The most promising strains based on the β-galactosidase activity screening (showing the highest intensity of color change) were selected for enzyme activity quantification. The selected Antarctic strains (×4) were cultivated in lactose broth (meat extract 0.3%, peptone 0.5%, lactose 0.5%) at 15 °C for 48 h; then, cells were lysed by sonication. The cell lysate was centrifuged at 4000 rpm for 30 min at 4 °C, and the supernatant was used as the enzymatic extract [24 (link)]. 20 µL of the enzymatic extract was added to a 22 mM solution of o-nitrophenyl-β-d -galactopyranoside (ONPG) (Sigma-Aldrich, Gillingham, UK). The reaction was carried out for 15 min at 15 °C and pH 6.5. The amount of released o-nitrophenol (ONP) after ONPG hydrolysis was measured at 420 nm. One unit of β-galactosidase activity (U) was defined as the amount of enzyme required to release 1 µmol of ONP per minute from ONPG under the experimental conditions described above. Additionally, differences in β-galactosidase activity were evaluated in the presence and absence of IPTG (Sigma-Aldrich, Gillingham, UK). All determinations were performed in triplicates.
O nitrophenyl β d galactopyranoside onpg
Manufactured by Merck Group
Sourced in United States, Germany
O-nitrophenyl-β-D-galactopyranoside (ONPG) is a chromogenic substrate used for the detection and quantification of β-galactosidase activity. When cleaved by β-galactosidase, ONPG releases a yellow-colored o-nitrophenol that can be measured spectrophotometrically.
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Lab products found in correlation
P nitrophenyl phosphate pnpp, by Merck Group (2 mentions)
Dithiothreitol (dtt), by Nacalai Tesque (2 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (1 mentions)
P nitrophenyl α d glucopyranoside pnpg, by Merck Group (1 mentions)
Simplicity uv water purification system, by Merck Group (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Protein assay dye reagent concentrate, by Bio-Rad (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Fluorescein isothiocyanate (fitc), by Thermo Fisher Scientific (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Hemin, by Merck Group (1 mentions)
Rpmi 1640, by Lonza (1 mentions)
Fluconazole flc, by Merck Group (1 mentions)
Potassium chloride (kcl), by ITW Reagents (1 mentions)
Mncl2, by Merck Group (1 mentions)
Fk506, by Cayman Chemical (1 mentions)
Nuclear dye hoechst 33342, by Merck Group (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
Ethyl acetate, by Thermo Fisher Scientific (1 mentions)
Milli q water purification system, by Merck Group (1 mentions)
22 protocols using o nitrophenyl β d galactopyranoside onpg
1
Quantitative Analysis of Antarctic Strains' β-Galactosidase Activity
2
Measuring Enzymatic Activities in Yeast
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To measure β-galactosidase or β-glucuronidase activity cultures were grown in YPD overnight, shifted to SC media with 3% glycerol, 2% ethanol, 3% acetate or without any added carbon source at OD600 0.1 to induce transcription and grown further to OD600 = 0.8 to 1.6. Cells were harvested and glass bead-disrupted in β-galactosidase buffer (5.0 mM Tris-HCl pH7.8; 5% glycerol; 10 mM KCl) or β-glucuronidase buffer (10 mM β-mercaptoethanol; 10 mM Na2EDTA; 0.1% sodium lauroyl sarcosinate; 0.1% Triton X-100; 10 mM sodium phosphate pH 7.0), respectively. Substrates were 4 mg/ml O-nitrophenyl-β-D-galactopyranoside (ONPG (Sigma), molar extinction coefficient ε = 4.5 x 106 M-1cm-1 at 420 nm) for the measurement of β-galactosidase activity and 4 mg/ml p-nitrophenyl-α-D-glucopyranoside (PNPG (Sigma), ε = 8800 M-1cm-1 at 415 nm [58 (link)]) for the measurement of β-glucuronidase activity. The kinetics of product formation over time were followed and the specific enzyme activities [mU/mg protein] were calculated by (ΔE/min x 106) / (ε [M-1cm-1] x protein concentration [mg]).
