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Related Concept Videos

Reporter Genes02:11

Reporter Genes

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Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
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Enhanced small green fluorescent proteins as a multisensing platform for biosensor development.

Guo-Teng Liang1,2,3,4, Cuixin Lai1,2,3, Zejun Yue1,2,3,5

  • 1School of Life Sciences, Westlake University, Hangzhou, Zhejiang, China.

Frontiers in Bioengineering and Biotechnology
|November 3, 2022
PubMed
Summary
This summary is machine-generated.

New flavin-binding fluorescent proteins (FPs) offer enhanced brightness for live cell imaging under low-oxygen conditions. These miniGFPs are sensitive to copper ions and flavin mononucleotide, enabling versatile biosensor applications.

Keywords:
copperflavinfluorescent protein biosensorhypoxialive cell imaging microscopy

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Area of Science:

  • Biochemistry and Molecular Biology
  • Biophotonics and Imaging
  • Biosensor Development

Background:

  • Engineered light, oxygen, and voltage (LOV)-based proteins can fluoresce without oxygen, enabling imaging in hypoxic/anaerobic environments.
  • Flavin-binding fluorescent proteins (FPs) show promise for biosensors due to sensitivity to metal ions and flavins.
  • Existing flavin-binding FPs have limited brightness compared to GFP-like FPs, necessitating improvement.

Purpose of the Study:

  • To develop brighter flavin-binding fluorescent proteins (FPs) using directed molecular evolution.
  • To characterize and benchmark the developed FPs for live cell imaging applications.
  • To assess the potential of these FPs as biosensors for metal ions and flavins.

Main Methods:

  • Directed molecular evolution was employed to create novel flavin-binding FPs, named miniGFP1 and miniGFP2.
  • Systematic benchmarking was performed in *Escherichia coli* and mammalian cells against spectrally similar FPs.
  • Photochemical properties, sensitivity to Cu(II) and Cu(I), and flavin mononucleotide binding were evaluated.

Main Results:

  • MiniGFP1 and miniGFP2 exhibit cyan-green fluorescence (450/499 nm) with a small size (~13 kDa).
  • MiniGFPs demonstrated improved photochemical properties and enabled long-term live cell imaging under anaerobic and hypoxic conditions.
  • High sensitivity to Cu(II) (Kd ~67-68 nM) and potential for Cu(I) detection were observed, along with selective flavin mononucleotide binding.

Conclusions:

  • MiniGFPs represent a significant advancement in flavin-binding FP brightness and performance.
  • These FPs are suitable for live cell imaging in challenging low-oxygen environments.
  • MiniGFPs offer a versatile multisensing platform for *in vitro* and in-cell biosensor development.