Single cell-level detection and quantitation of leaky protein expression from any strongly regulated bacterial system

Abstract

Extremely low levels of "leaky" expression of genes in bacterial protein expression systems can severely curtail cell viability when expressed proteins are toxic. A general method for sensitive detection of such expression is lacking. Here, we present a method based on microscopic visualization of a fluorescent "reporter" protein (RFP-HU-A) constructed by fusing red fluorescent protein (RFP) to the N-terminus of a nucleoid-associated, histone-like DNA-binding protein, HU-A. Localization of RFP-HU-A within nucleoids facilitates detection, quantitation, and characterization of leaky expression at the single-cell level.

Keywords: Bacterial nucleoid staining; Fluorescence imaging; Fluorescent HU; Gene expression; Leaky repression; Negative regulation.