3
HPLC Analysis of Microbial Metabolites
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BHI (Brain Heart Infusion, OXOID, Basingstoke, UK) ); TSB (Tryptone Soya Broth, OXOID, Hampshire, England); TSA (Tryptone Soya Agar, OXOID, Hampshire, England), Bradford reagent (Protein Assay Dye Reagent Concentrate, Bio-Rad, München, Germany); β-nicotinoamide adenine dinucleotide hydrate (NAD, Sigma-Aldrich, Steinheim, Germany); fluconazole (FLC, Sigma-Aldrich, Poznan, Poland), hemin (Sigma-Aldrich, St. Louis, MO, USA); polymyxin B sulfate salt (PB, Sigma-Aldrich, Denmark); fluorescein isothiocyanate (FITC, ThermoScientific, Rockford, IL, USA); Hank’s Buffer with Ca2+, Mg2+ (HBSS, PAN Biotech, UK); RPMI 1640 (Lonza, Walkersville, MD, USA); o-nitrophenyl-β-D-galactopyranoside (ONPG, Sigma, Steinheim Germany), heat inactivated (56 °C, 1 h) FBS (Fetal bovine serum, Gibco Life Technologies, Grand Island, NY, USA).). HPLC chemicals: Acetonitrile (Sigma, München, Germany) for separation was HPLC far UV/gradient grade (J. T. Baker, Avantor™ Performance Material); 32 mM Na2SO4 solution for chromatographic usage was prepared with 4.5 g anhydrous sodium sulfate (POCh, Avantor™ Performance Material, Gliwice, Poland) and MiliQ (ultrapure water made with Simplicity UV Water Purification System, Merck Millipore, Saint-Quentin, France).
4
Transcriptional regulation of parDE2 in M. tuberculosis
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The 363-bp upstream region preceding the parD2 start codon (P363parD2) of M. tuberculosis H37Rv was PCR-amplified and fused to a promoter-less lacZ reporter gene (3,067 bp) in pSK6 plasmid (Kant et al., 2009 (link)). The 363-bp lacZ fusion fragment was subsequently cloned in an E. coli-Mycobacterium shuttle vector pMV261, using NotI and DraI sites, forming pMS2 (Supplementary Figure S2 ). A promoter-less lacZ gene cloned in pMV261 (pMS1) was used as negative control. For parDE2 transcriptional regulation studies, parD2 or parDE2 coding sequences were placed downstream of the groEL promoter of pMS2, at restriction sites EcoR1 and HindIII, to form pMS3 and pMS4, respectively (Supplementary Figure S2 ). All these recombinant constructs were electroporated into M. smegmatis mc2 155.
β-galactosidase activity in all the M. smegmatis recombinants was determined colorimetrically using O-nitrophenyl-β-D-galactopyranoside (ONPG; Sigma), as described earlier (Miller, 1972 ; Dellagostin et al., 1995 (link)). Absorbance was measured at 420 nm on a Cecil Aquarius CE 7500 Spectrophotometer. One unit of β-galactosidase is the amount of enzyme producing 1 nmol of O-nitrophenol per min at 30°C. Each experiment was performed thrice in triplicate.
β-galactosidase activity in all the M. smegmatis recombinants was determined colorimetrically using O-nitrophenyl-β-D-galactopyranoside (ONPG; Sigma), as described earlier (Miller, 1972 ; Dellagostin et al., 1995 (link)). Absorbance was measured at 420 nm on a Cecil Aquarius CE 7500 Spectrophotometer. One unit of β-galactosidase is the amount of enzyme producing 1 nmol of O-nitrophenol per min at 30°C. Each experiment was performed thrice in triplicate.
