Ampicillin

The mCherry coding sequence was amplified via PCR using plasmid CD3-960 (Nelson et al., 2007 (link)) as template and primers 5′-AAAAGCTAGCAAAGAATTCATGGTGAGCAAGGGCGAGG AG-3′ and 5′-TTTCTCGAGTTTGTACAGCTCGTCCATGC-3′ flanked by NheI and XhoI recognition sites. The PCR product and pET21b were digested with NheI and XhoI, gel-purified, and ligated to yield pET-mCherry-6xHis (pMS1034). pMS1034 was used as a template for PCR to add the coding sequence for the 1xHA motif via primers 5′-AAAAGGATCCTATCCGTATGATGTGCCAGATTATGCCT GAGATCCGGCTGCTAACAAA-3′ and 5′-TTTTGGATCC GTGGTGGTGGTGGTGGTGCTCGAG-3′ (BamHI sites are underlined). The resulting 6,140 bp PCR product was digested with BamHI and recircularized, giving rise to pET21b-mCherry-6xHis-1xHA (pMS1090). mCherry-6xHis-1xHA was expressed in ER2566 cells in LB-medium (supplemented with 100 μg/mL ampicillin (K029.2, Carl Roth)) after inducing the expression with 0.5 mM IPTG at 37°C for 4 h and purified under denaturing conditions as described previously (Hammel et al., 2018 (link)) using nickel-charged resin. The concentration of recombinant mCherry-6xHis-1xHA was determined using the Pierce BCA protein assay kit following the manufacturer’s instructions.

5x KCM buffer (0.5M KCl, 0.15M CaCl₂, 0.25M MgCl₂), Escherichia coli strain K12 CJ236 (NEB, E4141), SOC outgrowth medium (ThermoFisher Scientific, 15544034), LB-agar plates supplemented with 100 µg/ml ampicillin (Roth, K029.2).

The bacterial strains used in this study are listed in Table 1. The E. coli strains were grown in Luria–Bertani (LB) broth medium. When required, antibiotics (Carl Roth, Karlsruhe, Germany; Sigma-Aldrich, St. Louis, MO, USA) were added to the cultures at the following concentrations: 75 μg mL⁻¹ ampicillin, 50 μg mL⁻¹ kanamycin, 50 or 120 μg mL⁻¹ hygromycin, and 50 μg mL⁻¹ apramycin (Carl Roth, Karlsruhe, Germany; Sigma-Aldrich, St. Louis, MO, USA). E. coli GB05-red [21 (link)] was employed in Red/ET recombineering experiments [22 (link)].
For conjugation, the Streptomyces albus J1074, del9 and del10 [19 (link)] strains were grown on oatmeal or mannitol soy (MS) agar [23 ] for sporulation.

The gene encoding Region II (amino acids 141–756) of the EBA-140 ligand (GenBank: AF 332918_1) was cloned from genomic DNA of P. falciparum clone Dd2 (ATCC, MR4, Manassas, VA, USA) with forward primer: CAA TAT ACG TTT ATA CAG AAA CGT ACT CAT TTG TTT GCT and reverse primer: TAT ATC GTG TTT TGT TTT AGG ATA TTT A. Polymerase Taq (Fermentas) was used in 35 cycles of amplification (94°C, 30 s; 54°C, 30 s; 72°C, 2 min 30 s) after a hot start at 94°C for 5 minutes and 10 minutes of final extension at 72°C. The PCR product was purified using a gel extraction kit (Qiagen) and cloned into a pDrive cloning vector (Qiagen) using a T4 DNA ligase (Fermentas). The resulting pDrive-RII recombinant plasmid was transformed into E. coli XL1 Blue competent cells and selected on LB-agar with 100 µg/ml ampicillin (Roth). The sequence was confirmed by restriction fragment analysis and DNA sequencing and used for obtaining recombinant baculovirus.

Zanamivir (GlaxoSmithKline, Brentford,
UK), ampicillin (Carl-Roth GmbH, Karlsruhe, Germany), and rifampicin
(Sigma-Aldrich, St. Louis, MO, USA) were used as references. Stock
solutions were prepared in water (Zanamivir: 10 mM; ampicillin: 10
mg/mL) or DMSO (rifampicin: 10 mg/mL).

Bacteria were grown at 37 °C and 150 rpm in LB medium containing 12.5 ng/µL tetracycline (Fisher BioReagents, Geel, Belgium) and 100 ng/µL ampicillin (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) to an optical density per mL measured at a wavelength of 600 nm (OD₆₀₀/mL) of 0.4 to 0.8. dsRNA expression was induced by the addition of 0.4 mM IPTG. Cells were grown for another 4 h at 37 °C and 150 rpm, harvested, and further treated according to the different food preparation protocols.