Glycerol-3-phosphate dehydrogenase (GlpD) reporter enzyme assay for Escherichia coli
Abstract Glycerol-3-phosphate dehydrogenase (GlpD) is a recently introduced reporter enzyme for E. coli [1]. GlpD calalyzes the oxidation of Glycerin-3-phosphate (G3P) to dihydroxyacetone-phosphate (DHAP). The oxidation is coupled to the reduction of the artificial yellow substrate tetrazol-3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid (MTT) to a blue-violet formazan, which is mediated by the electron carrier phenazin-methanosulfate (PMS). This leads to an increase in absorption at 570 nm, which is measured to quantify the GlpD activity. To avoid background, it is best to use an E. coli strain with a deletion of the chromosomal glpD gene, which is available e.g. at the Keio collection [2]. The glpD gene can be used for transcriptional fusions, e.g. to characterize promoters, and also for translational fusions, e.g. to study translational regulation. The assay was adapted to the microtiter plate format to enable the parallel handling of a large number of samples. The “procedure” (see below) describes an application with the glpD gene in a translational fusion with the gene of interest cloned under the control of the inducible arabinose promoter PBAD.