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.
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This work was supported by US Office of Naval Research Multidisciplinary University Research Initiative grant no. N00014-16-1-2388 (T.H.S.-S., E.D.S., and C.A.V.), US National Institutes of Health National Institute of General Medical Sciences Center for Integrated Synthetic Biology grant no. P50-GM098792 (T.H.S.-S. and C.A.V.), US National Institutes of Standards and Technology grant no. 70-NANB16H164 (T.H.S.-S. and C.A.V.), US National Science Foundation Synthetic Biology Engineering Research Center grant no. EEC-0540879 (C.A.V.), and a Fannie and John Hertz Fellowship (T.H.S.-S.).
The authors declare no competing financial interests.
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Segall-Shapiro, T., Sontag, E. & Voigt, C. Engineered promoters enable constant gene expression at any copy number in bacteria. Nat Biotechnol 36, 352–358 (2018). https://doi.org/10.1038/nbt.4111
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