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Intracellular directed evolution of proteins from combinatorial libraries based on conditional phage replication

Abstract : Directed evolution is a powerful tool to improve the characteristics of biomolecules. Here we present a protocol for the intracellular evolution of proteins with distinct differences and advantages in comparison with established techniques. These include the ability to select for a particular function from a library of protein variants inside cells, minimizing undesired coevolution and propagation of nonfunctional library members, as well as allowing positive and negative selection logics using basally active promoters. A typical evolution experiment comprises the following stages: (i) preparation of a combinatorial M13 phagemid (PM) library expressing variants of the gene of interest (GOI) and preparation of the $Escherichia\ coli$ host cells; (ii) multiple rounds of an intracellular selection process toward a desired activity; and (iii) the characterization of the evolved target proteins. The system has been developed for the selection of new orthogonal transcription factors (TFs) but is capable of evolving any gene—or gene circuit function—that can be linked to conditional M13 phage replication. Here we demonstrate our approach using as an example the directed evolution of the bacteriophage $\lambda$ cI TF against two synthetic bidirectional promoters. The evolved TF variants enable simultaneous activation and repression against their engineered promoters and do not cross-react with the wild-type promoter, thus ensuring orthogonality. This protocol requires no special equipment, allowing synthetic biologists and general users to evolve improved biomolecules within ∼7 weeks.
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Contributor : Bruno Savelli Connect in order to contact the contributor
Submitted on : Tuesday, October 16, 2018 - 11:06:42 AM
Last modification on : Tuesday, January 25, 2022 - 3:31:23 AM

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Andreas Brödel, Alfonso Jaramillo, Mark Isalan. Intracellular directed evolution of proteins from combinatorial libraries based on conditional phage replication. Nature Protocols, Nature Publishing Group, 2017, 12 (9), pp.1830 - 1843. ⟨10.1038/nprot.2017.084⟩. ⟨cea-01896489⟩



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