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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 (1,2,3,4,5,6,7,8). 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 micro-plate fluorometer (FluoroCount™; Packard BioScience), using as a blank reference the assay on a cell-free culture medium sample. Arbitrary units of β-gal activity (MUG units) were calculated as follows:
where F360/460, t, and A595 are sample fluorescence at the end of the reaction, time of reaction in minutes, and absorbance of the cell suspension, respectively.
As shown in (Figure 1)A, we examined a selection of both E. coli (ER100) and Pseudomonas putida (MAD1, MAD2) reporter strains carrying a chromosomal fusion of the lacZ gene with the P. putida Pu promoter activated by the toluene-responsive XylR regulator (10,11), and expressing a wide range of β-gal activities. In MAD1, which contains the wild-type XylR regulator, we detected a strong 70- to 150-fold stimulation of Pu activity upon toluene addition. In MAD2, we observed the constitutively high Pu activity mediated by the XylRΔA variant. In E. coli ER100, where XylR was expressed from a plasmid, we detected a higher basal level of Pu expression that increased approximately 2.5-fold upon toluene addition. Importantly, strains isogenic to those described here either lacking the Pu-lacZ fusion (ER99 and KT2442) or the activator gene (ET99) showed negligible fluorescence upon MUG addition ((Figure 1)A), indicating that the majority of fluorescence in the assay derived from MUG hydrolysis by β-gal. Furthermore, MAD2 cells showed high β-gal activity even in the absence of toluene, indicating that the increase in β-gal expression after toluene addition in ER100 and MAD1 was not an artifact due to cell permebilization by toluene. This was also confirmed by the 2.5-fold induction of Pu expression upon 2-methylphenol addition to E. coli ET100 ((Figure 1)A), a strain expressing the XylR-related and methylphenol-responsive activator TouR (12). Thus, the β-gal activity assay based on MUG hydrolysis is an effective method for measuring reporter gene activity in bacteria without the need for a cell lysis step. The reproduc-ibility and sensitivity of this in vivo assay is at least as good as the one displayed by the classic Miller's test (see Supplementary Figure S1 available online at www.BioTechniques.com).
