Green fluorescent protein as a quantitative and real time indicator of gene induction during oxidative stress in Escherichia coli

This work defines and validates an innovative system for the study of gene regulation. Pivotal to this system, is the relatively new reporter gene green fluorescent protein (GFP). Using gfp as the reporter gene allowed for non-invasive in vivo monitoring of gene expression in real time. Results are presented on the correlation between GFP protein concentration and fluorescence intensity. In these experiments, fluorescence was quantitatively measured both by a spectrofluorimeter and an on-line sensor developed by the Rao lab. The on-line sensor utilized a blue light emitting diode (LED) to excite GFP and monitored the green fluorescence during fermentation on-line via computer. The sensor was validated with a Perkin-Elmer spectrofluorimeter (R2 = 0.996) and then used to demonstrate that simply measuring GFP fluorescence from a cell suspension was equivalent to a western blot measurement of a heterologous protein. Employment of the on-line sensor and first derivative calculations of GFP fluorescence overcame the inherent drawbacks (stabilization, lag time) of GFP. The application of GFP as a generic toot for bioprocess development is discussed.; To study gene induction, a library of oxidative “stress probes” was constructed. Promoter regions from eight genes were fused to gfp coding sequence. Genes selected were soxS, sodA, zwf , and acnA from the SoxRS regulon, katG, ahpC, and dps from the OxyR regulon and recA from the SOS response of Escherichia coli. When exposed to oxidative stress, the stress probes responded by the emission of green fluorescence. These stress probes responded with specificity to various oxidative stresses (superoxide generation, hydrogen peroxide and hyperoxia). The differential expression of the oxidative stress genes was visualized in a straightforward and simplistic manner. In this work, data is presented on the kinetics and dose dependent responses of the selected genes. In addition, these stress probes may be used as indirect measurements of reactive oxygen species (ROS) which is beneficial for environmental monitoring Along this thought, cultures harboring the stress probes were subjected to various anti-tumor drugs to screen for ROS generation to identify their potential mode of action.