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Related Experiment Video

Updated: May 6, 2026

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells
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One-pot cloning and protein expression platform for genetic engineering.

Wakana Sato1, Judee Sharon1, Brock Cash1

  • 1Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN US.

Biorxiv : the Preprint Server for Biology
|September 5, 2025
PubMed
Summary
This summary is machine-generated.

We developed a one-pot cloning and protein expression platform for rapid genetic variant screening. This method streamlines synthetic biology workflows by combining Golden Gate cloning with cell-free systems, enabling faster protein engineering.

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Area of Science:

  • Synthetic Biology
  • Molecular Biology
  • Biotechnology

Background:

  • Traditional cloning and protein expression methods are often laborious, requiring multiple steps like bacterial transformation and purification.
  • High-throughput screening of genetic variants is crucial for protein engineering and synthetic biology applications.
  • Developing streamlined, efficient platforms is essential to accelerate research and development in these fields.

Purpose of the Study:

  • To present a novel one-pot platform integrating mutagenesis, plasmid assembly, and protein expression.
  • To demonstrate the efficiency and versatility of this platform for generating and screening genetic variants.
  • To establish a generalizable and automatable method for high-throughput cloning and protein engineering.

Main Methods:

  • Integration of Golden Gate cloning with cell-free transcription-translation systems.
  • One-pot reaction design eliminating intermediate purification and bacterial amplification steps.
  • Validation using fluorescent proteins, luciferase enzymes, antibiotic-converting enzymes, and the violacein biosynthetic pathway.

Main Results:

  • Efficient generation and screening of genetic variants in a single reaction.
  • Demonstrated versatility across various applications including single- and multi-site mutagenesis, library creation, and metabolic pathway engineering.
  • Successful application in multiplexed reactions and construction of multi-cistronic constructs.

Conclusions:

  • The developed one-pot platform significantly streamlines cloning and protein expression workflows.
  • This approach is a versatile, generalizable, and automatable tool for high-throughput protein engineering.
  • The method holds significant potential for advancing synthetic biology research and applications.