5
High-Pressure CO2 Extraction and Anti-Inflammatory Assay
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CO2 pure grade (99.95%, Air Liquide, Lisbon, Portugal) and EtOH (96%) were used for high pressure extraction experiments. Solvents used for conventional extractions purification steps and chromatographic analysis included ethyl acetate (99.98%, Fisher scientific U.K. Limited, Loughborough, UK), methanol (99.8%, Fisher scientific U.K. Limited, Loughborough, UK), dichloromethane (Honeywell Riedel-de Haën, Germany), distilled water, and ultrapure water purified with a Milli-Q water purification system (Merck Millipore, Billerica, MA, USA). For the inflammation assays, the inflammatory stimulus was carried out by the usage of MnCl2 (Merck, Darmstadt, Germany), and the anti-inflammatory positive control was carried by FK506 (Cayman Chemicals, Ann Arbor, MI, USA). Cellular lysis was preformed using Yeast Protein Extraction Reagent (Y-PER; ThermoFisher Scientific, Scientific, Rockford, IL, USA). To quantify the expression of reporter gene lacZ, we employed a solution buffer composed of Na2HPO4 (ROTH, Karlsruhe, Germany), NaH2PO4·H2O (Merck, Buchs, Switzerland), KCl (Panreac, Barcelona, Spain), and MgSO4·7H2O (Merck, Buchs, Switzerland), containing o-nitrophenyl β-D-galactopyranoside (ONPG; Sigma–Aldrich®–Poole, Dorset, UK). Nuclear staining was carried out using nuclear dye Hoechst 33342 (Sigma, Buchs, Switzerland).
6
Wnt Signaling Pathway Luciferase Assay
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For reporter gene assays, 105 DLD1 or SW480 cells were seeded per well (12 well plate). Cells were transfected with the indicated siRNAs using Lipofectamine 2000 (Life Technologies). 24 hours later, the 8×TOPflash reporter gene construct (a generous gift from Randall T. Moon, University of Washington) as well as the plasmid pCH110 encoding β-galactosidase (GE Healthcare, Freiburg, Germany) were transfected into these cells. 72 hours after the siRNA transfection, cells were harvested, lysed with reporter lysis buffer (Promega) and luciferase activities were measured using a luciferase assay reagent (Promega) and a luminometer (Orion II, Berthold Detection Systems, Wildbad, Germany). β-galactosidase activity was determined by standard methods using o-Nitrophenyl β-D-galactopyranoside (ONPG, Sigma-Aldrich, Taufkirchen, Germany) as a control for transfection efficiency.
7
Baculovirus-Insect Cell Expression System
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T. niembryonic cell line BTI-Tn5B1-4 (High Five) (
Granados et al. 1994
) and
S. frugiperdaovarian cell line Sf-9 (
Pasumarthy and Murhammer 1994 (link)
) were provided by Dr. Blissard, Boyce Thompson Institute of Cornell University.
T. niembryonic suspension cell line QB-Tn9-4s was established and preserved in our laboratory (
Meng et al. 2008
).
Autographa californicamultiple nucleopolyhedrovirus (AcMNPV-1A) (
Wood 1980 (link)
) and its β-galactosidase expressing recombinant strain AcMNPV-β-gal (
Wickham et al. 1992 (link)
) and secreted alkaline phosphatase (SEAP) expressing recombinant strain Ac-MNPV-SEAP (
Davis et al. 1992
) were kindly provided by Dr. Granados of Cornell University. All of the viruses were amplified and titrated following the plague assay method described by
Wood (1977)
using Sf-9 cells.
TNM-FH insect medium was prepared by supplementing Grace medium (Invitrogen,
www.lifetechnologies.com ) with 0.3% lac-talbumin hydrolyzate (BD,
www.bd.com ), 0.3% yeast extract (BD) and 10% fetal calf serum (FBS) (Thermo Scientific,
www.thermoscientific.com ). Serumfree Sf-900 III medium was from Invitrogen and serumfree EX-CELL 420 medium was from SAFC Biosciences (Sigma-Aldrich,
www.sigmaaldrich.com ). O-nitrophenyl-β-D-galactopyranoside (ONPG) and p-nitrophenyl phosphate (PNPP) were from Sigma-Aldrich.
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T. niembryonic cell line BTI-Tn5B1-4 (High Five) (
Granados et al. 1994
) and
S. frugiperdaovarian cell line Sf-9 (
Pasumarthy and Murhammer 1994 (link)
) were provided by Dr. Blissard, Boyce Thompson Institute of Cornell University.
