Isopropyl β d 1 thiogalactopyranoside iptg

E. coli and Aeromonads were grown with aeration in Luria–Bertani (LB-Miller, BD-Difco) broth. Vibrio strains were grown in LB medium with 3% NaCl. All strains were grown at 30 °C. Strains used in the study are listed in Supplementary Table 4. Unless otherwise noted, antibiotics, were used at the following concentration: 100 μg ml⁻¹ ampicillin (Sigma), 50 μg ml⁻¹ kanamycin (GoldBio) and 5 μg ml⁻¹ chloramphenicol (Sigma). Inducers were used as follows: E. coli: 200 μM isopropyl β-d-1-thiogalactopyranoside (IPTG, GoldBio) and 50 ng ml⁻¹ aTc (Clontech); Vibrios: 500 ng ml⁻¹ ciprofloxacin (Sigma) and 50 ng ml⁻¹ aTc; Aeromonads: 1 μg ml⁻¹ ciprofloxacin and 5 ng ml⁻¹ aTc. C4-HSL was supplied at a final concentration of 10 μM except for the experiment shown in Fig. 3b, in which it was administered at specified doses, as indicated in the figure.

Escherichia coli strains were grown in Luria-Bertani broth (LB; Difco) at 24 C and 240 rpm with the following selective antibiotics and inducing reagents used at the indicated concentrations: carbenicillin, 50 μg/ml; chloramphenicol, 20 μg/ml; isopropyl β-d-1-thiogalactopyranoside (IPTG; Gold Biotechnology), 1.0 mM or 0.1 mM; arabinose (Alfa Aesar), 0.2% (wt/vol).

l-Canavanine, tris(2-carboxyethyl)phosphine
(TCEP), l-Arg, sodium phosphate monobasic, and imidazole
were acquired from Sigma (St. Louis, Missouri). Kanamycin, isopropyl
β-d-1-thiogalactopyranoside (IPTG), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES), and Ni-NTA agarose beads were purchased from GoldBio
(St. Louis, Missouri). 2OG was purchased from Fluka. EDTA was purchased
from Invitrogen. 50% PEG 3350, PEG 400, PEG MME 550, and 50% ethylene
glycol were purchased from Rigaku or Hampton. All other chemicals
were reagent grade or better.

The Y82E9BR.3-specific RNAi clone, which was prepared previously^{21 (link)}, was cultured overnight in LB broth containing 50 μg/ml ampicillin (USB, Santa Clara, CA, USA). To induce double-stranded RNA, 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG; Gold Biotechnology, St. Louis, MO, USA) was added to plates for incubation at room temperature for 1 day before usage.

Transformed BL21 cells were cultured in Luria-Bertani (LB) broth (MB Cell, Los Angeles, CA, USA) containing 30 μg/mL of kanamycin (LPS solution, Daejeon, Korea) for 3 h at 37 °C, then incubated with 0.1 mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Goldbio, St. Louis, MO, USA) at 18 °C overnight. After IPTG induction, cells were centrifuged and resuspended in lysis buffer containing 50 mM of Tris-HCl (Welgene, Daegu, Korea), 0.5 M of NaCl (Welgene), and 10 mM of imidazole (Biosesang, Seongnam, Korea) with 1 mM of phenylmethylsulfonyl fluoride (Biobasic, Toronto, ON, Canada). Cell suspension was lysed by sonication for 40 × 10 s with 10 s intervals on ice. The lysate was centrifuged at 8000 rpm for 30 min at 4 °C, and His-tagged Mx1-9R was purified by Ni-NTA resin (Incospharm, Daejeon, Korea). Then, purified Mx1-9R fusion proteins were desalted using Amicon Ultra-15 Centrifugal Filter Units (Millipore Sigma, Darmstadt, Germany). Supernatant, pellet, flow through, and eluent were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the gel was stained with 0.05% Coomassie Blue R-250 (LPS solution). The amounts of recombinant proteins were quantified by Bradford assay or NanoDrop (Thermo Fisher, Waltham, MA, USA) measurements.