Strains, plasmids, and primers used in this study are listed in Table S4 in the supplemental material. L. pneumophila Philadelphia-1 laboratory-derived strain Lp02, a thymidine auxotroph, was cultured at 37°C in AYE broth and on ACES-buffered charcoal (Fisher)-yeast extract (Becton, Dickinson) (CYE) agar supplemented with 100 µg/ml thymidine (Sigma), 400 µg/ml cysteine (Fisher), and 135 µg/ml ferric nitrate (J. T. Baker) (51 (link)). thymidine was omitted when culturing thymidine prototroph strains. When necessary for antibiotic selection of mutants or plasmids, media were supplemented with kanamycin (Sigma) (10 µg/ml); chloramphenicol (Fisher) (5 µg/ml); or gentamicin (Gibco) (10 µg/ml). Where indicated, gene expression was induced by adding IPTG (Gold Biotechnology) to reach a final concentration of 200 µM. For all experiments, colonies were first inoculated into broth, incubated overnight, and diluted to an optical density at 600 nm (OD600) of 0.05 to 0.2 and then cultured to the E phase (OD600 of 1.0 to 2.0) or PE phase (OD600 of 3.7 to 4.0), as indicated.
Ferric nitrate
Manufactured by Thermo Fisher Scientific
Ferric nitrate is an inorganic chemical compound with the chemical formula Fe(NO3)3. It is a crystalline solid that is soluble in water and commonly used as a reagent in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Chloramphenicol, by Thermo Fisher Scientific (1 mentions)
Thymidine, by Thermo Fisher Scientific (1 mentions)
Cysteine, by Thermo Fisher Scientific (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by GoldBio (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Thymidine, by Merck Group (1 mentions)
Nitrilotriacetic acid, by Merck Group (1 mentions)
Human apo tf, by Merck Group (1 mentions)
Apo tf, by Merck Group (1 mentions)
Potassium chloride (kcl), by Thermo Fisher Scientific (1 mentions)
Nitric acid, by Thermo Fisher Scientific (1 mentions)
Magnesium chloride, by Thermo Fisher Scientific (1 mentions)
Lead acetate, by Thermo Fisher Scientific (1 mentions)
Cobalt nitrate, by Thermo Fisher Scientific (1 mentions)
Nickel nitrate, by Thermo Fisher Scientific (1 mentions)
Magnesium nitrate, by Thermo Fisher Scientific (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Milli q system, by Merck Group (1 mentions)
3 protocols using ferric nitrate
1
Culturing and Maintaining Legionella
2
Measuring Iron Uptake by Apo-Transferrin
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The uptake of iron by Human apo-Tf was measured by the change of transferrin absorbance at 462 nm (molar extinction increase for saturated human transferrin = 4860) (Frieden and Aisen 1980 (link)). Measurements were done on a Shimadzu-UV-1601 recording spectrophotometer (Shimadzu Scientific, Columbia, MD).
Human apo-Tf (Sigma, St. Louis, MO, Product # T-1147), was dissolved in 0.05 M Tris buffer, pH 7.4, with 20 mM sodium bicarbonate. It contained < 1% iron content, as determined by iron assay with ferrozine reagent.
FPC (Rockwell, Lot #4004), and food-grade iron pyrophosphate (Dr. Paul Lohmann GmbH KG, Emmerthal, Germany) were dissolved in Tris/bicarbonate buffer. Ferric-nitrilotriacetate (Fe-NTA) was prepared from ferric nitrate (Fisher Scientific, Pittsburgh, PA) and nitrilotriacetic acid (Sigma), following the protocol of Bates et al. (Bates et al. 1967 (link)).
The apo-Tf solution (1 ml) was equilibrated at 37 °C in a quartz cuvette, the iron-complex solution (20 μL) was added, and kinetic measurements started within 10 s of mixing. Data were recorded digitally at 1–s intervals, and the reaction was monitored until steady-state measurements were achieved.
Human apo-Tf (Sigma, St. Louis, MO, Product # T-1147), was dissolved in 0.05 M Tris buffer, pH 7.4, with 20 mM sodium bicarbonate. It contained < 1% iron content, as determined by iron assay with ferrozine reagent.
FPC (Rockwell, Lot #4004), and food-grade iron pyrophosphate (Dr. Paul Lohmann GmbH KG, Emmerthal, Germany) were dissolved in Tris/bicarbonate buffer. Ferric-nitrilotriacetate (Fe-NTA) was prepared from ferric nitrate (Fisher Scientific, Pittsburgh, PA) and nitrilotriacetic acid (Sigma), following the protocol of Bates et al. (Bates et al. 1967 (link)).
The apo-Tf solution (1 ml) was equilibrated at 37 °C in a quartz cuvette, the iron-complex solution (20 μL) was added, and kinetic measurements started within 10 s of mixing. Data were recorded digitally at 1–s intervals, and the reaction was monitored until steady-state measurements were achieved.
3
Sensitive Metal-Ion Detection Using DNAzymes
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