T. niembryonic suspension cell line QB-Tn9-4s was established and preserved in our laboratory (
Meng et al. 2008
).
Autographa californicamultiple nucleopolyhedrovirus (AcMNPV-1A) (
Wood 1980 (link)
) and its β-galactosidase expressing recombinant strain AcMNPV-β-gal (
Wickham et al. 1992 (link)
) and secreted alkaline phosphatase (SEAP) expressing recombinant strain Ac-MNPV-SEAP (
Davis et al. 1992
) were kindly provided by Dr. Granados of Cornell University. All of the viruses were amplified and titrated following the plague assay method described by
Wood (1977)
using Sf-9 cells.
TNM-FH insect medium was prepared by supplementing Grace medium (Invitrogen,
8
Measuring Promoter Activity via β-Galactosidase
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Promoter activity was analyzed by measuring the β-galactosidase activity using the pTCV plasmid54 (link). Cells were grown to stationary phase (OD600 ∼ 3) in BHI medium at 37 °C, with agitation. Collected samples (1 mL) were centrifuged and pellets were flash-frozen. Pellets were resuspended in 1 mL of Z Buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 50 mM β-mercaptoethanol, pH 7.0) and OD600 was measured. Cells were permeabilized with 0.5% toluene and 4.5% ethanol for 5 min at 30 °C in a water bath. To determine the β-galactosidase activity, 200 µL of Z buffer containing 4 mg/mL o-nitrophenyl-β-d -galactopyranoside (ONPG, Sigma-Aldrich) was added to each sample, followed by incubation at 30 °C in a water bath. Reactions were stopped by adding 500 µL 1 M NaCO3, and the time was recorded. Absorbance at 420 nm was measured after centrifugation for 5 min at 21,000 × g. The β-galactosidase activity (Miller units) was calculated as (1000 × A420)/(T × V × OD600). T, reaction time (in minutes); V, volume of bacteria (in mL).
9
Quantifying β-Galactosidase Activity in Cellular Extracts
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Cells (3 mg dry weight) were collected, harvested, washed twice with ice-cold 0.2% KCl, resuspended in 1 ml of lysis buffer (15 mM Tris tricarballylate, 4.5% glycerol, 0.9 mM MgCl2, 0.2 mM DTT; pH 7.2), transferred into screw-capped tubes containing 0.1 g of glass beads and disrupted using a FastPrep-24 instrument (MP Biomedicals) (six 30 s cycles at 6.5 m/s with 1 min on ice in between). The supernatant containing soluble proteins was analysed immediately. All measurements were carried out on three biological replicates with six technical replicates for each.
The total protein content of the cell extracts was determined by the Bradford method with bovine serum albumin as the protein standard. β-Galactosidase activity was determined by the colorimetric method using O-nitrophenyl-β-d -galactopyranoside (ONPG; Sigma) as substrate. Protein activity was determined using the rate of ONP production at 30°C (measured from the absorbance at 420 nm using a SpectraMax plus spectrophotometer) and expressed as a specific activity (mmol/min/g) using the total protein concentration of the sample. For each quantification, at least two independent samples were extracted and six technical replicates were analysed at different dilutions.
The total protein content of the cell extracts was determined by the Bradford method with bovine serum albumin as the protein standard. β-Galactosidase activity was determined by the colorimetric method using O-nitrophenyl-β-
10
Measuring tRNA Endonuclease Processing Activity
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The ability of each tRNA endonuclease to process a BHB-lacZ, restoring the β-galactosidase activity, was determined. To measure the activity quantitatively, the cells were grown in liquid cultures to mid-log phase at 30 °C in EMM containing appropriate supplement and 4 mM thiamine. The cells were then washed with thiamine-free medium and re-inoculated into EMM in the absence of thiamine and grown to mid-log phase. The assay of the β-galactosidase activity was performed as described in [32 (link),33 ] using o-nitrophenyl β-D-galactopyranoside (ONPG, Sigma, Saint Louis, MO, USA) as substrate.
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