A genetic toolkit for stable episomal transgenesis in the anaerobic gut parasite Blastocystis ST7-B

Blastocystis is among the most prevalent microbial eukaryote in the human gut, yet it has remained largely inaccessible to functional genetics. Here, we report a combinatorial toolkit for Blastocystis ST7-B that enables stable episomal transgene maintenance under antibiotic selection and recovery of colony-derived transgenic lines. Guided by a proteomics-informed candidate screen, we identified endogenous promoter-terminator pairs and benchmark their activity using NanoLuc luciferase (Nluc), defining near-background, weak, intermediate, and robust expression tiers. We optimise square-wave electroporation and establish conditions that balance DNA delivery with culture viability, providing a practical operating regime for routine transfection. Using resazurin-based viability assays alongside culture outgrowth validation, we identified puromycin and trimethoprim as the most reliable selectable systems. A three-stage workflow combining liquid enrichment, solid-phase selection, and liquid culture expansion supports recovery of colony-derived transgenic lines that can be cryopreserved and revived with retained growth, antibiotic resistance, and reporter expression. Finally, bicistronic constructs incorporating a codon-optimised P2A peptide supported selection-linked expression of anaerobic-compatible reporters (UnaG, smURFP, and SNAP-tag). Results showed reporter-dependent performance consistent with constraints such as chromophore availability and substrate permeability. Together, these make Blastocystis ST7-B markedly more amenable to genetic engineering.