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Intracellular directed evolution of proteins from combinatorial libraries based on conditional phage replication.

Andreas K Brödel1, Alfonso Jaramillo2,3,4, Mark Isalan1

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|August 11, 2017
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Summary
This summary is machine-generated.

This study introduces a novel intracellular directed evolution protocol for improving protein functions. The method enables efficient selection of protein variants within cells, leading to enhanced biomolecules in approximately seven weeks.

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Area of Science:

  • Synthetic Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Directed evolution is a key strategy for protein engineering.
  • Existing methods face limitations in selection stringency and efficiency.
  • Intracellular selection offers a promising alternative for protein improvement.

Purpose of the Study:

  • To present a novel protocol for intracellular directed evolution of proteins.
  • To demonstrate the advantages of this method over established techniques.
  • To apply the protocol for evolving orthogonal transcription factors.

Main Methods:

  • Construction of a combinatorial M13 phagemid library for gene variants.
  • Intracellular selection rounds to achieve desired protein activity.
  • Characterization of evolved protein variants and their functions.

Main Results:

  • Developed a versatile intracellular directed evolution system.
  • Successfully evolved bacteriophage lambda cI transcription factor variants.
  • Evolved variants exhibited orthogonal activity on synthetic promoters, enabling simultaneous activation and repression.

Conclusions:

  • The presented protocol offers a powerful and accessible method for directed protein evolution.
  • This approach minimizes artifacts and allows for precise selection logics.
  • The system is broadly applicable for engineering various gene and gene circuit functions.