title: In-depth characterization of the fluorescent signal of HyPer, a probe for hydrogen peroxide, in bacteria exposed to external oxidative stress.
abstract: Genetically encoded, fluorescent biosensors have been developed to probe the activities of various signaling molecules inside cells ranging from changes in intracellular ion concentrations to dynamics of lipid second messengers. HyPer is a member of this class of biosensors and is the first to dynamically respond to hydrogen peroxide (H2O2), a reactive oxygen species that functions as a signaling molecule. However, detailed characterization of HyPer's signal is not currently available within the context of bacteria exposed to external oxidative stress, which occurs in the immunological response of higher organisms against invasive pathogenic bacteria. Here, we performed this characterization, specifically in Escherichia coli exposed to external H2O2. We found that the temporal behavior of the signal does not correspond exactly to peroxide concentration in the system as a function of time and expression of the sensor decreases the peroxide scavenging activity of the cell. We also determined the effects of cell number, both before and after normalization of externally added H2O2 to the number of cells. Finally, we report quantitative characteristics of HyPer's signal in this context, including the dynamic range of the signal, the signal-to-noise ratio, and the half saturation constant. These parameters show statistically meaningful differences in signal between two commonly used strains of E. coli, demonstrating how signal can vary with strain. Taken together, our results establish a systematic, quantitative framework for researchers seeking to better understand the role of H2O2 in the immunological response against bacteria, and for understanding potential differences in the details of HyPer's quantitative performance across studies.
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Building E19-502C, Cambridge, MA 02139, USA.
authors.forename: Joseph B
authors.initials: JB
authors.lastname: Lim
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Building E19-502C, Cambridge, MA 02139, USA.
authors.forename: Kimberly A
authors.initials: KA
authors.lastname: Barker
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Building E19-502C, Cambridge, MA 02139, USA.
authors.forename: Beijing K
authors.initials: BK
authors.lastname: Huang
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Building E19-502C, Cambridge, MA 02139, USA. Electronic address: sikes@mit.edu.
authors.forename: Hadley D
authors.initials: HD
authors.lastname: Sikes
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D004926:Escherichia coli / Q000187:drug effects* / Q000502:physiology; D005456:Fluorescent Dyes / Q000032:analysis; D006861:Hydrogen Peroxide / Q000032:analysis*; D018384:Oxidative Stress; D013997:Time Factors
chemical_list: D005456:Fluorescent Dyes; D006861:Hydrogen Peroxide
keywords: Escherichia coli; HyPer; Hydrogen peroxide; Quantitative redox biology; Reactive oxygen species