A Molecular Device for Tuneable Evolution

Evolution is a unique property of biological systems and a challenge for Engineering Biology. It can
play a double role: On one hand it can be undesirable, as evolution may lead to mutations
inactivating an engineered function. On the other hand, evolution creates variation which can lead
to improving functions of interest or create new ones.

In this project we will exploit this dual role to create a molecular tool that can operate in a cell to
both prevent mutations resulting in loss of function and generate diversity for directed evolution
on demand. This will be achieved by using retrons. Retrons are DNA elements that encode for a
reverse transcriptase and a single-stranded DNA/RNA hybrid (msDNA) 1 . Once the retron is
transcribed, the reverse transcriptase generates the msDNA and a fragment of it gets integrated
back into the lag strand of the bacterial chromosome by recombination.
Retrons can be engineered and used to target a region of interest in the DNA of bacteria. They
have been used for DNA storage 2 , as they generate a record of cellular events in the form of
permanent modifications to the DNA. Moreover, because the msDNA sequence can harbour up to
80 bp of a cargo DNA, retrons can be used for direct mutagenesis of a target sequence 3 or to edit a
site of interest by recombination. We have preliminary results demonstrating the in vivo
mutagenesis in Escherichia coli of the rpoB gene encoding the beta subunit of the RNA
polymerase. Our results show that a wild-type rpoB can be replaced by a retron-borne segment
conferring resistance to the antibiotic rifampicin. Conversely, we have shown that mutations in the
gene rpoB can be rescued with a retron carrying the wildtype sequence of the gene

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.