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Quantifying bacterial cell lysis using GFP based fluorimetric assay.

Meenakshi Sharma1, Jaya Lakshmi Tyagi1, Krishna Mohan Poluri2

  • 1Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247667, Uttarakhand, India.

International Journal of Biological Macromolecules
|July 30, 2019
PubMed
Summary

This study presents a green fluorescent protein (GFP) based fluorescence spectroscopy method for quantifying antimicrobial protein activity by measuring cell lysis. The assay accurately measures protein-induced bacterial cell lysis, offering a robust tool for antimicrobial potency determination.

Keywords:
Antimicrobial agentsCell lysisEndolysinFluorescence spectroscopyGreen fluorescent proteinTurbidimetric assay

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Area of Science:

  • Biochemistry and Molecular Biology
  • Microbiology
  • Spectroscopy

Background:

  • Accurate quantification of microbial cell lysis is crucial for determining the antimicrobial potency of various agents.
  • Fluorescence spectroscopy provides precise measurement capabilities through specific fluorophore emission profiles.
  • Existing methods for antimicrobial activity assessment can be enhanced with more sensitive and quantitative techniques.

Purpose of the Study:

  • To develop and validate a reliable green fluorescent protein (GFP) based fluorescence spectroscopy protocol for evaluating antimicrobial protein activity.
  • To establish a quantitative method for measuring protein-induced bacterial cell lysis.
  • To demonstrate the application of this method using bacteriophage endolysins against E. coli.

Main Methods:

  • Utilized green fluorescent protein (GFP) expressing E. coli cells.
  • Employed fluorescence spectroscopy to measure GFP emission intensity, correlating it with cell lysis.
  • Validated the GFP-based fluorimetric assay results with turbidimetric assays and fluorescence microscopy.

Main Results:

  • The GFP-based fluorescence spectroscopy protocol reliably quantified cell lysis induced by T4 and T7 endolysins.
  • The method allowed absolute quantification of endolysin-mediated cell lysis under varying conditions (pH, salt, metal ions).
  • Results from the fluorimetric assay were consistent with traditional methods, confirming its accuracy and robustness.

Conclusions:

  • A robust and quantitative GFP-based fluorescence spectroscopy method for assessing antimicrobial protein activity via cell lysis was established.
  • This fluorimetric technique offers a precise way to measure antimicrobial potential by quantifying bacterial cell lysis.
  • The protocol is versatile and can be adapted for various GFP-expressing microbial strains and antimicrobial agents.