One-Step High-Throughput Assay for Quantitative Detection of β-Galactosidase Activity in Intact Gram-Negative Bacteria, Yeast, and Mammalian Cells

摘要

BioTechniquesVol. 40, No. 4 BenchmarksOpen AccessOne-step high-throughput assay for quantitative detection of β-galactosidase activity in intact Gram-negative bacteria, yeast, and mammalian cellsFaustino Vidal-Aroca, Michele Giannattasio, Elisa Brunelli, Alessandro Vezzoli, Paolo Plevani, Marco Muzi-Falconi & Giovanni BertoniFaustino Vidal-ArocaUniversità degli Studi di Milano, Milano, Italy, Michele GiannattasioUniversità degli Studi di Milano, Milano, Italy, Elisa BrunelliUniversità degli Studi di Milano, Milano, Italy, Alessandro VezzoliUniversità degli Studi di Milano, Milano, Italy, Paolo PlevaniUniversità degli Studi di Milano, Milano, Italy, Marco Muzi-FalconiUniversità degli Studi di Milano, Milano, Italy & Giovanni Bertoni*Address correspondence to Giovanni Bertoni, Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Via Celoria 26, 20133, Milan, Italy. e-mail: E-mail Address: [email protected]à degli Studi di Milano, Milano, ItalyPublished Online:21 May 2018https://doi.org/10.2144/000112145AboutSectionsView ArticleSupplemental MaterialPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail View article The capability to monitor gene expression represents an invaluable tool to understand cell regulation. The activity of β-galactosidase (β-gal), the product of the lacZ gene of Escherichia coli, is one of the most widely used reporters of gene expression. β-Gal is a versatile reporter since its activity can be monitored using a variety of chromo-genic and fluorogenic substrates in both prokaryotes and eukaryotes. However, efficient access of these substrates to β-gal enzyme is hindered by cell-surface barriers. Routine protocols for the quantitative determination of the in vivo expression of β-gal activity in any system require cell permeabilization or destruction of cell integrity to obtain crude extracts (1–8). This makes β-gal determination less straightforward than, for instance, green fluorescent protein (GFP)- or luciferase-based reporter systems, which do not require cell lysis or permeabilization. In general, besides being time- and material-consuming, multiple step manipulations before the addition of the β-gal substrate can hinder the development of high-throughput protocols, which are essential in genome-wide approaches. It would be desirable to develop simple one-step assays that could be automated for parallel screening of large numbers of samples. In this work, we report a fast method, functional in a broad range of organims, which makes β-gal determination as straightforward as other reporter systems mentioned here. In this system, β-gal activity is determined through the simple addition of the fluorogenic substrate 4-methy-lumbelliferyl β-D-galactopyranoside (MUG) (8,9) to intact cells of bacteria, yeast and mammalia. We show that, since it does not require centrifugation or cell lysis steps, this assay can be applied to high-throughput screenings, such as mutagenesis or two-hybrid, based on the differential expression of the lacZ gene.First, we evaluated MUG as β-gal substrate for quantitative assays on intact bacterial cells. Bacterial cultures were grown overnight in 96-well microplates, 20 L of each culture were transferred with a Multiprobe® IIEx Robotic Liquid Handling System (Packard BioScience, Meriden, CT, USA) to 96-well microplates (black/ clear Optilux™ flat bottom; BD Falcon™; BD Biosciences, San Jose, CA, USA) containing 80 L of Z-buffer (3), and the corresponding cell density was evaluated by determining the A595 in a Model 550 Microplate Reader (Bio-Rad Laboratories, Hercules, CA, USA). Twenty-five microliters 1 mg/mL MUG in dimethyl sulfoxide (DMSO) were then added to each well, and the samples were incubated at room temperature for 15 min. The reaction was stopped with 30 L 1 M Na2CO3. The amount of fluorescence generated by β-gal-dependent MUG hydrolysis was quantitated in a mi