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2 protocols using 3 n morpholino propane sulfonic acid

1

Enzymatic Activity Assays for Oxidative Stress

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Antibiotics, 5-aminolevulinic acid hydrochloride (5-ALA), ß-mercaptoethanol, ß-NADH, bovine xanthine oxidase, casein acid hydrolysate, cytochrome c from equine heart, deamino-NADH, desferoxamine mesylate (DFO), diethylenetriamine pentaacetic acid (DTPA), 2,2’-dipyridyl (DIP), ethyl acetate, ferric chloride, ferrous ammonium sulfate, 30% hydrogen peroxide, 8-hydroxyquinoline-5-sulphonic acid, E. coli manganese-containing superoxide dismutase, manganese (II) chloride tetrahydrate, 2-[N-morpholino]ethanesulfonic acid (MES), o-dianisidine dihydrochloride, o-nitrophenyl-ß-D-galactopyranoside (ONPG), potassium ferricyanide, potassium cyanide, tricine, protoporphyrin IX, and xanthine were purchased from Sigma. Ethylenediamine tetraacetic acid (EDTA), guanidine hydrochloride, hydrochloric acid, and 3-(N-morpholino) propane-sulfonic acid (MOPS) were purchased from Fisher Scientific; Coomassie protein assay reagent and albumin standard, from Thermo Scientific; sodium dithionite, from Fluka; and glacial acetic acid, from J.T.Baker.
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2

Cultivation and Genetic Manipulation of Bacterial Strains

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All bacterial strains and plasmids used in this study are listed in Table 1. Y. pestis CO92/pCD1 (YP6) was cultivated on brain heart infusion (BHI) agar (BD Biosciences, Bedford, MA) at 26°C for 48 h and in BHI broth cultures grown with aeration at 26°C or 37°C. E. coli strains were cultivated on Luria-Bertani (LB) agar (BD Biosciences) at 37°C overnight and in liquid cultures with aeration at 37°C or 26°C. When indicated, bacteria were grown in BHI broth that was adjusted and buffered to the appropriate pH. BHI broth was buffered with 100 mM MES [2-(N-morpholino)ethanesulfonic acid; Sigma] and then adjusted to pH 6.3 or 6.7, or it was buffered with 100 mM MOPS [3-(N-morpholino)propanesulfonic acid; Fisher Scientific] and then adjusted to pH 7.3 and filter sterilized. When necessary, antibiotics were added to the growth medium at the following concentrations: kanamycin (Kan), 50 μg/ml; carbenicillin (Carb), 100 μg/ml; and irgasan (Irg), 2 μg/ml. For the expression of genes cloned into pMWO-005, 50 ng/ml anhydrous tetracycline (ATc) was added to the liquid medium when strains were subcultured.
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