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Directed evolution of a synthetic phylogeny of programmable Trp repressors

Abstract

As synthetic regulatory programs expand in sophistication, an ever increasing number of biological components with predictable phenotypes is required. Regulators are often ‘part mined’ from a diverse, but uncharacterized, array of genomic sequences, often leading to idiosyncratic behavior. Here, we generate an entire synthetic phylogeny from the canonical allosteric transcription factor TrpR. Iterative rounds of positive and negative compartmentalized partnered replication (CPR) led to the exponential amplification of variants that responded with high affinity and specificity to halogenated tryptophan analogs and novel operator sites. Fourteen repressor variants were evolved with unique regulatory profiles across five operators and three ligands. The logic of individual repressors can be modularly programmed by creating heterodimeric fusions, resulting in single proteins that display logic functions, such as ‘NAND’. Despite the evolutionarily limited regulatory role of TrpR, vast functional spaces exist around this highly conserved protein scaffold and can be harnessed to create synthetic regulatory programs.

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Fig. 1: Evolution and function of bacterial regulatory programs.
Fig. 2: Evolution of effector specificity by compartmentalized partnered replication.
Fig. 3: Evolution of a regulatory array of effector and DNA operator specificities.
Fig. 4: Orthogonality matrix of evolved TrpR variants for effectors and operators.
Fig. 5: Intramolecular tethering drives enhanced regulatory logic and modularity.

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Acknowledgements

We would like to thank funding from the Air Force Office of Scientific Research (FA9550-14-1-0089) and the Welch Foundation (F-1654). M.P.L. was supported by a National Science Foundation Graduate Research Fellowship (Grant No. NSF/DGE-1346837).

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J.W.E. conceived the project and performed all experiments with assistance from M.P.L. A.D.E., M.P.L., and J.W.E. wrote the paper.

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Correspondence to Jared W. Ellefson or Andrew D. Ellington.

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Ellefson, J.W., Ledbetter, M.P. & Ellington, A.D. Directed evolution of a synthetic phylogeny of programmable Trp repressors. Nat Chem Biol 14, 361–367 (2018). https://doi.org/10.1038/s41589-018-0006-7

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