Engineered promoters enable constant gene expression at any copy number in bacteria

Thomas H. Segall-Shapiro, Eduardo D. Sontag, Christopher A. Voigt

    Research output: Contribution to journalArticlepeer-review

    125 Scopus citations

    Abstract

    The internal environment of growing cells is variable and dynamic, making it difficult to introduce reliable parts, such as promoters, for genetic engineering. Here, we applied control-theoretic ideas to design promoters that maintained constant levels of expression at any copy number. Theory predicts that independence to copy number can be achieved by using an incoherent feedforward loop (iFFL) if the negative regulation is perfectly non-cooperative. We engineered iFFLs into Escherichia coli promoters using transcription-activator-like effectors (TALEs). These promoters had near-identical expression in different genome locations and plasmids, even when their copy number was perturbed by genomic mutations or changes in growth medium composition. We applied the stabilized promoters to show that a three-gene metabolic pathway to produce deoxychromoviridans could retain function without re-tuning when the stabilized-promoter-driven genes were moved from a plasmid into the genome.

    Original languageEnglish (US)
    Pages (from-to)352-358
    Number of pages7
    JournalNature Biotechnology
    Volume36
    Issue number4
    DOIs
    StatePublished - Apr 1 2018

    ASJC Scopus subject areas

    • Biotechnology
    • Bioengineering
    • Applied Microbiology and Biotechnology
    • Molecular Medicine
    • Biomedical Engineering

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