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

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.

The 2016 UK Roadmap Bio-design for the Bio-economy highlighted the substantial impact that synthetic biology can bring to the UK and global economies by developing: frontier science and technology; establishing a healthy innovation pipeline; a highly skilled workforce and an environment in which innovative science and businesses can thrive. Synthetic biology promises to transform the UK Bio-economy landscape, bringing bio-sustainable and affordable manufacturing routes to all industrial sectors and will ensure society can tackle many contemporary global Grand Challenges including: Sustainable Manufacturing, Environmental Sustainability Energy, Global Healthcare, and Urban Development. Whilst synthetic biology is burgeoning in the UK, we now need to build on the investments made and take a further lead in training next generation scientists to ensure sustained growth of a capable workforce to underpin the science base development and growth in an advanced UK bio-economy.
This training provided by this CDT will give students from diverse backgrounds a unique synthesis of computational, biomolecular and cellular engineering skills, a peer-to-peer and industrial network, and unique entrepreneurial insight. In so doing, it will address key EPSRC priority areas and Bioeconomy strategic priorities including: Next-generation therapeutics; Engineered biomaterials; Renewable alternatives for fuels, chemicals and other small molecules; Reliable, predictable, and scalable bioprocesses; Sustainable future; Lifelong health & wellbeing.
Advances created by our BioDesign Engineering approach will address major societal challenges by delivering new routes for chemical/pharma/materials manufacture through to sustainable energy, whilst providing clean growth and reductions in energy use, greenhouse gas emissions and carbon footprints. Increased industry awareness of bio-options with better civic understanding will drive end-user demand to create market pull for products. The CDT benefits from unrivalled existing academic-industry frameworks at the host institutions, which will provide direct links to industrial partners and a direct pathway to early economic and industrial impact.

This CDT will develop 80-100 next-generation scientists and technologists (via the funded cohort and wider integration of aligned students at the three institutions) as adept scientists and engineers, instilled with technical leadership, who as broadly trained individuals will fill key skills gaps and could be expected to impact internationally through leadership roles in the medium term. Importantly the CDT addresses key skill-gaps identified with industry, which are urgently required to create and support high value jobs that will enable the UK to compete in global markets. Commercialisation and entrepreneurship training will equip the next generation of visionaries and leaders needed to accelerate and support the creation of new innovative companies to exploit these new technologies and opportunities.

The UK government identified Synthetic Biology as one of the "Eight Great Technologies" that could be a key enabler to economic and societal development. This CDT will be at the forefront of research that will accelerate the clean growth agenda and the development of a resilient circular bioeconomy, and will align with key EPSRC prosperity outcomes including a productive, healthy and resilient nation. To foster wider societal impact, the CDT will expect all students to contribute to public outreach and engagement activities including: open days, schools visits, and science festival events: students will participate in an outreach programme, with special focus on widening participation.

This CDT will contribute to the development of industrial strategy through the Synthetic Biology Leadership Council (SBLC), Industrial Biotechnology Leadership Forum (IBLF), and wider Networks in Industrial Biotechnology and Bioenergy and Professional Institutes.