Recombinant methods in protein and whole-cell biosensing

R. S. Shetty, L. L.E. Salins, S. Ramanathan, S. Daunert

Research output: Contribution to journalConference article

1 Scopus citations

Abstract

In this paper, we investigate the use of fluorescently-labeled binding proteins and genetically engineered bacterial cells for sensing of phosphate, glucose, and L-arabinose. To optimize the performance of the labeled binding proteins for biosensing purposes, a few key considerations were taken into account. A site-selective labeling protocol of the fluorescent reporter to the protein was used to ensure that the probe reported from a specific domain of the protein. The labeling sites chosen were hypothesized to undergo a physicochemical change when the biorecognition element binds the analyte. Cysteine mutations were introduced into the binding proteins by site-directed mutagenesis using the polymerase chain reaction (PCR). The residues selected were all in close proximity to the binding cleft, a region that is affected the most by the conformational change that accompanies ligand binding. The cysteine residues were then labeled with environment-sensitive fluorophores and changes in the fluorescence properties of the conjugates were monitored and related to the amount of ligand present. The application of microorganisms in sensing systems represent new advances in the development of novel analytical techniques for the detection of a target analyte. In these systems, a genetically engineered organism generates an analytically useful signal when it encounters a specific target substance due to selective recognition and binding properties towards that particular compound. This concept has been demonstrated using an optical bacteria-based sensing system capable of detecting the monosaccharide L-arabinose that employed the green fluorescent protein as a reporter protein.

Original languageEnglish (US)
Pages (from-to)98-107
Number of pages10
JournalProceedings of SPIE - The International Society for Optical Engineering
Volume3858
DOIs
StatePublished - 1999
EventProceedings of the 1999 Advanced Materials and Optical Systems for Chemical and Biological Detection - Boston, MA, USA
Duration: Sep 21 1999Sep 22 1999

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Condensed Matter Physics
  • Computer Science Applications
  • Applied Mathematics
  • Electrical and Electronic Engineering

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