Sybr gold nucleic acid stain

For the Michaelis–Menten analysis, reactions were performed under similar conditions to those above. Final substrate concentrations were: 0.5, 1, 2, 4, 6, 8 and 10 μM. The final enzyme concentration was 40 nM. Samples were separated and analysed by denaturing PAGE alongside a series of dilutions of the substrate at concentrations of 0, 6.25, 12.5, 25, 50, 100 and 500 nM. Initial rates of product formation were calculated from time points in the linear phase of the reaction using the calibration curve of the serial dilutions. These rates were then fitted by non-linear regression to the Michaelis–Menten equation as shown in Equation 1:
where v represents the initial rate, [E] represents the total enzyme concentration, k_cat represents the enzyme turnover number, [S] represents the initial substrate concentration and K_M represents the Michaelis constant.
For the single-turnover experiment, final substrate and enzyme concentrations were 5 nM and 200 nM, respectively. Samples were taken at the following time points: 0, 5, 15, 30, 60 and 120 s, and separated by denaturing PAGE, as described above. Gels were stained with SYBR^®Gold Nucleic Acid Stain (Thermofisher) and visualized on a Fujifilm FLA-5000 scanner with a Y510 filter and the excitation laser set to 478 nm.

Di-sodium ethylenediaminetetraacetic acid (EDTA-Na₂), malondialdehyde (MDA), 2-thiobarbituric acid (TBA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were acquired from Sigma Aldrich Co. (St. Louis, MO, USA). SYBR gold nucleic acid stain and trichloroacetic acid (TCA) were purchased from Thermo Fisher Scientific Co. (San Jose, CA, USA). Reagents and media for the cell line included a trypan blue dye, trypsin-EDTA, fetal bovine serum (FBS), penicillin-streptomycin and Dulbecco’s Modified Eagle Medium (DMEM) were purchased from Gibco BRL, Life Technologies Inc. (Rockville, MD, USA). Low melting point agarose (LMA), dimethyl sulfoxide (DMSO) and Triton-X was purchased from Amresco Inc. (Solon, OH, USA).

Bst DNA polymerase large fragment, magnesium sulphate and deoxynucleotide phosphates (dNTPs) were purchased from NEB (Ipswich, MA, USA). Thermo Pol Buffer was provided with the enzyme. Betaine, hypoxanthine and sodium bicarbonate were brought from Sigma-Aldrich (St. Louis, MO, USA). AlbuMax I was purchased from Gibco, Grand Island, NY, USA, and SYBR Gold nucleic acid stain (10,000x concentration) was purchased from Thermo Fischer Scientific (Waltham, MA, USA). RPMI 1640 media was obtained from Invitrogen, Carlsbad, CA, USA. All other materials used were of analytical grade and commercially available.

GreenB1 was folded in PBS/5 mM MgCl₂ at 1 μM concentration as described previously, 10% of FBS was added to the aptamer, and the mixture was incubated in a heat block at 37°C. Samples were taken after 1, 2, 3, 6, 24, and 48 h (n = 3) and kept at −20°C until further use. From each sample, 1 μL was mixed with loading dye and water and loaded on 3% agarose gel stained with SYBR Gold nucleic acid stain (S11494, Thermo Fisher Scientific). The gel was run at 100 V for 40 min.

Oligo ADP-ribosylation experiments were conducted in a buffer solution composed of 50 mM Tris-Cl at pH 8.0, 50 mM NaCl, and 5 mM ETDA. The reactions were carried out in a 10 µL reaction volume at 37 °C, lasting 30 min for T. aquaticus DarT2 and 60 min for S. coelicolor ScARP. In the case of T. aquaticus DarT2, 1 µM of the enzyme was exposed to 3 µM oligonucleotides, using an excess of β-NAD⁺ (500 µM). For S. coelicolor ScARP, 0.1 µM of the protein was added to 10 µM oligonucleotides, along with an excess of β-NAD⁺ (3 mM). The ADP-ribosylation reaction was stopped for oligo de-modification by hydrolases by heating the samples for 15 min at 95 °C. Subsequently, the samples were either incubated with buffer for control purposes or with 1 μM of the specified hydrolase at 37 °C for 30 min.
The reaction products were separated on denaturing polyacrylamide gels in 1 × TBE buffer after the addition of 10 µL urea loading dye (10 mM TRIS pH 8.0, 10 mM EDTA, 4 M urea), followed by a 3 min incubation at 95 °C. Then, 10 µL of the treated samples were loaded onto the gel, and the oligos were visualised under UV light (340 nm) after staining with ethidium bromide or SYBR Gold nucleic acid stain (Thermo Scientific (US)). The results of all experiments are representative of a minimum of three biological replicates.

Cells in the co-cultures were sampled at various times during growth. Samples of 1 ml were centrifuged (11,300 g, 10 min), and the supernatants were discarded. Cell pellets were washed three times with 0.1 M phosphate buffer (PBS; pH 7.2) before being fixed in 4% paraformaldehyde. Flow cytometry was performed using a BD Influx Fluorescence-Activated Cell Sorter (BD Biosciences, San Jose, CA, USA). To disrupt cell aggregates, the cultures were treated with 100 mM Na₂EDTA (Sigma Aldrich), passed through a 25-gauge needle 25 times, and then vortexed. The samples were then stained with SYBR Gold nucleic acid stain (ThermoFisher, Waltham, MA, USA). Optimization and calibration were performed before each flow cytometry analysis using 3.6-mm Ultra Rainbow fluorescent particles (Spherotech, Lake Forest, IL, USA). Forward and side scattering were used to gate out cellular debris, and the 488-nm argon laser was used to excite SYBR Gold while measuring emissions at 542/27 nm. Gating and median calculations for 20,000 cells were done using Flow Jo software (Tree Star, Ashland, OR). The ratios of the two distinct populations of cells within a mixed microbial community were identified from 20,000 recorded cells using size and complexity gates within FCS Express (Los Angeles, CA, USA) flow cytometry software.