Parallel on-chip gene synthesis and application to optimization of protein expression

Area of Science:

• Synthetic Biology
• Biotechnology
• Molecular Biology

Background:

• Precise control of protein expression and cost-effective gene synthesis are crucial for advancing synthetic biology.
• Current methods face limitations in throughput and cost for large-scale gene synthesis and variant screening.

Purpose of the Study:

• To develop an integrated on-chip technology for high-throughput gene synthesis and protein expression.
• To enable precise control over protein expression levels for diverse applications.

Main Methods:

• On-chip DNA oligonucleotide synthesis using inkjet printing.
• Isothermal oligonucleotide amplification and parallel gene assembly on a microchip.
• Mismatch-specific endonuclease for error correction, achieving an error rate of ~0.19 errors per kb.

Main Results:

• Successful synthesis of thousands of codon-usage variants of lacZα and 74 Drosophila protein antigens.
• Screening in Escherichia coli yielded sequences expressed at levels ranging from 0% to 60% of total cell protein mass.
• Demonstrated high-throughput capability in a single round of synthesis and screening.

Conclusions:

• The developed on-chip gene synthesis technology offers a low-cost, high-throughput solution for synthetic biology.
• This platform facilitates systematic investigation of protein translation and the engineering of complex biological systems.
• Enables rapid design, construction, and evolution of macromolecular machines, metabolic networks, and synthetic cells.