Allegra 25r centrifuge

Liquid cultures of VRE isolates ST80 and ST117 were grown overnight (16 h) in LB at 37 °C with shaking at 180 rpm. Bacteria were removed by centrifugation (5,525 g, 4 °C, 5 min) in the Allegra™ 25R centrifuge with a TS-5.1–500 rotor (Beckman Coulter), supernatants were collected and sterile filtered using a 0.2 µm syringe filter (Corning, Corning, USA). A sterile control was set up on LB agar.

Three biological replicates of Synechocystis wild type and ΔHoxE-H strains were grown for 5 days under continuous light or darkness as described above. Cells were collected by 5000 × g centrifugation for 10 minutes at 4 °C (Allegra^® 25R Centrifuge, Beckman Coulter), frozen in liquid nitrogen, and stored at −20 °C. For protein extraction, the cells were washed twice with 50 mM HEPES-KOH, pH 7.5 and then suspended in the presence of 1 mM PMSF. The cells were centrifuged and the pellet was suspended in equal volumes of extraction buffer (50 mM HEPES-KOH, 0.1% SDS, 0.1% Triton X-100, 1 mM PMSF, pH 7.5) and glass beads (150–212 μm diameter). Then the cells were homogenized at 3500 rpm in a Mini Bead Beater (Biospec Products). Intact cells and glass beads were removed by 1000 × g centrifugation for 5 minutes (Mikro 22R, Hettich) and the supernatant was incubated in 2% SDS for 30 minutes to facilitate the extraction of membrane proteins. Samples were centrifuged again at 12 000 × g for 20 minutes at 4 °C, and the supernatant was stored at −20 °C until further use. Protein concentration was determined by Bradford method (Bradford Reagent, Sigma-Aldrich). The proteins were precipitated in ice-cold acetone (1:4, v/v) at −20 °C overnight and stored at −80 °C.

The various instruments used are summarized in this section. 5L Bioreactor (BiotronLiflus GX 7 L, Gimpo-si, Gyeonggi-do, Korea), centrifuge (Allegra™ 25R Centrifuge-Beckman Coulter™, California, Massachusetts, USA), sonicator (QSonica, Newtown, USA), Rotary evaporator (STRIKE 300, Stereo Glass, San Martino in Campo-Perugia, Italy), UV–VIS spectrophotometer (Thermo Scientific™ GENESYS™ 10S), Autoclave (Hirayama HV-50, Japan).

The liver from a newborn pig (5 weeks old) was obtained from a market in Shenzhen City, Guangdong Province, PR China. The total protein content of the liver was 19.8%. The liver was cut into pieces and washed under running tap water, then drained. First, the drained liver pieces were dissolved in water at a liver to water ratio of 1:2.8 (m/V). After homogenization, the liver solution was frozen at −20 °C for 6 h and ultrasonicated with 1200DT ultrasonic cell crusher (Biosafer Technologies Co., Ltd., Beijing, PR China) at a power of 300 W for 15 min. We repeated these processes five times. The soluble fraction (extracted liver proteins from newborn piglet (LPNP) were centrifuged using Allegra™ 25R centrifuge (Beckman Coulter, Inc., Bremerhaven, Germany) at 8694×g for 10 min followed by filtration. The supernatants were lyophilized by FDU-1200 lyophilizer (Tokyo Rikakikai Co., Ltd., Tokyo, Japan) and stored at −20 °C.