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 l 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.© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. the chances of propagating nonfunctional library members because of multiple infections. A GOI with the desired characteristics upregulates gene VI expression on the AP, completing the phage life cycle. For example, a randomized TF library member that activates an artificial promoter upstream of gVI will increase its own phage production (Fig. 4a). In this way, a protein with novel desired properties can be selected after several rounds of reinfection. Applications of the methodThe method has been used to evolve a set of dual activator-repressor switches for orthogonal logic gates, based on bacteriophage λ cI variants, and multi-input promoter architectures, and these switches have been successfully applied in downstream synthetic gene circuits 19 . In general, the method is capable of evolving any gene-or gene circuit function-on the PM that can be linked to pVI production. This is analogous to previous uses of phageassisted continuous evolution (PACE) 22 (Fig. 4). With PACE, a wide range of medically and biotechnologically relevant biomolecules, including polymerases 22 , proteases 23 and genome-editing proteins 10 , as well as protein-protein interactions 24 , were linked to conditional M13 phage propagation. In principle, any application in which directed evolution approaches have been...

Accelerated evolution of a minimal 63–amino acid dual transcription factor

“…To obtain a set of small transcriptional activators derived from  Cro, we converted our phagemid-based batch selection system (12,15) for accelerated continuous evolution in a manner similar to phageassisted continuous evolution (PACE) (16,17). One difference between our system and classic PACE is that we use phagemids rather than phage to facilitate screening large libraries.…”

Programmable T7-based synthetic transcription factors

“…Considerable effort has been directed to expanding the pool of orthogonal bacterial repressors through mutagenesis, bioinformatics-driven parts mining and directed evolution ( 13 ). This can provide repressors that bind new inducers ( 14–17 ) as well as bind new operator sites ( 18 , 19 ).…”

Intein-assisted bisection mapping systematically splits proteins for Boolean logic and inducibility engineering

“…The limited performance of the two inducible intein from this study could also be improved via directed evolution 23, 32, 49, 58, 59 , which can mutate these inteins such they splicing with better efficiencies and with lower basal activities, and allow them to perform sufficiently well when inserted into other host proteins. We foresee a powerful combination between domain-insertion/acVHH-assisted bisection mapping and directed evolution, where the former identifies the optimal sites for differential activities.…”