This protocol presents methods for isolating and identifying novel nucleic acid-binding proteins. We focus on bacterial DNA-binding proteins, although the methods can be readily adapted to identify nucleic acid-binding proteins of eukaryotes or archaea, and proteins that bind to RNAs. Briefly, the DNA sequence of interest is affixed to beads and then incubated with bacterial cytoplasmic extract. Washes with buffers containing nonspecific DNA and low salt concentrations will remove non-adhering and low-specificity DNA-binding proteins, while subsequent washes with higher salt concentrations will elute DNA-binding proteins that have greater specificity. Eluted proteins are then identified by standard proteomic techniques, such as mass spectrometry. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Lysis procedure Basic Protocol 2: DNA affinity chromatography: Borrelia burgdorferi and Leptospira interrogans Basic Protocol 3: Visualization and identification of DNA-binding proteins.
Allosteric transcription factors (aTFs) regulate gene expression in response to small molecules. Engineering aTFs with new ligand specificities expands their utility in environmental monitoring and diagnostics. Here, we present protocols for improving the responsiveness of an aTF, SRTF1, to its non-native ligand, cortisol, based on the SELIS (Seamless Enrichment of Ligand-Inducible Sensors) methodology. First, an aTF library is generated by randomizing the ligand binding pocket. These libraries are then subjected to a selection process to remove protein variants that have lost the ability to repress transcription. The libraries are then plated onto solid media containing the target ligand. The most fluorescent colonies, signifying the most responsive aTFs, are cultured and assayed for the fold change in fluorescence upon induction with the target ligand. Colonies with the highest fold change are cultured, and the plasmid DNA is purified and sequenced. The gene variants are then subcloned into new expression vectors and assayed in a dose-response assay with the target and non-target ligands to have complete characterization of the sensor. Through one round of these protocols, we improved the response of SRTF1 to cortisol by seven-fold; however, the protocols outlined here are applicable to virtually any protein:effector pair. © 2025 The Author(s) Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Library design Basic Protocol 2: Library construction Basic Protocol 3: Selection and screening Basic Protocol 4: Sequencing and validation of hits Support Protocol 1: Predicting protein:effector structure Support Protocol 2: Design of mutagenesis primers Support Protocol 3: Preparation of chemically competent E. coli for single isolate transformations.
Blastocystis spp. is a widely prevalent anaerobic protozoan of uncertain pathogenicity found in the gastrointestinal tracts of over 1 billion people worldwide. Despite its potential significance in health and disease, Blastocystis spp. remains challenging to culture axenically due to its anaerobic nature and the diversity of its genetic subtypes. This manuscript presents a standardized toolkit for culturing Blastocystis spp. in xenic and axenic conditions, detailing protocols for the preparation of appropriate liquid and solid media, cryopreservation, and inoculation. By providing a comprehensive set of tools and methodologies, this work aims to streamline research on Blastocystis spp., enabling reproducibility, subtype characterization, and advancements in understanding its role in the gut microbiome and host health. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Establishment of xenic Blastocystis culture from stool samples in liquid medium Basic Protocol 2: Subculturing from existing Blastocystis liquid culture Support Protocol 1: Preparation of modified Jones' medium for Blastocystis culturing Support Protocol 2: Preparation of trypticase-yeast extract-serum-gastric mucin-9 (TSGYM-9) medium for Blastocystis culturing Support Protocol 3: Preparation of liver extract-yeast extract-serum-gastric mucin (LYSGM) medium for Blastocystis culturing Basic Protocol 3: Subculturing xenic Blastocystis cultures in diphasic medium Support Protocol 4: Preparation of Robinson's medium for Blastocystis culturing Support Protocol 5: Preparation of Ringer's agar slants for Blastocystis culturing Basic Protocol 4: Axenization of Blastocystis cultures Basic Protocol 5: Subculturing axenic Blastocystis cultures in diphasic medium Support Protocol 6: Preparation of Boeck and Drbohlav's Locke-egg serum (LES) medium for Blastocystis culturing Support Protocol 7: Preparation of Boeck and Drbohlav's diphasic modified medium (BDMM) for Blastocystis culturing Basic Protocol 6: Establishment of axenic Blastocystis cultures in Iscove's modified Dulbecco's medium (IMDM) Basic Protocol 7: Establishment of axenic Blastocystis cultures in soft IMDM agar Basic Protocol 8: Establishment of axenic Blastocystis cultures on solid IMDM agar Basic Protocol 9: Optimized method for establishing axenic Blastocystis cultures on solid IMDM agar Basic Protocol 10: Establishment of axenic Blastocystis cultures in semi-solid Locke's agar Basic Protocol 11: Cryopreservation of xenic Blastocystis cultures Basic Protocol 12: Cryopreservation of axenic Blastocystis cultures Basic Protocol 13: Inoculation of liquid medium with xenic Blastocystis cultures from frozen stocks Basic Protocol 14: Inoculation of liquid medium with axenic Blastocystis cultures from frozen stocks.
Cryptococcus neoformans is an opportunistic fungal pathogen that heads the Fungal Priority Pathogen List published by the World Health Organization (WHO) in 2022. This pathogen is a primary cause of death for immunocompromised individuals (e.g., those with HIV/AIDS, the elderly, immunotherapy recipients), causing approximately 118,000 deaths yearly worldwide. C. neoformans relies on virulence factors that include a polysaccharide capsule, melanin, extracellular enzymes, and thermotolerance to initiate and sustain host infection. Additionally, similar to other fungal pathogens (e.g., Candida albicans), C. neoformans may develop a biofilm organization linked to more persistent cryptococcal infections. Cryptococcal biofilms are highlighted in cases of cryptococcal meningitis, in which biofilm-like structures form that are highly resistant to host immune response and to antifungal therapies. In this regard, fungal biofilm formation has become an important area of study as a means to improve our understanding of the mechanisms regulating biofilm formation and infection and to advance the discovery of antibiofilm therapeutics. To assess biofilm properties and compare across treatments, quantification and evaluation of cell viability are important. Herein, we describe a standardized method to establish a cryptococcal biofilm and quantify total biomass and cell viability. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culturing and biofilm formation Basic Protocol 2: Biofilm quantification Alternate Protocol: Biofilm viability assay.
Aspergillus fumigatus is an opportunistic human fungal pathogen that is widely spread in the environment. The need to screen a large number of environmental or clinical isolates for phenotypic and/or genotypic purposes can be challenging and time consuming when using the protocols that are currently available. We have developed a novel approach that allows one to inoculate cultures, grow individual A. fumigatus isolates, and subsequently isolate DNA of sufficient quality for PCR and/or whole genome sequencing, all in a 96-well format. Compared to currently available methods, our protocols reduce both cost and labor significantly and are compatible with any experimental setup that uses a 96-well format. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: 96-well-format mini slant culture and preparation of spore suspension for Aspergillus fumigatus Basic Protocol 2: PCR-quality DNA isolation Basic Protocol 3: High-purity DNA isolation after mini mycelial mat culture.
