Light-responsive building blocks for synthetic biology

Synthetic biology is an emerging area of research that aims to mimic, modify or redesign natural systems. Its embodiments range from expanding the genetic code with orthogonal synthetic bases, designing synthetic genetic circuits, and metabolic engineering, to building artificial cells and designing synthetic molecular machines from modified natural components. A key element in a number of these endeavours is the creation of 'bioparts'; robust, well-characterized modules that encapsulate particular functions and can be combined by known rules to design a more complex component. Interactions between proteins and DNA and related molecules underpin many of the goals of synthetic biology, whether the target nucleic acid strand is used to encode genes or as a designed structural or recognition element. Therefore, a clear need exists for DNA-binding bioparts that can be combined with other modules to provide nuclease or transcription factor like behaviour. We believe a light-responsive DNA-binding biopart would be of great utility in providing environmental sensing, guiding or the ability to pattern or direct the action of synthetic biology constructs.

We will base our design on the solution of the structure of the bacterial protein EL222, which binds to DNA only in its light activated state. EL222 consists of a light-sensitive LOV domain and a C-terminal DNA-binding domain. LOV domains are found in a variety of blue-light sensing proteins in bacteria, fungi and plants. They bind a molecule of FMN, which serves as the light-sensing chromophore. Absorption of blue light by this molecule is conducive to reaction between FMN and an amino acid of the protein part forming a covalent photo-adduct, which in turn activates a C-terminal effector domain. Irradiation with blue light releases the C-terminal domain that can then bind to DNA.

We will design a hybrid between EL222 and the catalytic domain of the restriction endonuclease FokI, a well understood biopart which has been linked to zinc finger proteins and transcription like effector (TALE) domains to create highly specific designer endonucleases, thereby creating a photo-responsive nuclease that can be activated in cells with high spatial and temporal resolution. We will adapt an existing directed evolution assay to provide a tool to optimize the effectiveness of such fusions. With further work, such photonucleases will also be valuable tools to study the function of genes in live cells and have huge potential clinical utility to correct mutations in human disease in a spatially defined fashion improving biosciences underpinning health.

Interactions between proteins and DNA are vital for regulating many processes of life and are equally important in modifying or redesigning these processes. We wish to create a 'biopart' that binds to DNA when illuminated with blue light, and to modify it so that it can be readily combined with other off-the-shelf domains to create proteins with new functions for top-down or bottom-up synthetic biology approaches.

Recently, hybrid restriction enzymes comprised of the FokI DNA cleavage domain and a designed zinc finger protein for sequence specific DNA recognition have been developed as highly versatile reagents for genetic modification. We propose to exemplify the usefulness of our light-responsive DNA-targeting domain by combining it with FokI endonucleases to induce sequence specific DNA double-strand breaks. As well as having the potential for the efficient photo-directed gene disruption in cells for light-controlled gene therapy, adding nuclease activity permits the use of robust directed evolution experiments to optimize existing properties or incorporate new ones.

To provide additional information for rational design of photoactive fusion proteins, we will solve the first structure of a photoactivated state of a DNA-binding LOV domain protein by X-ray diffraction; we will focus on EL222, which upon irradiation with blue light undergoes a structural change to form a DNA binding dimer. Due to its short half-life, no structure of the photoactivated state has so far been reported. Preliminary work in our lab has shown that replacement of the cofactor FMN with 5-deazaFMN leads to the photoactivated state being stable for several weeks whilst retaining its ability to bind to DNA in a 2:1 protein:DNA complex.

This structural work will reveal the spatial relationship of the DNA binding domains of the dimeric state, which we will to help investigate the scope for modulating the DNA binding selectivity of the fusion proteins.

A wide range of academic researchers will benefit from this work, as detailed under 'academic beneficiaries'. In addition, the Researcher Co-I, student and technician working on the project will gain excellent training, providing scientific knowledge and skills as well as wider transferable skills suitable for future employment in a range of sectors.

Synthetic biology has been identified as a strategic priority by the BBSRC and our research would also be complementary to other sections of the 'Exploiting new ways of working' strand. Systems approaches to bioscience might be improved by providing data input to systems biology studies, e.g. by photomodulation of metabolic flux to calibrate mathematical models, and this work clearly represents a technology development for the biosciences. Synthetic biology itself has a pathway to impact set out by the RCUK Synthetic Biology Roadmap for the UK document; in particular bioparts and other enabling tools and construction rules are required to be developed in the 2012-2015 timeframe. This roadmap is geared towards a national capability to translate synthetic biology research into reducing healthcare costs through improved biotechnological production methods and smarter pharmaceuticals and to address issues relating to food security and future oil shortages.

We will obtain fundamental knowledge of structural changes to EL222 on photoactivation, and apply this knowledge to the creation of a modular biopart that can be combined with other 'off-the-shelf' domains in a rational manner to create more complex assemblies. The UK economy will benefit most obviously from commercialization of products and systems arising from this research, as demonstrated by the successful commercialization of zinc finger nucleases and TALENs. By disseminating the results from our research directly to relevant biotechnological companies in the UK, this will give UK companies a competitive edge over their global competitors and should help attract pharmaceutical research and development back to the UK. This in turn will increase both employment and tax revenue. There is also the potential for more subtle benefit to the economy, as improved healthcare will reduce losses in productivity.

The creation of other photocontrollable enzymes and proteins will have applications in a range of biotechnological sectors including health (through improved medicines and drug delivery platforms, particularly as spatial and temporal control of a biopharmaceutical's activity would be expected to reduce off-target effects and therefore help ease passage through clinical trials), agriculture (through new methods of genetic modification of crop plants and the potential for photocontrollable crop treatment agents, which would reduce environmental impact) and chemical manufacturing including bioenergy (through greater control of biocatalytic processes and resulting improvements in efficiency and waste reduction). Photonucleases themselves may find application as treatments for cancer, by specifically targeting mutated oncogenes. The ability to induce sequence specific DNA double-strand breaks using photonucleases may also have applications in light-controlled gene therapy.

The wider public will also benefit through improvements to healthcare technologies, benefitting patients and their families, and through job creation in the biotechnology industries, which will increase access to skilled and therefore more highly paid employment. Financial benefit to the UK as a whole through tax revenues arising from commercialization of this research will lead to improvements to public services. In addition, the planned outreach activities will provide information on how publicly funded research in academia affects biotechnology, a topic of interest to many.