Hygromycin

L. tarentolae T7-TR strain (Cat.-No. EGE-1410, Jena Bioscience, Germany), were cultivated on brain heart infusion (BHI) medium (Merck, Germany), with the addition of 15 μg/L of hemin (Jena Bioscience), 50 IU/mL of penicillin, and 50 μg/mL of streptomycin (Jena Bioscience). As used L. tarentolae is T7-TR, the inducible host, two more antibiotics, hygromycin (50 µg/ml) and nourseothricin (50 µg/ml; Jena Bioscience), were added. Cells were cultivated in 50-ml ventilated flasks (Orange, USA) at 26 °C in two styles: static and agitated culture. The suspension culture of L. tarentolae was propagated by dilution ratio of 1:10 to 1:100 into a fresh medium when it reached the stationary growth phase. After dilution, the number of cells was typically 10⁷/ml.

Each gene of the H and M loci was overexpressed in BPK026/0 cl5 after cloning in pLEXSY-hyg2 (Jena Bioscience) (Table S2). All genes except the MRPA gene were PCR amplified from BPK282 DNA using Phusion high-fidelity polymerase (NEB) with overhang primers (Table S2) for subcloning in pGEMT (Promega) before final cloning in pLEXSY-hyg2. MRPA was amplified using iProof high-fidelity DNA polymerase (Bio-Rad) with GC buffer plus 10% dimethyl sulfoxide (DMSO) and directly cloned in the pLEXSY-hyg2 using the In-Fusion HD cloning kit (Clontech). Every PCR-amplified sequence was sequenced at the VIB Genetic Service Facility (VIB Genetic Service Facility, Antwerp, Belgium). Promastigotes of BPK026/0 cl5 were then transfected using GenePulser Xcell (Bio-Rad) as described previously (37 (link)). Transfected parasites were selected with 50 µg·ml⁻¹ of hygromycin (Jena Bioscience) and then maintained with 100 µg·ml⁻¹.