Gene pulser micropulser electroporation cuvettes

Both plasmids were then transfected into Dd2 P. falciparum parasites cell lines, a multi-drug resistant strain derived from an Indochina isolate (Reilly et al., 2007 (link)). Transfection was performed by electroporation in uninfected erythrocytes (Hasenkamp et al., 2012 (link)). First, erythrocytes are washed with cytomix [10 mM/L K²HPO⁴/KH²PO⁴, 120 mM/L KCl, 0.15 mM/L CaCl₂, 5 mM/L MgCl₂, 25 mM/L HEPES, 2 mM/L EGTA, adjusted with 10 M/L KOH to pH 7.6]. Afterward, 300 µL of erythrocytes were added to the plasmid solution, which contains 50 µg of plasmid and up to 200 µL of cytomix. This was transferred to a Gene Pulser/MicroPulser™ Electroporation Cuvettes, 0.2 cm gap (Bio-Rad Laboratories, Inc) and eletric shocked on the Gene Pulser Xcell™ (Bio-Rad Laboratories) electroporator. The mixture was washed twice in 5 mL of malaria culture medium (MCM) [RPMI, 1640 (Gibco) with 2 mM L-glutamine, 200 µM hypoxanthine, 0.25 µg/mL gentamycin, 25 mM HEPES, 0.2% NaHCO3, and 0.25% Albumax II (Life Technologies)] to remove lysed erythrocytes. The transfected erythrocytes were then inoculated with P. falciparum Dd2 strain parasite erythrocytes at the trophozoite stage and 5 mL of MCM.

Transformation of E. coli NCM3722 was carried out via electroporation. For preparing electrocompetent cells, overnight culture was sub-cultured in fresh LB medium at 1/100 dilution factor without antibiotics, until it reached an OD₆₀₀ of approximately 0.40. Bacterial culture was cooled on ice for 15 min, followed by centrifugation at 4,000 X g for 5 min at 4°C. Supernatant was removed and the cell pellet was washed with 25 mL of 10% glycerol solution for every 50 mL of bacterial culture. Cells were centrifuged at 3,000 X g for 5 min. The washing step was repeated twice, and the final cell pellet was resuspended in 250 μL of 10% glycerol solution for every 50 mL of bacterial culture. Cells were aliquoted into 1.5 mL microcentrifuge tubes and stored at −80°C. Forty micro liter of chemically competent cells were pre-mixed with 10 ng of DNA for E. coli NCM3722. Cells were transferred into a Gene Pulser/MicroPulser Electroporation Cuvettes, 0.1 cm gap (Biorad, 1,652,083), and electroporation was carried out using MicroPulser Electroporator (Biorad, 652,100), with the “Bacteria” setting. Immediately after electroporation, cells were transferred into pre-chilled, 800 μL of SOC. Transformed cells were recovered in SOC for 1 h, at 37°C, with shaking. Twenty μL to 30 μL of recovered cells were plated onto LB agar with the appropriate antibiotic(s) and incubated at 37°C for 16 h.

A codon-optimized gene of wild-type CalB was synthesized for optimal expression in E. coli (GenScript) and cloned into expression vector pET22b+ (MilliporeSigma). A C-terminal 6-histidine Tag was designed to facilitate the purification process of all CalB variants. Using the same codon-optimized WT CalB sequence, three previously reported mutants of CalB displaying increased activity to bulky substrates in nonaqueous conditions were also synthesized: S47L, I189A, and double mutant I189A-L278P^{38 (link),39 (link),46 (link)}. Overexpression of the CalB enzyme from IPTG-inducible vector pET22b+ was tested using 5 different E. coli strains: Rosetta-Gami (DE3), Rosetta (DE3), Rosetta 2 (DE3) PLacI, Origami 2 (DE3), and BL21 (DE3) (MilliporeSigma). The selection markers employed were a combination of kanamycin and chloramphenicol (Rosetta-Gami (DE3)), chloramphenicol (Rosetta (DE3) and Rosetta 2 ((DE3)-PLacI), and streptomycin (Origami 2 (DE3)). Carbenicillin was used as resistance marker for expression vector pET22b+, which was electro-transformed into each strain using 0.1-cm Gene Pulser/MicroPulser electroporation cuvettes (Bio-Rad) and an ECM 630 electroporator (BTX Harvard Apparatus). Cells were recovered in SOB medium⁶² supplemented with 20 µL of 2 M glucose (filter-sterilized) and 2 M magnesium (1 M MgSO₄ and 1 M MgCl₂, autoclave-sterilized).