Tecan Microplate Reader for Quantifying Bacterial Fluorescence

Protocol Overview

To demonstrate the versatility of this instrument, I quantified the concentration of a bacterially produced small molecule using a dual-color fluorescent biosensor. This P. aeruginosa biosensor strain expresses YFP in response to this molecule, and expresses mKate constitutively for normalization purposes. Equal volumes of the biosensor strain was added to the wells of a black, flat, clear bottom, Corning 96-well plate (30 µL, mid-log phase, LB broth) and added 30 µL of a bacterial culture supernatant from a strain that produces the molecule of interest, or a control mutant that does not (grown in LB broth). In the Tecan software, I set up a default experiment and selected the appropriate 96-well plate and well coordinates. I set up a kinetic experiment that would last for a total of 3 hours at a temperature of 37°C. I selected the measurement of of YFP and mKate by inputing the optimal excitation and emission (500/540 nm and 590/645 nm) and also selected an absorbance measurement of 600 nm to measure culture density. I chose to read every 20 min, and selected a shaking speed of 400 rpm between timepoints. Values of YFP/mKate were plotted over time for the two strains (n = 5).