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Seamless insert-plasmid assembly at sub-terminal homologous sequences.

Anna-Sophia Krebs1, Tobias Bierig2, Gabriella Collu2

  • 1Laboratory of Nanoscale Biology, Division of Biology and Chemistry, Paul Scherrer Institute, CH-5232 Villigen PSI, Switzerland.

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Summary

This study introduces improved DNA assembly methods using homologous sequences (HS) for protein engineering. These techniques enhance molecular biology workflows for creating fusion proteins, advancing protein nanotechnology and synthetic biology.

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Area of Science:

  • Molecular Biology
  • Protein Engineering
  • Synthetic Biology

Background:

  • Seamless DNA assembly with domain boundary variations is crucial for protein engineering and nanotechnology.
  • Existing molecular biology workflows can be inefficient for complex DNA assembly tasks.

Purpose of the Study:

  • To evaluate sub-terminal homologous sequences (HS) for improved DNA assembly via co-transformation cloning and in vitro assembly.
  • To quantify the impact of HS-to-ends distances on cloning efficiency.
  • To develop a streamlined method for integrating fusion proteins into receptors with varied linker boundaries.

Main Methods:

  • Designed a blue-white-pink screening system to assess cloning efficiency.
  • Utilized T5 exonuclease and Phusion DNA polymerase for in vitro DNA assembly.
  • Employed co-transformation cloning with sub-terminal HS and primer 5'-overhangs for fusion protein integration.

Main Results:

  • Both co-transformation cloning and in vitro assembly methods are effective with HS-to-ends distances up to 10 base pairs.
  • Successfully integrated a fusion protein into a G-protein-coupled receptor (GPCR) with nine distinct linker boundaries.
  • Demonstrated a single-plasmid linearization approach for complex fusion protein construction.

Conclusions:

  • Sub-terminal homologous sequences significantly enhance DNA assembly efficiency for protein engineering.
  • The developed molecular cloning approach offers a versatile tool for protein nanotechnology and synthetic biology.
  • This method expands the utility of DNA assembly strategies for creating novel fusion proteins.