Quantifying bacterial cell lysis using GFP based fluorimetric assay

Abstract

Quantitative measurement of cell lysis against a given microbial strain is essential to calculate the antimicrobial potency of protein/peptide/nanomaterial based formulations. Fluorescence spectroscopy based measurements offer precise quantification of a process via selected flurophore emission profile. In this context, we elucidate a reliable and robust green fluorescent protein (GFP) based fluorescence spectroscopy protocol to evaluate the antimicrobial activity of proteins. The technique is based on the fact that the intensity of the GFP emission released from cells correlates with cell lysis and henceforth the antimicrobial potential of the chosen agent. The technique was demonstrated with two different families of bacteriophage endolysins (T7 and T4 endolysins) using GFP expressing E. coli cells. The GFP based method allowed the absolute quantification of T4 and T7 endolysins cell lysis characteristics at different pH, salt concentrations, and metal ions. The results obtained from GFP based fluorimetric assay were substantiated with turbidimetric assay and fluorescence microscopy. This fluorimetric method in conjugation with different GFP expressing microbial strains and antimicrobial agents can be efficiently applied as a quantification technique to precisely measure cell lysis.