Kanamycin

Triple gene vector, pGA482-12ER having Cry1Ac-Cry2Ab-EPSPS cassettes was transformed into Agrobacterium strain LBA4404, and clones were confirmed through colony PCR. The confirmed clone was further used for the transformation of cotton var. Coker312. Hypocotyls were excised from 7 days old plantlets of Coker312 and were immersed into Agrobacterium culture of the pGA482-12ER plasmid. These hypocotyl cuttings were dipped in bacterial culture and were occasionally shaken. Afterward explants were dried on a blotting paper, followed by their transfer onto co-cultivation MS medium (MSP09-100LT, Caisson Labs-USA), where for two days; these were incubated in dark at 26 ± 2 °C. Then for callus initiation, these hypocotyls were further shifted to the callus induction medium. After callus formation, the calli were placed on an embryo maturation medium. Once embryos started maturing, these were shifted to germination medium followed by shifting onto shooting and rooting media in jars. Throughout the process, the shooting and rooting medium was supplemented with 50 mg/L kanamycin (Carl ROTH) and 200 mg/L cefotaxime. The plantlets with fully established roots were shifted to pots containing peat moss and then after hardening was shifted to pots having soil.

To investigate the role of sRNA knock-outs on cea and cel gene expression on a longer time-scale, experiments were performed with the Fluostar Optima Plate Reader (BMG Labtech). A 500-µl aliquot of a starter culture at OD₆₀₀ 0.2 was induced with the appropriate MitC concentration as described above. To prevent cultures from drying out, the plate was sealed with an O₂ permeable foil. Antibiotics were added as required, ampicillin at 100 µg/ml (Carl Roth, Germany), kanamycin at 50 µg/ml (Carl Roth, Germany) and chloramphenicol at 5 µg/ml (Carl Roth, Germany). Bacterial growth (absorbance) and YFP and CFP fluorescence development (representing cea and cel expression, respectively) was followed over a period of 16 h at 37 °C, with shaking at 300 rpm.

Neisseria gonorrhoeae was grown overnight at 37°C and 5% CO₂ on agar plates containing gonococcal base (GC) agar [10 g/l Bacto agar (BD Biosciences, Bedford, MA, United States), 5 g/l NaCl (Roth, Darmstadt, Germany), 4 g/l K₂HPO₄ (Roth), 1 g/l KH₂PO₄ (Roth), 15 g/l Proteose Peptone No. 3 (BD), 0.5 g/l soluble starch (Sigma-Aldrich, St. Louis, MO, United States)] supplemented with IsoVitaleX (IVX): 1 g/l D-Glucose (Roth), 0.1 g/l L-glutamine (Roth), 0.289 g/l L-cysteine-HCL × H₂0 (Roth), 1 mg/l thiamine pyrophosphate (Sigma-Aldrich), 0.2 mg/l Fe(NO₃)₃ (Sigma-Aldrich), 0.03 mg/l thiamine HCl (Roth), 0.13 mg/l 4-aminobenzoic acid (Sigma-Aldrich), 2.5 mg/l β-nicotinamide adenine dinucleotide (Roth) and 0.1 mg/l vitamin B₁₂ (Sigma-Aldrich). GC medium is identical to the base agar composition but lacks agar and starch.
E. coli was grown in LB (Lysogeny Broth, Roth) medium or on LB agar plates (15 g/l Bacto agar (BD Biosciences, Bedford, MA, United States) at 37°C.
For N. gonorrhoeae antibiotics were used at the following concentrations: 2.5–5 μg/ml erythromycin (Thermo-Fisher), 100 μg/ml streptomycin (Sigma-Aldrich), 10 μg/ml chloramphenicol (Sigma-Aldrich). For E. coli antibiotics were used at the following concentrations: 50 μg/mL kanamycin (Roth).

Terrific broth medium (TB medium) was prepared in advance (4× concentrated and autoclaved). This medium was diluted in water (1 L) and the medium containing the cells and kanamycin (50 µg/mL) (Carl Roth, Karlsruhe, Germany) was pipetted at a volume of 10 mL. The cells were grown up to 1.5 of Density Optical (DO) (200 RPM, 37 °C). The shaker was set up at 18 °C, and the cells were grown up to 3.0 DO (180 RPM) and then induced overnight with 500 µM of Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Amresco, Solon, ME, USA). After, the cells were harvested at 8.700 g using centrifuge Sorval LyNX 6000-Thermo (Thermo Fisher Scientific, Waltham, MA, USA) and stored at −20 °C.

An overview of the experiments presented in this study is depicted in Supplementary Table S1.
After a number of passages, as shown in Supplementary Table S1, the main culture was started at a seed cell concentration of 0.2∙10⁶ cells mL⁻¹. PenStrep (stock with 10,000 Units mL⁻¹ penicillin and 10 g L⁻¹ streptomycin, Sigma-Aldrich, United States) or kanamycin (stock with 30 g L⁻¹, Carl Roth, Germany) was added to the culture, resulting in a concentration of 1% or 30 mg L⁻¹, respectively, for the final culture medium. Cultivation was carried out in 250-ml Corning® polycarbonate flasks with vent-cap (“single-use polycarbonate”) (Sigma-Aldrich, United States) or 250 ml Erlenmeyer glass flasks (Duran, Schott AG, Germany).

The TAT-MeCP2 construct consists of the sequences encoding from 5′ to 3′, the His-tags, TAT-peptide, human MeCP2 isoform B (498aa), and Strep-tag cloned into the expression plasmid pET-28a(+) (Novagen, Merck, Darmstadt, Germany). Protein expression was carried out in the Escherichia Coli strain BL21 (DE3) (#60300-2, Lucigen) in eight 300 mL LB Rich cultures containing 50 mg mL⁻¹ Kanamycin (#25389-94-0, Roth). Following induction with 0.5 mM isopropyl-b-D-1-thiogalactopyranoside (IPTG, #I6758, Sigma Aldrich), these cultures were incubated for 20 hours at 30 °C shaking at 250 RPM. Cells were harvested by centrifugation at 4200 g for 30 min; the pellets were stored at −80 °C.
The protein was purified in a step-wise process involving cell disruption via sonication, centrifugation to pellet cell debris, Strep-Tactin XT affinity chromatography (#2-4014-001, IBA, Göttingen), gel filtration chromatography and LPS removal using Triton X-114 (X114, Sigma-Aldrich). The purified TAT-MeCP2 protein samples were stored at −80 °C.
The TAT-MeCP2-eGFP construct was designed, expressed and purified in a similar fashion to its TAT-MeCP2 counterpart, with the only difference being the presence of eGFP coding sequence between the sequences encoding for MECP2 and the Strep-Tag. The coding sequences of both constructs can be provided upon request.