Assaying β-Lactamase and dUTPase Activities

β-Lactamase assays, using nitrocefin as substrate, were performed with cells in exponential growth phase as described^{13 (link)}, using a Thermomax (Molecular Devices) microtiter plate reader. β-Lactamase activities were recorded as initial slopes divided by cell density (maximum velocity (V_max))/OD_650nm.
dUTPase activity was assayed using His₆–dUTPase proteins purified after expression in E. coli. Enzyme assays were performed using the EnzCheck Pyrophosphate Assay Kit (Molecular Probes), as previously reported^{14 (link)}.

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Tormo-Más M.Á., Mir I., Shrestha A., Tallent S.M., Campoy S., Lasa Í., Barbé J., Novick R.P., Christie G.E, & Penadés J.R. (2010). Moonlighting bacteriophage proteins derepress staphylococcal pathogenicity islands. Nature, 465(7299), 779-782.

Publication 2010

Assay Cells Devices Dutpase E coli Enzyme assays Molecular probes Nitrocefin Proteins Pyrophosphate β lactamase

Corresponding Organization : Universidad Cardenal Herrera CEU

Other organizations : Instituto Valenciano de Investigaciones Agrarias, Virginia Commonwealth University, Universitat Autònoma de Barcelona, Universidad Publica de Navarra, Agrobiotechnology Institute, University Medical Center, New York University

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Variable analysis

independent variables

Growth phase of cells (exponential growth phase)

dependent variables

β-Lactamase activities (initial slopes divided by cell density (V_max/OD_650nm))
DUTPase activity (using purified His6–dUTPase proteins)

control variables

Nitrocefin as substrate for β-Lactamase assays
Thermomax (Molecular Devices) microtiter plate reader for β-Lactamase assays
EnzCheck Pyrophosphate Assay Kit (Molecular Probes) for dUTPase activity assay

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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