Caution: Plasmid DNA topology affects luciferase assay reproducibility and outcomes

LightSwitchTM Luciferase Assay System

A Redox-Sensitive Luciferase Assay for Determining the Localization and Topology of Endoplasmic Reticulum Proteins

Correct localization and transmembrane topology are crucial for the proteins residing and functioning in the endoplasmic reticulum (ER). We have developed a rapid and convenient assay, based on the redox-sensitive luciferase from Gaussia princeps (Gluc) and green fluorescence protein (GFP), to determine the localization or topology of ER proteins. Using the tandem Gluc-GFP reporter fused to dif...

Heterogeneous oxygen availability affects the titer and topology but not the fidelity of plasmid DNA produced by Escherichia coli

BACKGROUND Dissolved oxygen tension (DOT) is hardly constant and homogenously distributed in a bioreactor, which can have a negative impact in the metabolism and product synthesis. However, the effects of DOT on plasmid DNA (pDNA) production and quality have not been thoroughly investigated. In the present study, the effects of aerobic (DOT ≥30% air sat.), microaerobic (constant DOT = 3% air sa...

A plasmid-based lacZ gene assay for DNA polymerase fidelity

A significantly improved DNA polymerase fidelity assay, based on a gapped plasmid containing the lacZα reporter gene in a single-stranded region, is described. Nicking at two sites flanking lacZα, and removing the excised strand by thermocycling in the presence of complementary competitor DNA, is used to generate the gap. Simple methods are presented for preparing the single-stranded competitor...

Expression and Purification of the luciferase enzyme and in Vivo ATP Assay

Introduction: Gene expression and purification of luciferases from the firefly, Lampyris turkestanicus, and optimization of cellular ATP measurements were performed. Methods: cDNA encoding luciferases from Lampyris turkestanicus was transferred from pQE30 vector into pET28a expression vector and pLtu28 was built. Newly constructed vector was expressed in E. coli XL1 Blue and the recombinant l...