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Single-step linker-based combinatorial assembly of promoter and gene cassettes for pathway engineering

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Abstract

A linker-based approach for combinatorial assembly of promoter and gene cassettes into a biochemical pathway is developed. A synthetic library containing 144 combinations, with 3 promoters and 4 gene variants, was constructed for the ackA and pta genes of the acetate utilization pathway in E. coli. The in vitro isothermal assembled library was then introduced into E. coli mutant (acs-, pta-, ackA-) and selected for restoration of acetate utilization. 81% of the colonies screened contained the complete functional pathway. Thirty positive clones were analyzed and accounted for 10% of the 144 promoter–gene combinations.

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Acknowledgment

This work was supported by Synthetic Genomics, Inc (SGI).

Conflict of interest

H.O.S. is Co-Chief Scientific Officer, a member of the Board of Directors of SGI, and holds stock in SGI.

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Authors and Affiliations

  1. J. Craig Venter Institute, Synthetic Biology Group, 10355 Science Center Drive, San Diego, CA, 92121, USA

    Adi Ramon & Hamilton O. Smith

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  1. Adi Ramon
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  2. Hamilton O. Smith
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Correspondence to Adi Ramon.

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Ramon, A., Smith, H.O. Single-step linker-based combinatorial assembly of promoter and gene cassettes for pathway engineering. Biotechnol Lett 33, 549–555 (2011). https://doi.org/10.1007/s10529-010-0455-x

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  • DOI: https://doi.org/10.1007/s10529-010-0455-x

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