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Genome-scale promoter engineering by coselection MAGE

Nature Methods volume 9, pages 591–593 (2012)Cite this article

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Abstract

Multiplex automated genome engineering (MAGE) uses short oligonucleotides to scarlessly modify genomes; however, insertions >10 bases are still inefficient but can be improved substantially by selection of highly modified chromosomes. Here we describe 'coselection' MAGE (CoS-MAGE) to optimize biosynthesis of aromatic amino acid derivatives by combinatorially inserting multiple T7 promoters simultaneously into 12 genomic operons. Promoter libraries can be quickly generated to study gain-of-function epistatic interactions in gene networks.

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Figure 1: CoS-MAGE strategy for enriching highly modified genomes.
Figure 2: Indigo production from synthetic T7 promoter–regulated operons.

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Acknowledgements

We thank J. Keasling, A. Juminaga, P. Carr, S. Kosuri, J. Aach and T. Gianoulis for helpful discussions and data interpretation, H. Salis (Pennsylvania State University) for providing pJ401, and C. Voigt (Massachusetts Institute of Technology) for providing pN249. This work was funded by multiple programs from the US National Science Foundation (SynBERC, SA5283-11210), the Department of Energy (Genome to Life Center, DE-FG02-03ER6344) and the Wyss Institute for Biologically Inspired Engineering. Additional funding was from the Next-Generation BioGreen 21 Program (SSAC, PJ008109), Rural Development Administration and the Intelligent Synthetic Biology Center of Global Frontier Project funded by the Ministry of Education, Science and Technology (2011-0031956), Korea. H.H.W. is supported by the Wyss Institute Technology Development Fellowship and the US National Institutes of Health Director's Early Independence Award (grant DP5OD009172). J.J. is supported by the National Junior research fellowship from the National Research Foundation of Korea (2012-0000391).

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Author notes

  1. Harris H Wang and Hwangbeom Kim: These authors contributed equally to this work.

Authors and Affiliations

  1. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, USA

    Harris H Wang, Le Cong & George M Church

  2. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA

    Harris H Wang

  3. Department of Chemistry, Yonsei University, Seoul, Korea

    Hwangbeom Kim, Jaehwan Jeong & Duhee Bang

  4. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA

    Le Cong & George M Church

  5. Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, Massachusetts, USA

    Le Cong

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  1. Harris H Wang
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  2. Hwangbeom Kim
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Contributions

H.H.W., H.K., D.B. and G.M.C. designed the study. H.H.W., H.K., L.C. and J.J. performed the experiments. H.H.W., H.K. and D.B. analyzed the data and prepared the initial manuscript. All authors edited and revised the final manuscript.

Corresponding authors

Correspondence to Harris H Wang or Duhee Bang.

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Competing interests

G.M.C. has advisory roles, royalties and/or equity holding in several synthetic biology companies.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Tables 1 and 3, Supplementary Note (PDF 911 kb)

Supplementary Table 2

Summary of coselection markers available in EcNR2 (MG1655 derivative). (XLSX 13 kb)

Supplementary Table 4

Oligo and primer sequences used in the study. (XLSX 14 kb)

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Wang, H., Kim, H., Cong, L. et al. Genome-scale promoter engineering by coselection MAGE. Nat Methods 9, 591–593 (2012). https://doi.org/10.1038/nmeth.1971

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  • DOI: https://doi.org/10.1038/nmeth.1971

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