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A systematic approach to inserting split inteins for Boolean logic gate engineering and basal activity reduction.

Trevor Y H Ho1,2,3, Alexander Shao1,4, Zeyu Lu1

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We developed a new method, intein-assisted bisection mapping (IBM), to identify split sites in proteins for synthetic biology. This approach overcomes limitations in split protein ligation and expands the functional sequence space for protein engineering.

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Area of Science:

  • Synthetic biology
  • Protein engineering
  • Molecular biology

Background:

  • Split inteins enable seamless ligation of synthetic split proteins.
  • Identifying split sites is challenging due to extein junction sequence requirements.

Purpose of the Study:

  • To develop a robust method for identifying split sites in proteins.
  • To expand the utility of split inteins in synthetic biology applications.

Main Methods:

  • Augmented a mini-Mu transposon-based screening approach.
  • Devised the intein-assisted bisection mapping (IBM) method.

Main Results:

  • IBM robustly identified split sites on five proteins.
  • Successfully converted split proteins into AND or NAND logic gates.
  • Demonstrated that inteins expand functional sequence space for protein splitting.

Conclusions:

  • The IBM method provides a generalizable and systematic route for creating split protein-intein fusions.
  • This approach facilitates protein engineering and synthetic biology applications.
  • Mitigated basal activities of highly active proteins by splitting them.