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Related Concept Videos

Translation01:31

Translation

133.6K
Lesson: Translation
Translation is the process of synthesizing proteins from the genetic information carried by messenger RNA (mRNA). Following transcription, it constitutes the final step in the expression of genes. This process is carried out by ribosomes, complexes of protein and specialized RNA molecules. Ribosomes, transfer RNA (tRNA), and other proteins produce a chain of amino acids—the polypeptide—as the end product of translation.
Translation Produces the Building Blocks of...
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Total Internal Reflection Fluorescence Microscopy01:05

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Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid surface with a higher refractive index, such as a glass coverslip. TIRF only allows fluorophores in proximity to the solid surface to be excited. When light from a medium with a lower refractive index (such as air) hits the glass coverslip at a critical angle, the light undergoes total internal reflection stead of passing through the glass.
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Related Experiment Video

Updated: May 1, 2026

Quantitative Immunofluorescence to Measure Global Localized Translation
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Cell-Free Translation Quantification via a Fluorescent Minihelix.

Jessica A Willi1,2, Ashty S Karim1,2, Michael C Jewett1,2,3

  • 1Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, United States.

ACS Synthetic Biology
|July 9, 2024
PubMed
Summary

This study introduces a new fluorescent minihelix reporter for quantifying nonfluorescent proteins in cell-free gene expression systems. This method enables accurate real-time protein detection without staining, advancing synthetic biology applications.

Keywords:
cell-free gene expressionfluorescent reporterhigh-throughputin vitro transcription and translationprotein quantificationsynthetic biology

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Synthetic Biology

Background:

  • Cell-free gene expression systems are vital for applications like medicine production, diagnostics, and educational tools.
  • Quantifying nonfluorescent proteins in these systems presents a significant hurdle.

Purpose of the Study:

  • To develop and validate a novel method for accurate quantification of nonfluorescent proteins in cell-free expression systems.
  • To introduce a fluorescent minihelix reporter for real-time protein detection and analysis.

Main Methods:

  • Adaptation and utilization of an optimized tetra-cysteine minihelix as both a fusion protein and a standalone reporter with the FlAsH dye.
  • Encoding the fluorescent reporter on a primer pair for protein tagging via Polymerase Chain Reaction (PCR).
  • Detection using standard 96/384-well plate readers, RT-qPCR systems, or gel electrophoresis without staining.

Main Results:

  • The fluorescent minihelix reporter enables quantitative detection of tagged proteins and standalone reporters in cell-free reactions.
  • The fluorescent signal is stable, correlates linearly with protein concentration, and allows for precise quantification.
  • The reporter facilitates the study of cell-free expression dynamics and engineered ribosome activity.

Conclusions:

  • The developed fluorescent minihelix reporter system overcomes the challenge of quantifying nonfluorescent proteins in cell-free gene expression.
  • This technology offers a versatile and efficient tool for real-time monitoring and quantification, applicable to various synthetic biology endeavors.
  • The reporter is expected to significantly aid in the engineering of *in vitro* transcription and translation systems.