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Related Concept Videos

Reporter Genes02:11

Reporter Genes

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.
Commonly used reporter...

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Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence
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Codon-optimized fluorescent proteins designed for expression in low-GC gram-positive bacteria.

Inka Sastalla1, Kannie Chim, Gordon Y C Cheung

  • 1Laboratory of Bacterial Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 33, Bethesda, MD 20892-3202, USA.

Applied and Environmental Microbiology
|February 3, 2009
PubMed
Summary

Codon optimization significantly enhances fluorescent protein expression in low-GC bacteria like Bacillus anthracis. This breakthrough enables better protein expression and reporter applications in challenging bacterial hosts.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Microbiology

Background:

  • Fluorescent proteins are vital tools in biological research.
  • Expression challenges arise in bacteria with suboptimal codon usage and GC content.
  • Bacillus anthracis presents a low-GC content host with expression hurdles.

Purpose of the Study:

  • Analyze codon usage and GC content discrepancies for fluorescent proteins in Bacillus anthracis.
  • Investigate the reasons for undetectable fluorescence of certain proteins in B. anthracis.
  • Develop codon-optimized fluorescent protein genes for improved expression in low-GC bacteria.

Main Methods:

  • Comparative analysis of fluorescent protein gene sequences (GFP, CFP, YFP, PAGFP) against B. anthracis genome.
  • Gene expression analysis in B. anthracis and Staphylococcus aureus.
  • Construction and testing of codon-optimized fluorescent protein genes.
  • Utilizing optimized GFP as a reporter for promoter activity studies.

Main Results:

  • Significant GC content and codon usage mismatches identified for CFP, YFP, and PAGFP, but not GFP.
  • CFP and YFP showed undetectable fluorescence in B. anthracis due to expression failure, not transcription or degradation.
  • Codon-optimized genes (GFP, CFP, YFP, PAGFP) demonstrated high expression in B. anthracis and S. aureus.
  • Optimized GFP successfully reported on the activity of the B. anthracis protective antigen promoter.

Conclusions:

  • Codon optimization is crucial for high-level fluorescent protein expression in low-GC bacteria.
  • Optimized fluorescent proteins serve as effective reporters for gene expression studies in challenging hosts.
  • This strategy enhances the utility of fluorescent proteins in diverse bacterial systems, including virulence studies.