Gel extraction kit

Cells were collected for genomic DNA extraction using the QuickExtract DNA Extraction Solution (Epicenter, Chicago, IL), following the manufacturer’s protocol. In brief, the pelleted cells were re-suspended in the QuickExtract solution, vortexed for 15 s, incubated at 65 °C for 6 min, vortexed for 15 s and then incubated at 98 °C for 10 min. The genomic region around the PAM was PCR amplified with high-fidelity Herculase II DNA polymerases. The PCR primers were (forward 5′-GCTCCTGTCGGGTCCCAAGG-3′) and (reverse 5′-ACCTGGACTGGCTTTGGCCC-3′). The PCR products were separated in 2% agarose gel and purified with a gel extraction kit (Thermo Scientific) for Sanger DNA sequencing and NGS^{15 (link)}. DNA sequencing was performed by the MGH DNA core facility.

Chitosan (medium MW, 75–85% deacetylated), oleic acid, Kallichore P188 (Poly (ethylene glycol)-block-poly (propylene glycol)-block-poly (ethylene glycol), Avicel (microcrystalline cellulose), and trehalose were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Precirol^® ATO 5 (Glyceryl Di stearate NF/Glyceryl palmitostearate), and Gelucire 50/13 (Stearoyl polyoxylglycerides NF/Stearoyl macrogol glycerides EP) were kind gifts from Gattefosse (Lyon, France). Mannitol, lactose, and sucrose (El-Gomhoreya, Cairo, Egypt), Dulbecco’s Modified Eagle’s medium (DMEM, Gibco Invitrogen, Darmstadt, Germany), fetal bovine serum (FBS), Trypsin EDTA, penicillin and streptomycin (Biowest, Lakewood Ranch, FL, USA). DNaeay DNA extraction kit, Hot star taq PCR master mix (Qiagen, Hilden, Germany). pEGP miR-cloning and expression vector (Cell Biolabs, San Diego, CA, USA), fast digest BamHI and fast digest NheI enzymes, T4 ligase, Gel extraction kit, Plasmid miniprep, Turbofect transfection reagent (Thermo Scientific, Waltham, MA, USA). HI pure endotoxin-free plasmid maxiprep (Invitrogen, Waltham, MA, USA), MTT reagent (Sigma Aldrich, St. Louis, MO, USA). All other chemicals used were of pharmaceutical grade or the highest commercially available grade.

ScFvs were produced as Aga2p fusions^{57 (link)}. In brief, 4 µg of pPNL6 vector in Cut Smart buffer was digested using 1 µl of NheI HF and BamHI HF (New England Biolabs) at 37 °C for 1 hour. Digested plasmid was then gel extracted using the Thermo Fisher Scientific Gel Extraction Kit. Equimolar aliquots of each scFv plasmid were pooled, and the resultant pool was amplified using primers that annealed to the hexa-his tag (reverse primer) or signal peptide (forward primer) and had a 50-bp overlap with the pPNL6 vector digested with NheI and BamHI. The pooled amplification was gel extracted to ensure that it was the correct size. Yeast were prepared by first streaking a YPAD plate and incubating for 2–3 days until single colonies were identifiable. A single colony was inoculated in 5 ml of YPAD with overnight shaking at 30 °C. Cultures were harvested into six tubes and pelleted. Yeast were resuspended in electroporation buffer (10 mM Tris base, 250 mM sucrose, 2 mM MgCl₂) containing the gel-extracted library amplification and digested pPNL6 vector. This mixture was then pulsed, and the electroporated yeast were recovered in SD-CAA media overnight (30 °C shaking). These yeast were then induced by a 1:10 dilution into SG-CAA media and grown at 20 °C shaking for 2–3 days.

Based on the observation of the reporter gene assay, sporulating oocysts (24 h) were superficially sterilised as described above. The oocyst pellet was mixed 1:1 with glass beads (diameter 0.5 mm) and strongly grinded on a vortex mixer for about 5 min. The successful disintegration was controlled by microscopy. RNA was extracted with the Roche High Pure RNA Isolation Kit. The glass beads/oocysts lysate was mixed with 400 μl 1xPBS 4 °C, then 800 μl binding buffer was added and mixed again. The supernatant was applied to the High Pure Filter Tube and we further followed the protocol supplied by the manufacturer. The cDNA synthesis was performed with the Thermo Scientific Maxima H Minus First strand cDNA Synthesis Kit according to the manufacturers instructions. The cDNA was purified from the PCR reaction assay with the Gelextraction Kit (Thermo Scientific) and eluted in ultrapure water. The transcripts were amplified with the primer pair #7 and #8 for COWP6 (Kappa Hifi polymerase (Peqlab), GC-Buffer) and with the primer pair #9 and #10 for COWP2 (Kappa Hifi polymerase (Peqlab), Fidelity buffer). The PCR-products with the expected size were purified from gel, cloned into pjet.1.2 (Clonejet, Thermo Scientific) and sequenced (Co. GATC, Konstanz). (Primer sequences are listed in Additional file 1).

The 16S ribosomal ribonucleic acid (rRNA) gene fragments of 6 lactic acid-producing isolates were amplified using the universal primers F-27 (5′-AGAGTTTGATCMTGGCTCAG-3′) and R1494 (5′-CTACGGYTACCTTGTTACGAC-3′) using PCR machine (Bio-rad T100 thermal cycler). PCR products were checked via agarose gel electrophoresis then purified using gel extraction kit (Thermo scientific, Lithuania) and sequenced by Macrogen, Koria.

The lyophilized standard strains (S. agalactiae, E. faecalis, G. vaginalis and C. albicans) and cryopreserved clinical isolates were grown on appropriate agar plate, after which single colonies were picked for rejuvenation. Particularly, C. trachomatis was mixed with Hela cells inoculated on 2 mL DMEM high glucose medium (GIBCO company) containing 10% fetal bovine serum and 50 μg/mL gentamicin (Macleans Biochemical Technology), and centrifuged to promote entry [49 ]. The 6-well plate was incubated in a 37 °C, 5% CO₂ incubator for 48 h, and the cells were collected to obtain C. trachomatis. The cultured pathogen solution, except C. trachomatis, was diluted 10 times gradient with sterile phosphate buffered saline (PBS) solution, and then quantified with colony forming units (CFU).
Considering the difficulty in C. trachomatis quantification, the gyrA gene fragments were amplified by PCR and the concentration of the purified PCR products (Gel extraction kit, Omega) was determined (Thermo NanoDrop 2000). Then the copy number was calculated according to the length of the target gene fragments (http://cels.uri.edu/gsc/cndna.html), and the ten-fold gradient dilution was used as the LAMP reaction template.