title: 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.
authors.affiliation: Center for Protein Science, Design and Engineering (CPSDE), Department of Biological Sciences, Indian Institute of Science Education & Research (IISER) Mohali, Knowledge City, Sector-81, SAS Nagar, Punjab 140306, India.
authors.forename: Kanika
authors.initials: K
authors.lastname: Arora
authors.affiliation: GE Healthcare Life Sciences, Wipro GE Healthcare Pvt Ltd, 7th Floor, Summit Tower B, 73/1, Brigade Metropolis, Mahadevapura, Bangalore 560048, India.
authors.forename: Sachin S
authors.initials: SS
authors.lastname: Mangale
authors.affiliation: Center for Protein Science, Design and Engineering (CPSDE), Department of Biological Sciences, Indian Institute of Science Education & Research (IISER) Mohali, Knowledge City, Sector-81, SAS Nagar, Punjab 140306, India. Electronic address: guptasarma@iisermohali.ac.in.
authors.forename: Purnananda
authors.initials: P
authors.lastname: Guptasarma
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D004926:Escherichia coli / Q000166:cytology* / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D008856:Microscopy, Fluorescence; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins
keywords: Bacterial nucleoid staining; Fluorescence imaging; Fluorescent HU; Gene expression; Leaky repression; Negative regulation