Electroporator

Transfection was performed by electroporation. Briefly, trophozoites in log phase were harvested and washed with phosphate buffer saline (PBS), followed by incomplete cytomix buffer (10 mM K₂HPO₄/KH₂PO₄ (pH 7.6), 120 mM KCl, 0.15 mM CaCl₂, 25 mM HEPES (pH 7.4), 2 mM EGTA, 5 mM MgCl₂]. The washed cells were then re-suspended in 0.8 ml of complete cytomix buffer (incomplete cytomix containing 4 mM adenosine triphosphate, 10 mM glutathione) containing 200 mg of plasmid DNA and subjected to two consecutive pulses of 3000 V/cm (1.2 kV) at 25 mF (Bio-Rad, electroporator). The transfectants were initially allowed to grow without any selection. Drug selection was initiated after 2 days of transfection in the presence of 10 µg ml⁻¹ G-418 for constructs with GFP or 10 µg ml⁻¹ of hygromycin B was used for tetracycline inducible constructs.

A single colony of wildtype C. glutamicum ATCC 13032 was inoculated into 3 mL of BHI medium and grown at 30 °C and 225 rpm. After overnight growth, 2 mL culture was transferred to 50 mL fresh BHI medium and grown to OD₆₀₀ of ~ 1.75. Cells were chilled on ice for 10 min and centrifuged for 5 min at 3500 rpm and 4 °C. The pellet was washed once with 50 mL of ice-cold 10% (v/v) glycerol containing 1 mM Tris (pH 7.5) in ultrapure water and once with 50 mL of ice-cold 10% (v/v) glycerol, and was then resuspended in 1 mL ice-cold 10% glycerol. Aliquots (100 µL) were stored at − 80 °C. For electroporation, cells were thawed on ice (10 min), mixed with ~ 100 ng plasmid, and transferred to an electroporation cuvette (2 mm gap). Electroporation was performed with an electroporator (Bio-Rad) at 25 μF, 200 W and 2.5 kV, yielding a pulse duration of ~ 5 ms. Immediately after electroporation, cells were mixed with 1 mL pre-warmed BHI in the cuvette, and were transferred to a 2-mL microcentrifuge tube. Cells were heat-shocked at 46 °C for 6 min in a water bath, transferred to a 14-mL culture tube (VWR), incubated for 2 h at 30 °C, and plated on LB-agar plates containing 25 mg/L kanamycin. Positive clones were validated by colony PCR, plasmid miniprep, and gene sequencing (Genewiz).

E14Tg2a embryonic stem cells were maintained in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 15% fetal calf serum (FCS), 2 mM L-glutamine, 1 mM sodium pyruvate, 1% penicillin/streptomycin, 100 μM non-essential amino acids (all from Invitrogen), 100 μM β-mercaptoethanol (Sigma) containing leukemia inhibitory factor (LIF) in the presence of 1 μM PD-0325901 and 3 μM CT-99021 (“2i”) at 37 °C with 5% CO₂. ESCs (5 × 10⁶) were electroporated with 40 μg linearized targeting vector (Rosa26-CAG-NLS-TIR1-IRES-puro) or co-electroporated with 20 μg linearized targeting vector (Ash2l-, Wdr82-AID), 15 μg gRNA plasmid and 5 μg NLS-FLAG-linker-Cas9 expression vector using a Bio-Rad electroporator (250 V, 500 μF) and selected for 7 days with 1 μg/ml puromycin (Sigma) or with 200 μg/ml G418 (Invitrogen), respectively. Double targeted ES cells were expanded under double selection (puromycin + G418) and induced with 500 μM indole-3-acetic acid (IAA, Sigma).

Electrocompetent cells of A. baylyi were prepared as published for E. coli [38 (link)] with modifications. Briefly, a logarithmic culture of A. baylyi was grown at 30 °C in LB to 2.5×10⁸ cells ml⁻¹. The cells were chilled, washed twice with ice-cold distilled water, and concentrated 500 times in 10 % ice-cold glycerol solution (v/v). A 40 µl aliquot was mixed with DNA (400 ng pJK2 or 200 ng RSF1010 DNA), transferred into a 2 mm gap electroporation cuvette and pulsed with 12.5 kV cm⁻¹ (25 µF, 200 Ω) using a BioRad electroporator. Next, the cells were suspended in 1 ml prewarmed SOC (2 % tryptone, 0.5 % yeast, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mM glucose) and aerated for 1 h at 30 °C. Appropriate dilutions were plated on LB (recipient titre) and LB containing antibiotics (transformants) and the plates were incubated at 30 °C for 16 to 20 h or 36 to 48 h, respectively.

E. histolytica strain HM1:IMSS trophozoites were grown axenically in TYI-S-33 medium [58 (link)]. The cells were maintained and grown in TYI-33 medium complemented with 15% adult bovine serum, 1X Diamond’s vitamin mix and antibiotic (125 μl of 250 U/ml benzyl penicillin and 0.25 mg/ml streptomycin per 90 ml of medium). Transfection was performed by carrying out electroporation as described previously [59 (link)]. Briefly, trophozoites in log phase were harvested and washed with PBS followed by incomplete cytomix buffer [10 mM K₂HPO₄/KH₂PO₄ (pH 7.6), 120 mM KCl, 0.15 mM CaCl₂, 25 mM HEPES (pH 7.4), 2 mM EGTA, 5 mM MgCl₂]. The washed cells were then resuspended in 0.8 ml of complete cytomix buffer (incomplete cytomix containing 4 mM adenosine triphosphate, 10 mM glutathione) containing 200 μg of plasmid DNA and subjected to two consecutive pulses of 3000 V/cm (1.2 kV) at 25 μF (Bio-Rad, electroporator). The transfectants were initially allowed to grow without any selection. Drug selection was initiated after two days of transfection in the presence of 10 μg/ml G-418 for constructs with luciferase reporter gene.

The plant transformation binary vector pCAMBIA1300, modified to include the maize ubiquitin (Ubi-1) promoter and renamed pSVP [30 (link)], was used to drive dsCHI expression. To construct dsCHI hairpin cassettes (Fig 1), a pyruvate orthophosphate dikinase (PDK) intron of 767 bp was introduced between the sense and antisense coding sequence of 501 bp of the Chilo partellus chitinase gene (MK560453) and synthesized by Bio Basic Inc. The cassette was introduced at the Kpnl restriction site of pSVP and transformed into competent Agrobacterium tumefaciens LBA4404 made by a Bio-Rad electroporator. One hundred microlitres of transformed cells was spread on YEP agar containing 25 μg/ml tetracycline and 50 μg/ml kanamycin and grown at 28°C for 48 hours. The positive clones were confirmed by colony PCR with dsCHI primers (Table 1) designed to amplify a 279 bp region of the dsCHI hairpin and streaked on YEP agar for subsequent infection. Transformation was performed by the immature maize embryo transformation protocol described by [31 (link)]. The transformants were maintained on media containing 100 mg/ml cefotaxime and 200 mg/ml carbenicillin to grow until 4 foliage leaves emerged. Then, they were moved into sterile pot soil and transferred to a screen house (S1 Fig in S1 File). Genomic DNA was isolated from the putative transformants and untransformed control for PCR confirmation.