A platform for rapid and precise DNA module rearrangements in Synthetic Biology

Recently, a new field of science has emerged called Synthetic Biology, which aims to apply engineering principles (for example, the use of modular components, and a "design-build-test-modify" approach to improvement) to the development of biological systems for useful purposes. One major target in Synthetic Biology is the creation of genetically modified microorganisms, to produce valuable chemical substances economically, in high yield and with low environmental impact, or to carry out beneficial chemical transformations such as neutralization of pollutants in waste water. To create these organisms, it is often necessary to introduce a set of new genes (encoded in DNA sequence) and assemble them in specified positions within the organism's long intrinsic DNA sequence ('genome'). The genetic techniques currently available for this 'assembly' task are still quite primitive and inadequate, and gene assembly is considered to be a serious bottleneck in the work leading to the development of useful microorganisms. The first main aim of our proposed research programme is to establish a sophisticated new methodology for this gene assembly process which will achieve a step-change in the speed and efficiency of creating new microorganism strains. For this purpose we will adapt a remarkable group of bacterial enzymes called the serine integrases, whose natural task is to carry out this kind of genetic rearrangement but which have hitherto been underused as tools for Synthetic Biology. We will design rapid, robust and efficient ways of making gene cassettes that can be slotted in (using serine integrases) to any one of a number of different specified positions ('landing pads') in genome DNA. By doing this we can assemble collections of genes to order within a particular microorganism. Furthermore we can choose where to place the genes in the genome and in what order, and replace any individual parts with different versions. This permits much easier optimization of complex genetic systems than is currently possible. Using our new methods we intend to engineer microbial cells to make next-generation biofuels, to make chemicals for the plastics industry by microbial fermentation instead of by using fossil fuel, and to synthesise new antibiotics.
A second major target in Synthetic Biology is to make 'smart cells' that can respond in clever ways to external signals (for example, light, high temperature, or a chemical in their environment), or that can 'remember' if they have been exposed to a particular signal and how many times. These smart cells could thus be switched on to perform a useful function only when we need it, or could be programmed to carry out an ordered series of tasks, rather like the wash-rinse-spin-dry cycles of a washing machine. The serine integrase-based tools that we will create for gene assembly lend themselves to the construction of simple yet highly effective intracellular devices for detecting and counting signals. So a second part of our programme is to show the way to the design and construction of these memory devices, and prove that they can work in the way we envisage.

We aim to establish a versatile, widely applicable site-specific recombinase-based platform to facilitate rapid DNA assembly and rearrangement. Bacteriophage-derived serine integrases will be used to establish a platform of DNA assembly tools that can be used to apply engineering principles to a wide range of microorganisms. Our platform will rely on the high efficiency, specificity and especially unidirectionality of the bacteriophage serine integrases. Our first key objective is to assemble a collection of up to 20 fully orthogonal serine integrase systems, along with their recombination sites (attP, attB, attL, attR) and recombination directionality factors (RDFs), the presence or absence of which specifies attL x attR or attP x attB recombination, respectively. Our approach will be to characterize these systems (a) from published examples that have been shown to have recombination activity, and (b) by identifying new temperate phages with serine integrases from environmental samples. Using these systems we will establish methodologies for precise assembly and substitution of genes and their regulatory components in vivo and/or in vitro, and to facilitate pathway assembly in industrially important microorganisms including E. coli, Streptomyces, Aspergillus and yeast species. Specifically, we aim to engineer a model pathway for carotenoid biosynthesis in E. coli, and, in collaboration with industrial partners, pathways for production of ethanol, polymer intermediates, and antibiotics. Pathway design will be supported by predictive mathematical modelling. We will also demonstrate the promise and potential of these systems for construction of genetic memory devices, including "binary" counting circuits based on integrase-mediated inversion, that can be coupled to make devices to count up to large numbers (>1000).

The first to benefit from the outputs of our research programme will be others working in the field of Synthetic Biology, both in the academic and industrial sectors. Our SIDR platform will provide new methodologies for metabolic engineering, allowing the rapid construction, testing and optimization of novel metabolic pathways for the production of high-value industrial and platform chemicals in microbial cell factories. We envisage that we will thereby dramatically enhance the speed and economic efficiency of research and development in this field, allowing industrial researchers to progress towards full-scale production much faster than is currently possible with existing gene assembly techniques, and to be able to afford much more advanced assembly projects than was hitherto feasible. Similarly, the platform will enable academic researchers to construct experimental systems and achieve their research aims much more quickly and effectively than before. BBSRC has identified Synthetic Biology as a field which could supply substantial economic benefits for the UK, and our technology will enable rapid progress of those seeking to realize these benefits. This programme will therefore have a directly beneficial effect on the competitiveness of the UK economy within a relatively short timescale (years rather than decades), by stimulating activity in the Synthetic Biology sector, as well as supporting global advances in economic activity in this area.
To achieve imaximum impact we will investigate the possibility of setting up a spin-out company using IP derived from this project, in order to optimize production and dissemination of the materials we generate through the SIDR platform.
The specific projects that we plan to undertake together with our industrial partners should lead directly to improved biosystems for production of ethanol, polymer precursors, and antibiotics, benefiting the Industrial Biotechnology sector. As the field of Synthetic Biology progresses, it is expected that intervention in cellular processes to achieve the desired outcomes will become more and more complex and sophisticated, and the biosensors and counting systems that we aim to develop in this programme are likely to become essential tools to reach the required levels of control. The economic benefits of these systems are likely to be slightly further in the future than the benefits of our pathway assembly tools, but ultimately may be very substantial, opening up new ways to control and actuate useful biological processes.
The project will have an impact on training in the new field of Synthetic Biology, as a significant number of people will be exposed to new enabling new technologies, and to how to apply them. Training and education will be at all levels; postdoctoral researchers, Ph.D students, undergraduates and schools. A major objective of our impact plan is to stimulate young scientists to take up biological science and in particular Microbiology as a career path by meetings, talks, iGEM projects, SAW projects etc. Outreach activities to the general public will also be undertaken with the aim of achieving a greater understanding of the potential of Synthetic Biology.
The ultimate beneficiaries will be the public UK and worldwide, who will gain access to novel, cheaper and cleaner products from industrial biosystems. By their nature, some of these products are likely to impact on the Healthcare sector; for example, pharmaceutical products and biomaterials for therapy. Biological production of certain chemicals will reduce our dependence on dwindling fossil hydrocarbon resources as feedstocks and allow for cleaner, environmentally friendly production methods. The UK public will also gain directly if we have a lead in this sector bringing economic wealth to the nation, as well as enjoying better health and new or cheaper consumer products.