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

Nature Biotechnology volume 29, pages 449–452 (2011)Cite this article

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Abstract

Low-cost, high-throughput gene synthesis and precise control of protein expression are of critical importance to synthetic biology and biotechnology1,2,3. Here we describe the development of an on-chip gene synthesis technology, which integrates on a single microchip the synthesis of DNA oligonucleotides using inkjet printing, isothermal oligonucleotide amplification and parallel gene assembly. Use of a mismatch-specific endonuclease for error correction results in an error rate of ∼0.19 errors per kb. We applied this approach to synthesize pools of thousands of codon-usage variants of lacZα and 74 challenging Drosophila protein antigens, which were then screened for expression in Escherichia coli. In one round of synthesis and screening, we obtained DNA sequences that were expressed at a wide range of levels, from zero to almost 60% of the total cell protein mass. This technology may facilitate systematic investigation of the molecular mechanisms of protein translation and the design, construction and evolution of macromolecular machines, metabolic networks and synthetic cells.

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Figure 1: The integrated on-chip oligo array synthesis, amplification and gene assembly process.
Figure 2: Expression of synthetic lacZα codon variants in E. coli.
Figure 3: Optimization of protein expression.

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Acknowledgements

We thank Z. Chen, K. Ma and Q. Wang for excellent technical contributions. The project was supported by the Beckman foundation, The Hartwell Foundation, and a Coulter-Duke translational partnership award to J.T.

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Author notes

  1. Jiayuan Quan and Ishtiaq Saaem: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA

    Jiayuan Quan, Ishtiaq Saaem, Nicholas Tang, Siying Ma, Hui Gong & Jingdong Tian

  2. Institute for Genome Sciences and Policy, Duke University, Durham, North Carolina, USA

    Jiayuan Quan, Ishtiaq Saaem, Siying Ma & Jingdong Tian

  3. Institute for Genomics and Systems Biology and Department of Human Genetics, The University of Chicago, Chicago, Illinois, USA

    Nicolas Negre & Kevin P White

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  8. Jingdong Tian
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Contributions

J.Q. performed gene library synthesis, expression screen and protein purification experiments. I.S. performed on-chip oligo and gene synthesis experiments. N.T. and J.Q. performed colony imaging and image analysis. S.M., I.S. and J.Q. performed error correction experiments. N.N. and K.P.W. provided wild-type transcription factor sequences and clones. H.G. and J.T. designed gene library sequences. J.T. designed overall strategy and supervised the project. All authors contributed to the manuscript preparation.

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Correspondence to Jingdong Tian.

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The authors declare no competing financial interests.

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Supplementary Tables 1–3, Supplementary Figs. 1–4, Supplementary Sequences and Supplementary Note (PDF 698 kb)

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Quan, J., Saaem, I., Tang, N. et al. Parallel on-chip gene synthesis and application to optimization of protein expression. Nat Biotechnol 29, 449–452 (2011). https://doi.org/10.1038/nbt.1847

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