Conjugative plasmid-deficient derivatives of E. faecalis strain E512 were obtained by plasmid curing using novobiocin (25 (link)). E. faecalis E512 harboring the conjugative plasmid pE512 was inoculated into BHI broth (~1 × 105 CFU/mL) with increasing concentrations (0 to 10 mg/L) of novobiocin (Sigma) and incubated at 37°C for 72 h. The culture that grew at the highest novobiocin concentration was serially diluted and plated onto BHA plates to obtain individual colonies. Randomly selected colonies were replica plated onto BHA plates and screened for the pE512-associated gene, pcfG, encoding the relaxase/mobilization nuclease domain-containing protein by PCR (Table S1). Plasmid profiles of the E. faecalis strain E512 and its derivatives were prepared and analyzed by agarose gel electrophoresis.
Novobiocin
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Switzerland, Sweden
Novobiocin is a laboratory reagent used in microbiology and biochemistry research. It is an antibiotic compound that inhibits bacterial DNA gyrase, an essential enzyme for bacterial cell replication and growth. Novobiocin is commonly used in culture media and assays to selectively inhibit the growth of Gram-positive bacteria.
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Lab products found in correlation
Nalidixic acid, by Merck Group (14 mentions)
Ampicillin, by Merck Group (9 mentions)
Erythromycin, by Merck Group (8 mentions)
Vancomycin, by Merck Group (8 mentions)
Streptomycin, by Merck Group (6 mentions)
Rifampicin, by Merck Group (6 mentions)
Tetracycline, by Merck Group (6 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (5 mentions)
Polymyxin b, by Merck Group (5 mentions)
Bacitracin, by Merck Group (5 mentions)
Penicillin g, by Merck Group (5 mentions)
Tryptic soy broth (tsb), by BD (5 mentions)
Lincomycin, by Merck Group (4 mentions)
Neomycin, by Merck Group (4 mentions)
Kanamycin, by Merck Group (4 mentions)
Gentamicin, by Merck Group (4 mentions)
Novobiocin, by BD (4 mentions)
Nalidixic acid, by BD (4 mentions)
Verapamil, by Merck Group (4 mentions)
Penicillin, by Merck Group (3 mentions)
93 protocols using novobiocin
1
Plasmid Curing of E. faecalis E512
2
Plasmid Curing and Antibiotic Resistance
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All the bacterial strains and plasmids used in this study are listed in Table 1 . Strains were cultured in BBL™ Muller Hinton II (Cation-Adjusted) Broth (MHB) or Trypic Soy Broth (TSB) (Becton Dickinson, Franklin Lakes, NJ). For solid media, broth was supplemented with 1.7% agar (Alfa Aesar, Waltham, MA). Where needed, strains were cured of their plasmid as described in Udo et al. (1997 (link)). Briefly, a culture was grown overnight shaking at 37°C and then diluted 1:500 in TSB. This culture was grown at 42°C for 48 h and dilutions were then plated on TSA and grown at 37°C to obtain single colonies. Plates containing isolated colonies were replica plated to MHA and MHA supplemented with 20 μg/mL of cadmium chloride and then grown at 37°C. Cadmium sensitive colonies were double purified and screened with the pC02 PCR screen described below to test for the presence of the large plasmid(s). Large plasmid cured strains were made resistant to rifampin and novobiocin as previously described by selecting mutant strains that were resistant to rifampin (Sigma-Aldrich, St Louis, MO) followed by subsequent passage in increasing concentrations of novobiocin (Sigma) (Udo et al., 1997 (link)).
3
Selective Medium for Detecting Pathogenic Salmonella
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Xylose lysine agar plates supplemented with Tergitol 4 (XLT4; Difco, Becton Dickson, Sparks, MD, USA) were poured after supplementing 20 mg/L nalidixic acid and 25 mg/L novobiocin (Sigma, St. Louis, MO, USA) as a selective medium to enumerate nalidixic acid and novobiocin-resistant marker pathogens including Salmonella typhimurium and Salmonella enteritidis. Stock solutions of each antibiotic were prepared by dissolving 200 mg nalidixic acid and 250 mg novobiocin in 10 mL sterile distilled water followed by filter-sterilization. Escherichia coli counts were enumerated using 3M Petrifilm E. coli/Coliforms Count Plates (3M Microbiology, St. Paul, MN, USA). Aerobic plate counts (APC) and psychrotrophs were performed using standard plate count agar (PCA; Difco, Becton Dickson, Sparks, MD, USA).
