Biomass samples for the estimation of the protein and RNA content were collected by centrifugation, washed with 0.9% NaCl, and stored at −70 °C before use.
The protein content in the biomass was estimated using a Bio-Rad DC Protein Assay kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions.
To separate two nucleic acids, RNA and DNA, the Schmidt−Tannhauser method was used [66 ]. At first, biomass samples were incubated with ice-cold 0.25 N HClO4 (1 mL) at 0 °C for 30 min to remove the low-weight cytoplasmic metabolites. During incubation, the test tubes containing the samples were gently mixed 2–3 times. Then, the biomass was collected at 8000× g for 3 min and incubated in 0.5 mL of 1 N KOH at 37 °C for 1 h, to hydrolyse the RNA to the monomers. The test tubes were mixed 2–3 times using a vortex. Then, the hydrolysed RNA was separated from the protein and DNA by adding ice-cold 3N HClO4 (0.5 mL) followed by centrifugation at 13,000× g for 5 min at 0 °C. The UV spectra of the obtained supernatants were recorded using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). The RNA concentration (g/L) in the solution was calculated using the following formula [67 ,68 (link)]:
where 0.19 is value of the (OD270−OD290) difference, which corresponds to nucleic acid hydrolysate with a nucleic acid phosphate concentration of 1 mg/L, and 10.3 is the average coefficient to transfer the phosphate amount to the ribonucleotides amount.
The protein content in the biomass was estimated using a Bio-Rad DC Protein Assay kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions.
To separate two nucleic acids, RNA and DNA, the Schmidt−Tannhauser method was used [66 ]. At first, biomass samples were incubated with ice-cold 0.25 N HClO4 (1 mL) at 0 °C for 30 min to remove the low-weight cytoplasmic metabolites. During incubation, the test tubes containing the samples were gently mixed 2–3 times. Then, the biomass was collected at 8000× g for 3 min and incubated in 0.5 mL of 1 N KOH at 37 °C for 1 h, to hydrolyse the RNA to the monomers. The test tubes were mixed 2–3 times using a vortex. Then, the hydrolysed RNA was separated from the protein and DNA by adding ice-cold 3N HClO4 (0.5 mL) followed by centrifugation at 13,000× g for 5 min at 0 °C. The UV spectra of the obtained supernatants were recorded using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). The RNA concentration (g/L) in the solution was calculated using the following formula [67 ,68 (link)]:
where 0.19 is value of the (OD270−OD290) difference, which corresponds to nucleic acid hydrolysate with a nucleic acid phosphate concentration of 1 mg/L, and 10.3 is the average coefficient to transfer the phosphate amount to the ribonucleotides amount.
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