Controlling de novo-designed protein-protein interactions within cells

Using rational and computational protein design methods (1,2,3,4), the Woolfson group has produced a tool box of de novo-designed protein-protein interaction (PPI) domains. To date, these have mostly been built, characterised and used in vitro. Recently, however, the Woolfson and Savery groups have collaborated to port these designs directly into living cells for applications in synthetic and cell biology. This project aims to build on this firm base with the overall objective of producing switchable PPI systems for in-cell applications.
Many PPIs that control cellular processes are regulated through post translational modifications (PTMs). In eukaryotes one of the most prevalent PTM is phosphorylation, the transfer of a phosphate group to specific amino acid residues. However, this type of PTM appears to be much less prevalent in prokaryotes (5), offering a potential opportunity to create orthogonal control systems in such organisms.
Recent work conducted by the Woolfson and Savery groups has demonstrated that phosphorylation can be used to successfully disrupt de novo-designed PPIs in vitro (unpublished). The work that I carried out thus far has aimed to transfer this system into bacterial cells, and we have promising initial results. Alongside this, various other PPIs with potential for interruption are being investigated and designed. Generally, this involves incorporating kinase and phosphatase recognition motifs within the PPI designs. The next step is to express the requisite kinase in E. coli and demonstrate that the system functions appropriately; i.e., that expression of the kinase leads to phosphorylation of the target de novo proteins, and subsequent disabling (or enabling) of the PPIs. We will also investigate any impact of kinase expression on growth rate and morphology of cells.
Following on from this, the next focus of the project will be to investigate various mechanisms for maintaining tight and selective control of kinase expression, and to integrate the kinase encoding cassette into the bacterial chromosome, using recombineering or CRISPR-based genome editing methods. The dynamics of this system will be thoroughly examined to ensure that it functions in a robust manner, likely utilising the liquid-handling robotic platforms available in the BrisSynBio Biosuite for high throughput testing. The overall aim of this aspect this aspect of the project will be to produce an E. coli strain that can be used to test promising switchable de novo PPI designs for a range of applications in cells.
The first application where we will implement this switchable PPI system is as an orthogonal transcription-control apparatus, progressing the on-going collaboration between the Savery and Woolfson groups. This offers an ideal starting point, given the system has already been well characterised in E. coli. Through the controlled expression of each component it should be possible to build up new signalling networks with useful and unique dynamics. Real-time testing of these systems will be possible using microfluidic devices. However, this is just one conceivable opportunity for such a system to be employed, and others will be explored throughout the project; e.g., the switchable assembly and disassembly of large, functional protein assemblies (e.g., microtubules and microcompartments) in cells. These ideas align with the joint directions of the Woolfson and Savery groups generally, as they are interested in forging a new field of de novo protein design in the cell. Switchable devices produced that demonstrate the required characteristics will find uses in the expanding field of synthetic biology, cell biology and biotechnology.
The interdisciplinary approach of this project, harnessing the discrete strengths of both the Woolfson and Savery groups, will result in novel research methodologies and output. This project falls within the EPSRC Synthetic Biology research area and aligns with its research objectives.

The emerging and dynamic field of Synthetic Biology has the potential to provide solutions to some of the key challenges faced by society, ranging across the healthcare, energy, food and environmental sectors. The UK government has recently a "Synthetic Biology Roadmap", which presents a vision and direction for Synthetic Biology in the UK. The report projects that the global Synthetic Biology market will grow from $1.6bn in 2011 to $10.8bn by 2016. It highlights that there is an urgent need for the UK to develop the interdisciplinary skills required to take advantage of the opportunities provided by Synthetic Biology.

The challenge to the academic and industrial research communities is to develop new translational approaches to ensure that these potential benefits are realised. These new approaches will range across the design and engineering of biologically based parts, devices and systems as well as the re-design of existing, natural biological systems across all scales from molecules to organisms. The techniques will encompass not only individual cells, but also self-assembled biomimetic systems, engineered microbial communities and multicellular organisms, combining multiple perspectives drawn from the engineering, life and physical sciences.

Realising these goals will require a new generation of skilled interdisciplinary scientists, and the training of these scientists is the primary goal of the SBCDT. Our programme will give the breadth of coverage to produce a "skilled, energized and well-funded UK-wide synthetic biology community", who will have "the opportunity to revolutionise major industries in bio-energy and bio-technology in the UK" (David Willetts, Minister for Universities and Science) in their future careers. This will be made possible through genuine inter-institutional collaboration in partnership with key industrial, academic and public facing institutions.

The potential impact of the SBCDT, and its potential national importance, are very therefore high, and the potential benefits to society are significant.