A data comparison between a traditional and the single-step β-galactosidase assay

This article describes reproducibility of a single-step automated β-galactosidase, and the equivalence of its data to the traditional assay (“Experiments in Molecular Genetics” [1]). This was done via a pairwise comparison of both methods using strains with Miller Unit [MU] values ranging from 0 to over 2000. The data presented in this article is associated with the research article entitled “A single-step method for mid to high throughput β-galactosidase assays in Escherichia coli using a microplate reader” [2].


β-Gal
Microplate reader a b s t r a c t This article describes reproducibility of a single-step automated βgalactosidase, and the equivalence of its data to the traditional assay ("Experiments in Molecular Genetics" [1]). This was done via a pairwise comparison of both methods using strains with Miller Unit [MU] values ranging from 0 to over 2000. The data presented in this article is associated with the research article entitled "A single-step method for mid to high throughput β-galactosidase assays in Escherichia coli using a microplate reader" [2]. &

Value of the Data
Validates a single-step automated assay to measure β-galactosidase activity in E. coli with a microplate reader.
Establishes the method provides consistent data, with a coefficient of variation similar to that of the traditional assay.
Student's t-test demonstrates equivalence of the single-step and the traditional assay data.

Data
The data presented in this article is a pairwise comparison of the MU activities of the traditional β-galactosidase assay and a single-step automated method [2]. 4 different strains with MU activities (0-10, 40-70, 500-900, and 1800-2400 respectively) were assayed using both methods.

Cell culture and sample preparation
Samples were grown overnight at 37°C in LB media. For the traditional assay, samples were treated as described by Miller [1]. For the single-step automated microplate assay, 80 μL of culture was transferred into 96-well Costar s flat bottom microplates (Sigma-Aldrich). 120 μL of a custom mix was added to each well used, including the blank (containing 80 μL LB). The custom mix consists of 60 mM Na 2 HPO 4 , 40 mM NaH 2 PO 4 , 10 mM KCl, 1 mM MgSO 4 , 36 mM β-mercaptoethanol, 166 mL/ml T7 lysozyme, 1.1 mg/ml ONPG, 6.7% PopCulture s Reagent. The Costar microtitre plates were then placed in a FLUOstar Omega Microplate Reader (BMG LABTECH).

Kinetic OD 600 and OD 420 measurements
The FLUOstar Omega Microplate Reader was set to 30°C, with OD 600 and OD 420 readings taken every 60 s for 1 h (20 flashes per well each cycle). Wells were set to shake at 500 rpm (double orbital shaking) between readings to ensure homogenisation of the sample and more efficient lysis. A script to run these parameters on a FLUOstar Omega Microplate Reader can be found in Supplementary Data A.

MARS Data Analysis
The MARS Data Analysis software package was used to process the OD 600 and OD 420 data generated by the by FLUOstar Omega Microplate Reader. Miller Units values are expressed as follows: The kinetic OD 420 readings were converted into the slope of OD 420 over time (OD 420 /min), multiplied by 5000, and adjusted for the initial OD 600 reading at the first time point. The obtained MU values were then compared to those of the traditional assay (Table 1). More detailed lab notes on running this assay and subsequent processing in MARS Data Analysis are available in Supplementary Data B.
2.4. Data comparison between the single-step β-galactosidase assay and the traditional assay See Table 1. Table 1 The single-step automated assay was consistent over multiple orders of magnitude and generated data equivalent to that of the traditional assay. The coefficients of variation of the Miller Unit [MU] measurements using the one-step and the traditional assay were similar for all β-galactosidase expressing strains suggesting that the two methods have comparable precision and reproducibility. Additionally, no statistically significant difference was observed between the single-step and traditional βgalactosidase assay (two-tailed Student's t-test assuming unequal variance; P40.05).

One-step assay
Traditional assay T-test