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Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors
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A Chimeric Two-Component Regulatory System-Based Escherichia coli Biosensor Engineered to Detect Glutamate.

Sambandam Ravikumar1, Yokimiko David2, Si Jae Park3

  • 1Department of Biotechnology and Bioengineering, Chonnam National University, 77 Yongbong-ro, Gwangju, 61186, Republic of Korea.

Applied Biochemistry and Biotechnology
|April 4, 2018
PubMed
Summary

Researchers developed novel biosensors using two-component regulatory systems (TCRSs) and green fluorescent protein (GFP) to efficiently screen microorganisms producing glutamate. This high-throughput system enables the discovery of new gene products and optimization of amino acid production pathways.

Keywords:
DegSEnvZEscherichia coliGlutamateTwo-component system

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Area of Science:

  • Microbiology
  • Biotechnology
  • Molecular Biology

Background:

  • Amino acid production is crucial for various industries.
  • Screening methods for microorganisms that produce specific amino acids, like glutamate, can be inefficient.
  • Development of high-throughput biosensors is needed to accelerate discovery and optimization.

Purpose of the Study:

  • To construct and validate novel amino acid biosensors for high-throughput screening of glutamate-producing microorganisms.
  • To utilize a chimeric two-component regulatory system (TCRS) fused with green fluorescent protein (GFP) as a reporter for glutamate detection.
  • To establish a system for single-cell level analysis of glutamate concentration.

Main Methods:

  • Construction of a chimeric DegS/EnvZ (DegSZ) TCRS by fusing domains from Planococcus sp. PAMC21323 and Escherichia coli.
  • Integration of the DegSZ TCRS with a green fluorescent protein (GFP) reporter gene under the control of the ompC promoter.
  • Validation of the biosensor's response to varying concentrations of glutamate.
  • Application of fluorescence-activated cell sorting (FACS) for single-cell glutamate quantification.

Main Results:

  • The chimeric TCRS-based biosensor demonstrated a significant 4-fold increase in fluorescent signal upon glutamate addition.
  • A clear linear correlation was established between fluorescence intensity and exogenous glutamate concentration.
  • The biosensor successfully determined glutamate concentration at the single-cell level using FACS.

Conclusions:

  • The developed chimeric TCRS-based biosensor is an effective high-throughput tool for screening glutamate-producing microorganisms.
  • This system facilitates the identification of novel gene products and the optimization of metabolic pathways for amino acid production.
  • The biosensor's ability to perform single-cell analysis opens avenues for detailed strain improvement and pathway engineering.