Combining in vitro peptide display and in vivo directed evolution to search for entirely de novo folds of DNA-binding domains

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This project will take recent advances in protein evolution methods to develop entirely novel DNA binding domains as a basis for new synthetic transcription factors in genetic regulation. The development of new-to-nature protein functionality remains a significant challenge even given recent computational advances in protein structure determination. To address this we will take an experimental approach that combines CIS peptide display for the in vitro expression and selection of massive libraries (Patel et al. Protein Engineering, Design & Selection 26, 307-315, 2013), with phage-based in vivo selection (Brodel et al. Nature Communications 7, 13858, 2016), which gives exponential amplification of genetically selectable traits.At 66 amino acids, lambda cro is perhaps the smallest known transcription factor that functions in E. coli, and it can be converted into an activator (Brodel et al. Science Advances 6: eaba2728, 2020). Therefore, providing an unstructured gene sequence coding for 70 - 100 aa, could in principle provide a reasonable starting point for de novo evolution of a transcription factor. The ~1091 - 10130 amino acid combinations seem daunting, but that is also why it is a fascinating question to ask whether the compounding advantages of evolution, which we will employ, will lead to an exponential selection trajectory. The project will take a staged approached to ensure viable progression and development of the techniques during the project. Initially, we will screen large libraries of structured but non-DNA binding sequences based on familiar DNA regulatory elements (e.g. helix-turn-helix), using in vitro CIS display. Subsequently, this will be expanded to create massive libraries of unstructured proteins to screen for novel DNA binding scaffolds. DNA binding domains from these studies will then be developed as transcription factors using further protein engineering and in vivo accelerated evolution using a phage-based microbial gene drive as well as a novel technique for accelerating protein evolution through gene-directed DNA damage. The outcomes will be further analysed through biochemical, computational and structural approaches.