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Scalable and cost-efficient custom gene library assembly from oligopools.

Chase R Freschlin1, Kevin K Yang2, Philip A Romero1,3

  • 1Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA.

Science Advances
|May 22, 2026
PubMed
Summary

OMEGA is a new, low-cost gene assembly method that enables high-throughput experimental validation of computationally designed proteins. This technique allows for the parallel assembly of thousands of genes, significantly reducing costs and increasing scalability for synthetic biology applications.

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Area of Science:

  • Synthetic Biology
  • Molecular Biology
  • Bioinformatics

Background:

  • Computational protein design and sequence space exploration are advancing rapidly.
  • Experimental validation of designed genes is limited by DNA synthesis cost and scalability.

Purpose of the Study:

  • To introduce OMEGA (Oligo-based Multiplexed Efficient Gene Assembly), a novel, cost-effective method for large-scale gene assembly.
  • To enable high-throughput experimental validation of computationally designed genes.

Main Methods:

  • OMEGA computationally fragments genes into short, high-fidelity oligonucleotides for pooled library synthesis.
  • Genes are assembled in parallel across multiplexed subpools using Golden Gate assembly.
  • Optimization of fragment number per gene and orthogonal ligation sites was performed.

Main Results:

  • OMEGA can assemble up to 2.6-kilobase constructs using up to 70 Golden Gate sites.
  • A library of 810 green fluorescent protein variants was assembled and screened with 94-97% recovery and high uniformity.
  • Per-gene costs were reduced to as low as $1.50.

Conclusions:

  • OMEGA provides a scalable and accessible solution for precision library construction.
  • This method bridges computational design with experimental validation in synthetic biology.
  • OMEGA is available as open-source software and a Colab notebook for widespread adoption.