Ht plate reader

First, pET-28a-sadC, pET-28a-ssuE, PET-28a-rutF, pET-28a-cogA, pET-28a-sutR, and pET-28a-psuK expressing E. coli BL21 (DE3) strains were cultured at 37°C and 200 rpm to OD₆₀₀ of 0.8–0.9. Then, the temperature was shifted from 37°C to 20°C and 0.2 mM IPTG was added. After induction for 8 h, the cells were harvested by centrifugation at 10,000 × g for 10 min and resuspended in PBS standard buffer. Then, the cells were sonicated and the supernatant was harvested after centrifugation at 10,000 × g for 15 min. Finally, FMN reductases were purified by using affinity chromatography using Ni²⁺-NTA columns.
The activity of FMN reductases was detected by calculating the decrease of NADPH (NADH), using FMN as the substrate, at 30°C for 10 min. The reaction was measured on a Biotek HT plate reader (Winooski, VT, USA) with an absorbance of 340 nm. One unit of FMN reductase activity was defined as the amount of NADPH (NADH) decrease under 1 µmol enzyme per minute at 30°C.

Norepinephrine enzyme-linked immunosorbent assays (ELISA) were performed according to the manufacturer instructions (Rocky Mountain Diagnostics, Colorado, CO USA). Plasma was collected using EDTA anti-coagulant. ELISAs were read with a Bio-Tek HT plate reader (Bio-Tek, Winooski, VT USA). Standard curve analysis was performed with GraphPadv6.0a software (GraphPad Software, Inc., La Jolla, CA USA) using a four-parameter logistic curve.

First, P. denitrificans DYTN-1 strains carrying reported plasmids were cultured at 30°C and 150 rpm to OD₆₀₀ of 0.4–0.6 ,then 50 µM cumate (4-isopropylbenzoic acid) was added. After 48 h of incubation, 20 µL of the activated cells was transferred to 96-well microtiter plates and diluted 10 times with phosphate-buffered saline (PBS) prior to fluorescence and OD₆₀₀ measurements. Green fluorescence measurements were performed using a Biotek HT plate reader (Winooski, VT, USA) with excitation at 485 nm, emission at 528 nm, while red fluorescence measurements were with excitation at 588 nm, emission at 633 nm.

About 2 mL overnight cultured strains was inoculated in 50 mL modified M9 medium (Supplementary Notes) which contained 100 mg/L SMX, 1 g/L NO₃⁻ or NH₄⁺, then cultured at 30°C for 72 h. Culture supernatant was obtained by centrifugation at 15,000 × g for 5 min and diluted for metabolite quantification. Nitrate and ammonia nitrogen were determined using LH-NO₃ Liquid and LH-NH₄ Liquid (Lianhua, Shanghai, China) kits on a Biotek HT plate reader (BioTek, Winooski, VT, USA) with emission at 410 and 420 nm, respectively.

MIA PaCa-2 cells were seeded at 10,000 cells/well into clear-bottomed, white-walled 96-well plates (Corning) and allowed to adhere overnight. The following day, wells were treated with PFK15, inhibitor cocktail (4 μM CCCP, 10 μM oligomycin, 200 μM bromopyruvate and 2 mM iodoacetate) or vehicle (DMSO). Global ATP was quantified using a Vialight plus assay (Lonza, Slough, UK), following the manufacturer’s instructions, using a Biotek HT plate reader.

First, pET-28a-sadC, pET-28a-ssuE, PET-28a-rutF, pET-28a-cogA, pET-28a-sutR, and pET-28a-psuK expressing E. coli BL21 (DE3) strains were cultured at 37°C and 200 rpm to OD₆₀₀ of 0.8–0.9. Then, the temperature was shifted from 37°C to 20°C and 0.2 mM IPTG was added. After induction for 8 h, the cells were harvested by centrifugation at 10,000 × g for 10 min and resuspended in PBS standard buffer. Then, the cells were sonicated and the supernatant was harvested after centrifugation at 10,000 × g for 15 min. Finally, FMN reductases were purified by using affinity chromatography using Ni²⁺-NTA columns.
The activity of FMN reductases was detected by calculating the decrease of NADPH (NADH), using FMN as the substrate, at 30°C for 10 min. The reaction was measured on a Biotek HT plate reader (Winooski, VT, USA) with an absorbance of 340 nm. One unit of FMN reductase activity was defined as the amount of NADPH (NADH) decrease under 1 µmol enzyme per minute at 30°C.