Split fluorescent protein-mediated multimerization of cell wall binding domain for highly sensitive and selective bacterial detection

Shirley Xu, Inseon Lee, Seok Joon Kwon, Eunsol Kim, Liv Nevo, Lorelli Straight, Hironobu Murata, Krzysztof Matyjaszewski, Jonathan S. Dordick

Research output: Contribution to journalArticlepeer-review

Abstract

Cell wall peptidoglycan binding domains (CBDs) of cell lytic enzymes, including bacteriocins, autolysins and bacteriophage endolysins, enable highly selective bacterial binding, and thus, have potential as biorecognition molecules for nondestructive bacterial detection. Here, a novel design for a self-complementing split fluorescent protein (FP) complex is proposed, where a multimeric FP chain fused with specific CBDs ((FP-CBD)n) is assembled inside the cell, to improve sensitivity by enhancing the signal generated upon Staphylococcus aureus or Bacillus anthracis binding. Flow cytometry shows enhanced fluorescence on the cell surface with increasing FP stoichiometry and surface plasmon resonance reveals nanomolar binding affinity to isolated peptidoglycan. The breadth of function of these complexes is demonstrated through the use of CBD modularity and the ability to attach enzymatic detection modalities. Horseradish peroxidase-coupled (FP-CBD)n complexes generate a catalytic amplification, with the degree of amplification increasing as a function of FP length, reaching a limit of detection (LOD) of 103 cells/droplet (approximately 0.1 ng S. aureus or B. anthracis) within 15 min on a polystyrene surface. These fusion proteins can be multiplexed for simultaneous detection. Multimeric split FP-CBD fusions enable use as a biorecognition molecule with enhanced signal for use in bacterial biosensing platforms.

Original languageEnglish
Pages (from-to)54-64
Number of pages11
JournalNew Biotechnology
Volume82
DOIs
StatePublished - 25 Sep 2024
Externally publishedYes

Keywords

  • Bacteria detection
  • Cell wall binding domain
  • Enzymatic biosensors
  • Split fluorescent protein

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