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Related Experiment Videos

A novel fluorescent protein-based biosensor for gram-negative bacteria.

Yan Y Goh1, Bow Ho, Jeak L Ding

  • 1Department of Biological Sciences, National University of Singapore, Singapore 117543.

Applied and Environmental Microbiology
|November 27, 2002
PubMed
Summary

Researchers engineered enhanced green fluorescent protein (EGFP) into a biosensor to detect endotoxin and gram-negative bacteria. This novel fluorescent biosensor offers dual functionality for environmental and clinical applications.

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Environmental Science

Background:

  • Endotoxins and gram-negative bacteria pose significant health and environmental risks.
  • Existing detection methods can be complex and time-consuming.
  • Development of rapid, sensitive biosensors is crucial for effective monitoring.

Purpose of the Study:

  • To engineer enhanced green fluorescent protein (EGFP) mutants as a fluorescence-based biosensor.
  • To detect both endotoxin (lipopolysaccharide) and live gram-negative bacteria.
  • To establish the binding affinities and sensing capabilities of the engineered EGFP mutants.

Main Methods:

  • Site-directed mutagenesis of EGFP based on rational computational design.
  • Construction and expression of EGFP mutants (EGFP(i)) with endotoxin binding motifs in E. coli.

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  • Purification of EGFP(i) proteins and testing for binding affinity to lipid A and sensing of gram-negative bacteria.

    • Main Results:

    • EGFP mutants (G10 and G12) demonstrated high binding affinities to lipid A (K(D) values of 7.16 x 10(-7) M and 8.15 x 10(-8) M, respectively).
    • Binding affinity remained stable across a wide range of pH and ionic strength.
    • EGFP(i) successfully detected gram-negative bacteria (E. coli, Pseudomonas aeruginosa) and bacteria in environmental water samples via fluorescence quenching.

    Conclusions:

    • Engineered EGFP mutants function as effective fluorescent biosensors for both endotoxin and gram-negative bacteria.
    • The dual detection capability offers a novel approach for environmental monitoring and diagnostics.
    • This work provides a foundation for developing advanced biosensing technologies.