins, which are classified into 10 groups (A–H). As a member of this family, hSox2 has been shown to be involved in various biological processes, but its specific function remains unclear. Previous studies have used eukaryotic expression systems, GST-tag purification, and bacterial inclusion body refolding techniques to produce Sox family proteins. However, these methods are often limited by issues such as low yield, incorrect folding, or inefficient purification, restricting their application in functional and structural studies. In this study, a prokaryotic expression system for the hSox2-HMG domain was constructed using the pET22b vector and Escherichia coli BL21(DE3) as the host strain. Protein expression was induced by IPTG, and initial purification was performed using Ni-NTA affinity chromatography, followed by ultrafiltration concentration and size exclusion chromatography to improve purity. By optimizing lysis and elution conditions, we successfully obtained hSox2-HMG protein with high expression levels and purity. This method provides a cost-effective and scalable strategy for hSox2-HMG production, ensuring high purity and correct folding of the protein. The optimized experimental protocol lays a foundation for structural and functional studies of hSox2-HMG.

Transposon-based genetic transformation enables stable transgene integration in avian genomes and is increasingly used in the development of transgenic chickens for enhanced disease resistance, productivity, and biopharmaceutical applications. Conventional transformation techniques in avian biotechnology, including viral vectors and primordial germ cell (PGC) manipulation, are limited by biosafety risks, low efficiency, and technical complexity. This protocol outlines a two-step cloning approach for generating transposon-compatible gene constructs suitable for chicken embryo microinjection. Topoisomerase-based (TOPO) cloning is used as the first step due to its ability to directly clone PCR-amplified products without the need for restriction site-engineered primers while simultaneously producing an insert flanked with EcoRI restriction sites. The insert is subsequently transferred into the transposon vector through EcoRI-mediated restriction digestion and ligation. This approach simplifies construct generation by integrating the speed of TOPO cloning with the precision of restriction cloning, while ensuring compatibility with transposon-mediated integration systems. The protocol is efficient, reproducible, and does not require specialized equipment, providing a practical and scalable tool for gene construct assembly in avian transgenesis research.

Protein–protein interactions facilitate cellular functions through the creation of networks and multi-protein complexes. Mapping the interactions within and between protein networks and elucidating the composition of protein complexes provides critical insight into biological processes. Interactions among soluble cytoplasmic proteins have been extensively investigated through the application of immunoaffinity capture as well as conventional nuclear two-hybrid testing. The integrated membrane yeast two-hybrid provides a method to investigate protein–protein interactions between integral membrane proteins in their native membrane environment. This procedure makes use of the ability of the amino-terminal fragment of ubiquitin (Nub) and the carboxyl-terminal fragment of ubiquitin (Cub) to refold reconstituting functional ubiquitin, which can be recognized by a ubiquitin peptidase. Appending a fusion protein composed of Cub fused to LexA and VP16 (CLV) to a candidate "bait" protein and Nub to candidate "prey" proteins allows a test of their interaction. If the two proteins interact closely, the CLV fragment is cleaved and enters the nucleus to activate the expression of reporter genes, signaling the interaction. When the bait and prey proteins are tagged with CLV and NubG, respectively, at their genomic loci, they are only copies of the bait and prey in the cell and are expressed under the regulation of their native promoters. This avoids overexpression artifacts that can occur if the tagged proteins are expressed from plasmids while the untagged chromosomally encoded copies of the bait and prey continue to be expressed.

Human coronavirus OC43 (HCoV-OC43) is an endemic “common cold” coronavirus widely used to study fundamental aspects of coronavirus biology and to test therapeutic interventions. Recently, we used a yeast-based reverse genetics strategy to create recombinant HCoV-OC43 and fluorescent reporter viruses. We assembled a DNA copy of the HCoV-OC43 genome from six linear dsDNA fragments and a linearized yeast centromeric plasmid/bacterial artificial chromosome (YCpBAC) vector in Saccharomyces cerevisiae using transformation-associated recombination (TAR). Reporter genes encoding mCardinal fluorescent protein or histone H2B fused to mClover3 (mClover-H2B) or mRuby3 (mRuby-H2B) were inserted into an intergenic region between the HCoV-OC43 M and N genes. Assembled full-length HCoV-OC43-encoding plasmids were delivered into permissive mammalian cells to initiate viral gene expression, genome replication, and production of infectious progeny. This technique allows for the precise mutagenesis of any area of the HCoV-OC43 genome using homologous recombination, yielding genetically defined reference plasmids for the future generation of HCoV-OC43 virus stocks.

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 Staphylococcus aureus and Staphylococcus epidermidis 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.

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.

The CRISPR-Cas system of Thermus thermophilus 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 T. thermophilus Cas6 proteins (Cas6-1 and Cas6-2) in E. coli. 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 T. thermophilus Cas6 proteins. This protocol is useful for researchers requiring purified T. thermophilus Cas6 for structural studies, biochemical characterization, and the development of CRISPR-based biotechnological tools.

Rice (Oryza sativa), a staple crop sustaining half of humanity’s caloric intake, is threatened by numerous insect-vector-transmitted diseases, such as rice stripe disease, caused by the rice stripe virus (RSV). Most genetic studies on plant antiviral defense mechanisms rely on natural or artificial infection and transgenic approaches, which require months of plant transformation. Here, we present a streamlined protocol that enables rapid analysis of RSV–host interactions within three days. The method encompasses three key phases: (1) polyethylene glycol (PEG)-based precipitation of RSV virions from infected plant tissues, (2) sequential purification through differential ultracentrifugation with glycerol cushion optimization, and (3) high-efficiency transfection of purified virions into rice protoplasts via PEG-mediated delivery. Viral replication is quantitatively assessed using RT-qPCR targeting viral RNA and immunoblotting with RSV nucleocapsid protein-specific monoclonal antibodies. This approach eliminates dependency on stable transgenic lines, allowing the simultaneous introduction of exogenous plasmids for functional studies. Compared with conventional methods requiring several months for transgenic plant generation, our protocol delivers analyzable results within three days, significantly accelerating the exploration of antiviral mechanisms and resistance gene screening.

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.

Endophytic actinomycetes, particularly Streptomyces species, have gained significant attention due to their potential to produce novel bioactive compounds. In this study, we isolated and characterized an endophytic Streptomyces sp. VITGV100 from the tomato plant (Lycopersicon esculentum), employing the direct streak method and whole-genome sequencing. A genome analysis was done to uncover its biosynthetic potential and identify indole-type compounds. The strain's secondary metabolite production was evaluated through GC–MS analysis, and its antimicrobial activity was tested against selected human pathogenic bacteria. Our protocol outlines a comprehensive approach, describing the isolation and extraction of metabolites and genome mining for indole-type compounds. This isolate has potential pharmaceutical applications, accelerating the discovery of novel indole-type bioactive compounds.