4
Cultivation of Single-Spore B. cinerea Isolate
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The single-spore B. cinerea isolate DAR83297 (Living Culture Collection at NSW Department of Primary Industries, Orange, NSW, Australia, 2020) used for these experiments was sub-cultured onto 25% potato dextrose agar (PDA) plus Novobiocin (Sigma, Darmstadt, Germany) and incubated at 23 °C for 14 days. The 25% PDA plus Novobiocin medium was prepared with 9.75 g/L PDA (BD Difco) and 11.25 g/L base agar (Gelita Grade J3) in distilled water (1 L) pre-autoclave. Post-autoclave, Novobiocin stock solution (0.0175 g/mL) was added at 6.65 mL/L. B. cinerea plugs at 4 mm in diameter were harvested from the outside growing edge of actively growing cultures and placed in the centre of 90 mm Petri plates containing 20 mL of fresh PDA plus Novobiocin. All petri plates were kept at 23 °C in the dark for a total of six days before being used in a detached leaf inoculation assay.
5
Alkaloid Extraction and Purification
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All chemicals used in this study to extract and purify alkaloids were research-grade. Verapamil, carbenicillin, novobiocin, and erythromycin were obtained from Sigma-Aldrich Chimie S.a.r.l (St. Quentin Fallavier, France).
6
Compound Treatment on Cell Lines
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HeLa, primary renal proximal tubule epithelial, SH-SY5Y, and HT29 human cells were obtained from American Type Culture Collection. Cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 25 mM glucose and supplemented with 2 mM glutamine, 1 mM pyruvate, 10% fetal bovine serum, and antibiotics. Cultures were brought to 50 to 70% confluence and exposed to different compounds. NAM dinucleotide (NAD+), NMN, NAM, and ADO were purchased by Sigma-Aldrich. NR was synthesized as described by Yang et al. (68 (link)). NAD+, NMN, NR, NAM, and ADO were dissolved in culture media. Apigenin was dissolved in DMSO (Sigma-Aldrich). PJ34, 6(5H)-phenanthridinone, AMPCP, and novobiocin were dissolved in water (Sigma-Aldrich). All results are expressed as the percentage of the control (untreated cells); each sample was normalized by the protein content.
7
Salmonella Enteritidis Culture Preparation
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The organism used was a poultry isolate of Salmonella enterica serovar Enteritidis (SE), bacteriophage type 13A, obtained from the USDA National Veterinary Services Laboratory (Ames, IA, United States). The isolate is resistant to 25 μg/mL of novobiocin (NO, catalog no.N-1628, Sigma) and was selected for resistance to 20 μg/mL of nalidixic acid (NA, catalog no.N-4382, Sigma) in our laboratory. In this study, 100 μL of SE from a frozen aliquot was added to 10 mL of tryptic soy broth (Catalog No. 22092, Sigma) and incubated at 37°C for 8 h, and passed three times every 8 h to ensure that all bacteria were in log phase as previously described (Lin et al., 1995 (link)). Post-incubation, bacterial cells were washed three times with sterile 0.9% saline by centrifugation at 1864 × g for 10 min, reconstituted in saline, quantified by densitometry with a spectrophotometer (Spectronic 20D+, Spectronic Instruments Thermo Scientific, Rochester, NY, United States), and diluted to an approximate concentration of 4 × 107 cfu/mL. Concentrations of SE were further verified by serial dilution and plated on brilliant green agar (BGA, Catalog No. 70134, Sigma) with NO and NA for enumeration of actual cfu used in the experiment.
8
Salmonella Enteritidis Phage Type 13A Isolation
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A primary poultry isolate of Salmonella enterica serovar Enteritidis bacteriophage type 13A, was obtained from the USDA National Veterinary Services Laboratory (Ames, IA, USA). This strain is resistant to 25 µg/mL of novobiocin (NO, catalog no. N-1628, Sigma) and was selected due to its resistance to 20 µg/mL of nalidixic acid (NA, catalog no. N-4382, Sigma) in our laboratory. The Salmonella Enteritidis culture was performed according to previous publications [19 (link)] to obtain approximate bacterial concentrations of 4 × 104 and 4 × 107 cfu/mL. Levels of S. Enteritidis were further verified by serial dilutions and plated on brilliant green agar (BGA, Catalog No. 70134, Sigma) with NO and NA for enumeration of actual cfu used in the experiment.
