Erlenmeyer flasks

FreeStyle™ 293-F cells (Catalog number R79007, Thermo-Fisher Scientific, Waltham, MA) in suspension were maintained in FreeStyle™ 293 expression media in 125 ml Erlenmeyer flasks on an orbital shaker (135 rpm) in 8% CO₂ at 37 °C according to the manufacturer’s instructions (Thermo-Fisher Scientific, Waltham, MA). Cells (30 ml at 1 × 10⁶ cells ml⁻¹) in flasks were transiently transfected with antibody expression vectors (pVITRO-Cat IgG-IgL; pVITRO-Cat IgG-IgK; or pVITRO-felinized mAb E) using FreeStyle™ MAX transfection reagent (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. Transfected cells were collected on day 7, centrifuged at 1,000 × g for 15 min, then the supernatants were filtered through 0.2 μm filters (Whatman, Shrewsbury, MA).
Recombinant antibodies were purified by loading Ig-containing supernatants onto a 1 ml HiTrap Protein A HP column. After washing in phosphate buffered saline (PBS), purified antibodies were eluted with 0.1 M glycine-HCl buffer (pH 2.7), and fractions were collected into tubes containing 1 M Tris-HCl (pH 9).

The M. smegmatis pooled CRISPRi screen was performed similar to the M. tuberculosis screens described above with two primary differences: 1) the growth media contains ADC (see Mycobacterial cultures); and 2) cultures were grown shaking at 120 rpm in vented 125 mL Erlenmeyer flasks (Thermofisher #4115-0125).

The endophytic fungi P. citrinum TDPEF34 and G. candidum TDPEF20 were isolated as described previously from the roots of healthy and Leaf Brittle-diseased (LBD) date palm, respectively [1 (link)]. Briefly, mycelia from pure cultures of endophytic fungi were inoculated into potato dextrose broth (PDB) medium in Erlenmeyer flasks (Thermo Fisher Scientific, Foster City, CA, USA) and incubated in the shaker-incubator for 3 weeks at 30 °C, 150 rpm. Subsequently, fungal cultures were filtered through Whatman no.1 filter paper (Thomas Scientific, Foster City, CA, USA) and the resulted filtrates were extracted with n-hexane, ethyl acetate (EtOAc) and methanol (MetOH). The extraction was performed trice and the extracts were dried by a rotary evaporator under vacuum at 45 °C and freezing dried to complete dryness. Mycelia of endophytic fungi cultures were rinsed in sterile water and dried in an oven at 60 °C for 48 h. Then, the mycelia were ultrasonically extracted for 30 min in absolute ethanol and extracted in a similar way. The obtained extracts were stored at 4 °C before being used for further study.