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Assembly and Tracking of Microbial Community Development within a Microwell Array Platform
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Precise and versatile microplate reader-based analyses of biosensor signals from arrayed microbial colonies.

Fabian S F Hartmann1, Tamara Weiß1, Louise L B Kastberg1

  • 1Section for Synthetic Biology, Department Bioengineering, Technical University of Denmark (DTU), Lyngby, Denmark.

Frontiers in Microbiology
|June 30, 2023
PubMed
Summary
This summary is machine-generated.

Microplate readers offer a sensitive alternative to imaging for analyzing microbial fluorescent biosensors. This method enhances phenotypic screening for metabolic engineering and systems biology applications.

Keywords:
Corynebacterium glutamicumEscherichia coliGFPMrx1-roGFP2Saccharomyces cerevisiaearrayed microbial coloniesgenetically encoded biosensorsmCherry

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Area of Science:

  • Microbiology
  • Biotechnology
  • Systems Biology

Background:

  • Genetically encoded fluorescent biosensors are crucial for microbial phenotypic screening.
  • Analyzing fluorescent signals from microbial colonies typically requires specialized imaging equipment with specific filters.
  • This can be challenging due to the diversity of biosensor properties.

Purpose of the Study:

  • To investigate the use of monochromator-equipped microplate readers as a versatile alternative to imaging for analyzing fluorescent biosensor signals from microbial colonies.
  • To assess the sensitivity and dynamic range of microplate reader-based analyses compared to traditional imaging methods.
  • To demonstrate the applicability of this technique for various microbial systems and biosensor types.

Main Methods:

  • Utilized monochromator-equipped microplate readers for fluorescence analysis of microbial colonies.
  • Applied the technique to analyze LacI-controlled mCherry expression in *Corynebacterium glutamicum* and GFP reporter activity in *Saccharomyces cerevisiae*.
  • Investigated ratiometric fluorescent reporter proteins (FRPs) for analyzing internal pH in *Escherichia coli* and redox states in *C. glutamicum*.

Main Results:

  • Microplate reader analysis demonstrated improved sensitivity and dynamic range compared to imaging for reporter gene expression.
  • High sensitivity was achieved for analyzing ratiometric FRPs, including pH-sensitive mCherryEA in *E. coli*.
  • Redox shifts were successfully measured in *C. glutamicum* colonies using Mrx1-roGFP2, highlighting the role of mycothiol (MSH) in maintaining redox state.

Conclusions:

  • Microplate readers provide a sensitive and versatile platform for analyzing diverse fluorescent biosensor signals from microbial colonies.
  • This approach facilitates comprehensive phenotypic screening, aiding in the development of new strains for metabolic engineering and systems biology.
  • The method enhances the analysis of cellular parameters like pH and redox state in microbial colonies.