9
Purification and Characterization of Topoisomerases
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E. coli DNA topoisomerase I was purified according
to our previously published procedure.37 (link)E. coli DNA gyrase subunit A and
subunit B were purified as previously described.34 (link)Variola DNA topoisomerase I was purified
as previously described.34 (link) A His-tagged
T5 exonuclease was purified from E. coli strain BLR(DE3) carrying plasmid pET28a(+)-His-T5E using a Ni-NTA
column followed by a Q Sepharose Fast Flow column. The His-tag may
be removed by TEV protease.
Plasmid pAB1, a derivative of pUC18,
was constructed as described previously.33 (link) (−) sc plasmids pAB1 and pUC18 were purified from E. coli cells harboring the plasmids (Top10/pAB1
or Top10/pUC18). Rx plasmid pAB1 was relaxed using variola DNA topoisomerase I or E. coli DNA
topoisomerase I and purified with phenol extraction and ethanol precipitation.
A double fluorophore-labeled oligomer HP1 5′-[FI]-CGCTGTCGAACACACGCTTGCGTGTGTTC-[TAMRA]-3′
was purchased from Eurofins Genomics LLC and used without further
purification. An extinction coefficient of 301,301 M–1 cm–1 was used to determine the concentration of
HP1.
Novobiocin, ciprofloxacin, ethidium bromide, and Hoechst
33258
were purchased from Sigma-Aldrich, Inc. SYBR gold, SYBR green, and
ethidium homodimer 1 (EthD1) were bought from ThermoFisher Scientific,
Inc. NSC compounds were obtained from NCI DTP program (https://dtp.cancer.gov ).
to our previously published procedure.37 (link)E. coli DNA gyrase subunit A and
subunit B were purified as previously described.34 (link)Variola DNA topoisomerase I was purified
as previously described.34 (link) A His-tagged
T5 exonuclease was purified from E. coli strain BLR(DE3) carrying plasmid pET28a(+)-His-T5E using a Ni-NTA
column followed by a Q Sepharose Fast Flow column. The His-tag may
be removed by TEV protease.
Plasmid pAB1, a derivative of pUC18,
was constructed as described previously.33 (link) (−) sc plasmids pAB1 and pUC18 were purified from E. coli cells harboring the plasmids (Top10/pAB1
or Top10/pUC18). Rx plasmid pAB1 was relaxed using variola DNA topoisomerase I or E. coli DNA
topoisomerase I and purified with phenol extraction and ethanol precipitation.
A double fluorophore-labeled oligomer HP1 5′-[FI]-CGCTGTCGAACACACGCTTGCGTGTGTTC-[TAMRA]-3′
was purchased from Eurofins Genomics LLC and used without further
purification. An extinction coefficient of 301,301 M–1 cm–1 was used to determine the concentration of
HP1.
Novobiocin, ciprofloxacin, ethidium bromide, and Hoechst
33258
were purchased from Sigma-Aldrich, Inc. SYBR gold, SYBR green, and
ethidium homodimer 1 (EthD1) were bought from ThermoFisher Scientific,
Inc. NSC compounds were obtained from NCI DTP program (
10
Quantifying Salmonella Enteritidis in Poultry
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The CCT collected in both experiments (n = 20 poults/group) were homogenized and diluted with saline (1:4 w/v). CCT homogenate samples were diluted by ten-fold serial dilutions and 100 µL were plated on brilliant green agar (BGA, Catalog no. 70134, Sigma St. Louis, MO, USA) plates containing 25 µg/mL novobiocin (NO, catalog no. N-1628, Sigma St. Louis, MO, USA) and 20 µg/mL of nalidixic acid (NA, catalog no. N-4382, Sigma, St. Louis, MO, USA), incubated at 37 °C for 24 h, then enumerated for total Salmonella enterica serovar Enteritidis cfu. Following plating to enumerate total Salmonella enterica serovar Enteritidis, the CCT homogenate samples were enriched with tetrathionate enrichment broth, 1× final dilution, and further incubated at 37 °C for 24 h. Enrichment samples were streaked onto XLT-4) selective media for confirmation of Salmonella presence. Plates that were negative on the enumeration method but were positive on enrichment were considered as 500 cfu/g as the limit of detection for SE viability.
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