(A) Schematic of the plasmid architecture used to assay promoters and terminators using Nluc as reporter. Coloured blocks/arrows denote interchangeable modules; P1-P8 (purple) represent the necessary primers to PCR amplify the DNA fragments for assembly and green fragments represent the ssOBs needed to complete the DNA assembly reaction. This configuration highlights the modularity of the approach. Plasmids were cloned and amplified in E. coli and then introduced into Blastocystis ST7-B, functioning in practice as an E. coli↔Blastocystis ST7-B shuttle vector. Robust expression and drug selection were obtained without adding a Blastocystis-specific or canonical eukaryotic origin of replication (e.g., 2µ in yeasts or SV40 ori in mammalian cells), indicating that sequences in the bacterial backbone were sufficient for plasmid maintenance in Blastocystis ST7-B. (B) A bicistronic construct model using P2A peptide to enable co-expression of a fluorescent reporter and a selectable marker under a single promoter-terminator pair. Reporters used were UnaG, smURFP, and SNAP-tag®; selection markers were Ecdhfr (trimethoprim) and pac (puromycin).

(A) Heatmap with hierarchical clustering (left) and boxplots (right) of promoter–terminator activity in Blastocystis ST7-B. Activity was measured by Nluc luminescence. For each construct, raw relative light units (RLU; n = 3 independent replicates) were normalized to the LeguP1 reference construct (external standard) to obtain relative strength. Values are displayed on a 0–1 scale using min–max scaling after normalization; on this scale, 1.0 corresponds to the LeguP1 reference. Promoter fragment lengths are indicated by the numbers in the labels. Unsupervised clustering resolved, into tiers: (I) robust, (II) moderate, (III and III′) weak, and (IV) negligible. In the adjacent boxplots, points represent individual replicate values. (B) Effect of square-wave voltage on Nluc luminescence (RLU; n = 3 independent replicates per condition) with pulse duration fixed at 30 ms. (C) Effect of single pulse duration on luminescence signal (RLU; n = 3 independent replicates per condition) voltage fixed at 300 V. For B and C, electroporation runs were performed with a single pulse while plasmid amount and cell density were held constant. Boxplots show log-transformed raw RLU values (not normalized). Group differences were assessed by Kruskal–Wallis followed by Dunn’s post hoc test (α = 0.05); brackets mark tested contrasts and *, **, *** indicate P < 0.05, 0.01, 0.001, respectively. Although the 20 ms pulse was not significantly different from 15, 25, or 30 ms, it was chosen for subsequent electroporation runs because it produced the narrowest interquartile range (least dispersion) while maintaining a high signal.

Genes from which endogenous promoters and terminators were cloned to drive transgene expression in Blastocystis ST7-B.

(A) Fluorescence micrographs of Blastocystis ST7-B transfected with LeguP1:UnaG::P2A::pac:LeguTer and imaged 3 days post-transfection before antibiotic selection using an Olympus IX81 fluorescence microscope. Images show phase-contrast overlaid with the UnaG channel. In the absence of exogenous BR, only weak signal is detected, consistent with reported intrinsic autofluorescence in Blastocystis (yellow arrows; Nagel et al., 2015). After incubation with 1 mM BR, UnaG-positive cells show strong fluorescence (red arrow), whereas neighbouring non-transformed cells remain at background. Scale bar, 10 µm. (B) Representative single-cell confocal images showing compartmentalised UnaG fluorescence in Blastocystis ST7-B after incubation with 50 µM unconjugated BR. WT and UnaG-expressing cells were imaged under identical acquisition settings. For display purposes, the lower threshold of the UnaG channel in the transgenic clone was set to the maximum intensity observed in the WT control, so that only signal above the WT autofluorescence range is visible. (C) smURFP fluorescence in Blastocystis ST7-B is limited by chromophore access and is unmasked by saponin permeabilisation. Live cells expressing cytosolic smURFP were incubated with 50 µM BV and imaged by confocal microscopy either without permeabilisation or after treatment with 0.1% saponin. No signal was detected above background without saponin. After permeabilisation, smURFP fluorescence became strong and broadly distributed throughout the cytoplasmic compartment, consistent with the untargeted construct design. (D) SNAP-tag® labelling in Blastocystis ST7-B is dependent on substrate availability and concentration and yields broadly distributed fluorescence. Live clones expressing an untargeted cytosolic SNAP-tag® were incubated with increasing concentrations of a cell-permeant benzylguanine fluorophore substrate and imaged by confocal microscopy. Signal was at or near background at low substrate but increased progressively with substrate concentration and became broadly distributed throughout the cytoplasmic compartment, consistent with efficient covalent labelling in live cells. Scale bars in B, C, and D represent 5 µm.

Antibiotic potency and selection-window determination in Blastocystis ST7-B.

Dose–response curves for puromycin, trimethoprim, and WR99210 were estimated from a resazurin-based viability assay (n = 3 independent replicates per drug per concentration); points show mean ± SD, and the insets list the estimated IC50 values with R²-values > 0.75 for all fits. Right panels: small culture validation using 1 x 10⁷ WT Blastocystis ST7-B cells per culture, assayed in triplicates across a wide range of concentrations. Cultures were incubated for 3 days, and viability was assessed by the phenol red pH indicator shift resulting from acidification of the medium, with yellow indicating growth and red indicating no growth. The concentration at the yellow-to-red transition formed the rational basis to develop a selection strategy for Blastocystis ST7-B transformants.

Stepwise selection and clonal propagation of Blastocystis ST7-B transformants with puromycin and trimethoprim monitored over days post-transfection (DPT).

Stage I: transfected cells are kept in 2 mL tubes inside anaerobic jars and exposed to increasing drug concentrations; values highlighted in green refer to the maintenance concentration. Stage II: enriched cultures are plated on solid medium incubated for 2 weeks, yielding well-isolated colonies that are picked at 22 DPT. At this stage, the drug concentrations are lowered to ensure that individual plated cells, which later form colonies, remain within their resistance capacity during early outgrowth on the plate. Stage III: colonies are re-established in liquid culture under the same stepwise antibiotic regime. Subsequently, volume expansion from 2 mL microcentrifuge tubes to 14 mL culture tubes proceeds through an intermediate 5 mL step to increase the success of culture establishment. The entire workflow takes up to 40 days.

Oligonucleotide sets used for expression vector cloning.

In cases where only the promoter fragment was changed, most oligonucleotides were reused; however, complete sets are shown here for clarity and completeness. Primers used for PCR amplification of DNA fragments are designated MRT-xxx, whereas single-stranded oligonucleotide bridges used for DNA assembly are termed ssOB-yyy. Oligonucleotides are grouped by plasmid, named pMRT-zzz.