Isogenic E. coli K-12 strains (see Table S1 in the supplemental material) were obtained from the Keio collection (52 (link)) or constructed using CRISPR genome editing technology (53 (link)). Plasmids and primers used for this work are listed in Table S4. E. coli strains were cultured by shaking at 200 rpm aerobically at 37°C in Luria-Bertani (LB) broth or by plating on LB agar incubated at 37°C. Yeast extract, tryptone, agar, and 5(6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) were obtained from Thermo Fisher Scientific Corp. (Waltham, MA, USA). Ciprofloxacin, ampicillin, and kanamycin were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). Vancomycin, erythromycin, lysine monohydrochloride, arginine hydrochloride, histidine hydrochloride, malonate, propidium iodide, proteinase K solution, 5× protein loading dye, dimethyl sulfoxide (DMSO), and 2.2′-bipyridyl were purchased from Sangon Biotech Inc. (Shanghai, China). Lipopolysaccharide (LPS) was bought from Beyotime Biotech Inc. (Jiangsu, China). Ceftriaxone (Roche, Shanghai, China), and meropenem (Shenghuaxi Pharmaceutical Co., Chongqing, China) were gifts from the Zhongshan Hospital (Xiamen, China) pharmacy.
Malonate
Manufactured by Sangon
Sourced in China
Malonate is a chemical compound that is commonly used in laboratory settings. It is a dicarboxylic acid with the chemical formula CH2(COOH)2. Malonate serves as a building block for various organic synthesis reactions and is often employed in biochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Yeast extract, by Thermo Fisher Scientific (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Tryptone, by Thermo Fisher Scientific (1 mentions)
Ciprofloxacin, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Sangon (1 mentions)
Propidium iodide, by Sangon (1 mentions)
Protein loading dye, by Sangon (1 mentions)
Lipopolysaccharide lps, by Beyotime (1 mentions)
Vancomycin, by Sangon (1 mentions)
Erythromycin, by Sangon (1 mentions)
Proteinase k solution, by Sangon (1 mentions)
2.2 bipyridyl, by Sangon (1 mentions)
Triclosan, by Sangon (1 mentions)
3 protocols using malonate
1
Isogenic E. coli Strain Generation
2
Antibiotic Bactericidal Assay Protocol
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Antibiotic bactericidal assay was performed as previously described.22 (link) In brief, VA-S, VA-RCAZ, ATCC33787, ΔnqrA, and ΔnqrF were cultured for 12 hours at 30°C. Samples were harvested at 8,000 rpm for 3 minutes, and washed three times with 30 mL sterile saline and resuspended in M9 minimal medium with NaAc (10 mM), MgSO4 (2 mM), and CaCl2 (0.1 mM) to 0.6 at OD600. Indicated concentrations of CAZ with or without furfual, malonate, NaN3, CCCP, or triclosan were added in a final volume of 5 mL. After incubation at 30°C for 6 hours, 100 µL aliquot samples were collected, serially diluted, and plated (10 µL aliquots) on LB medium containing 2.5% agar. The plates were cultured at 30°C for 8–10 hours. Only those dilutions generating 20–200 clones were counted to calculate CFU. Percent survival was determined by dividing the CFU obtained from a treated sample by the CFU obtained from control. For fatty acid-enabled killing of VA-S by CAZ, the bacteria were cultured in LB with 0.2 mM of palmitate or stearate for 12 hours. The collected cells were used for the antibiotic bactericidal assay described above. Furfual, malonate, triclosan palmitate, and stearate were purchased from Sangon Biotech, and Sigma-Aldrich provided NaN3 and CCCP. Experiments were repeated in three independent biologic replicates.
3
Evaluating Antibiotic Susceptibility of V. alginolyticus
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V. alginolyticus ATCC 33787 was grown at 30°C in Luria–Bertani (LB) broth (pH 7.0) supplemented with 3% sodium chloride. The overnight culture was diluted 1:100 (v/v) in fresh LB broth (supplemented with 0, 0.3, or 0.5 μg/ml OFX) at 30°C for 2 h. The bacteria were harvested by centrifugation at 8,000 rpm for 3 min, and the cells were washed and resuspended in sterile saline and adjusted to an optical density (OD600) of 1.0. To build a growth curve, the bacterial growth was examined by measuring the OD600 of the culture at the indicated time. To study the effect of antibiotics, inhibitors of pyruvate cycle, or fatty acid biosynthesis on bacterial growth, the bacterial growth was examined by measuring the OD600 of the bacterial cultures at 10 h in medium with OFX, malonate, or triclosan (Sangon Biotech, Shanghai, China). At least three biologic replicates were performed.
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