Harnessing Nature's ability to create membrane compartmentalisation through redesign of a protein machinery.

A biological cell can be thought of as a complex chemical reactor where vast numbers of interactions are simultaneously taking place; these are vital to its function and survival. To prevent unwanted cross-talk and interference within the "noise" of all these concurrent chemical pathways, a cell compartmentalises these processes localizing different functions within individual membrane-bound structures (organelles). Confinement of chemical processes also allows a cell to maintain incompatible environments that are optimal for each organelle's function, which would not be possible within a single "pot".
If we are to mimic this complexity within synthetic "nanoreactors", or even build synthetic cells capable of useful tasks within areas as diverse as medicine, environmental remediation, or energy capture and storage, we will need to develop ways of mimicking cellular compartmentalisation within synthetic man-made structures. This is the challenge we will address in this project.
Internal membrane bound compartments within living cells are normally generated by molecular machineries made up of 'protein' components. We will redesign the structure of proteins belonging to one of these types of machineries to control its usability with membrane vesicles made in the laboratory. Ultimately, we will use this protein-based biotechnology to create nanoreactors contained within the larger membrane vessel (organelle). Membrane-shaping machineries naturally occurring in higher organisms, such as humans, are composed of many proteins working in concert; obviously such complexity is not currently compatible with large scale laboratory engineering of compartmentalised architectures. Therefore the first aim of our project is to engineer a minimal membrane-shaping protein system working on higher organism membranes. Precedent for this biological simplicity has been observed in some ancient bacteria containing similar membrane-shaping machineries made up of very few protein components. We will mimic this simpler model by reducing the number of functional components of an already well-characterised higher organism complex, through genetic engineering. This protein-based technology will be used to develop multi-compartment architectures, similar to biological cells, with each compartment containing molecules of different sizes and chemical properties. We will learn to control the membrane shaping action of this biotechnology so that we are able to stop and restart the generation of new compartments. This will allow us to control the encapsulation of different components in specified compartments within the larger membrane structure.
A first proof of concept will consist of encapsulating a molecular machinery capable of converting genetic information into new protein molecules inside one single internal compartment, mimicking the function of the cell nucleus. Further work will be conducted to allow selective movement of chemicals between internal compartments within a single vessel. The ultimate product of this project will be a well-characterised, functional toolkit enabling researchers to create complex compartmentalised and communicating nanoreactors. These devices could be used as cell-sized vehicles that simultaneously produce, transport and deliver therapeutic cargo to specific targets in the body. Alternatively, they could be employed as chemical factories that can remove pollutants from water supplies.

In the short term, this work will develop professional researchers with organisational and analytical skills, together with knowledge of communication and scientific writing. Both PDRAs will undertake a range of highly specialised techniques and will be able to positively contribute to both academic research and student training and industry.
The long term impact of this research will be in facilitating development of novel synthetic biology technologies. The development of a minimal protein machinery to control compartmentalisation of different cargoes within membrane architectures will enable the creation of devices that mimics the highly coordinated functionalities of biological cells. These multivesicular architectures will enable encapsulation of parallel chemical processes within separate membrane compartments providing a high degree of structural complexity.
The ability to create smart membrane architectures will positively affect the many industrial sectors where synthetic biology has been projected to make a future impact. These include the pharmaceuticals, environmental monitoring and remediation, green energy and agricultural sectors. This wide-range of applications of synthetic biology technology is why the UK government considers this to be one of the eight great technologies important to the UK's future knowledge-based economy.
While not a direct aim, this research also has the potential to impact upon attempts to enable targeted therapeutic delivery using exosomes. Exosomes are natural nanoscale lipid vesicles that transport biochemical signals such as proteins and RNAs between cells. Recent development in the field of biopharmaceuticals, where biomolecules are proposed as novel drugs, means that there is significant efforts to develop efficient and targeted methods to deliver proteins and nucleic acids into cells. One strategy thought to be of promise is by loading biopharmaceuticals into patient-derived exosomes for delivery. Exosomes naturally form within the cell within the multivesicular bodies created by ESCRT proteins. Therefore our work in engineering ESCRT for compartmentalisation in synthetic biology may reveal some useful techniques that could be applied to the manufacture of artificial exosomes for medicine. If this proves to be the case, this is a potential area of future collaborative research to develop minimal ESCRTs for manufacture of artificial vectors for delivery of biomacromolecules. Exosomes are also implicated in cancer metastasis, therefore artificial exosomes could also be an important tool in cancer research.
Finally, as a new and potentially disruptive field of science, synthetic biology projects can have significant impact in public dissemination of science. The concept of engineering using biology to create new functionalities not naturally found in the biological realm will capture public imagination but also has the potential to raise ethical concerns. By engaging the public as a whole with a real, leading edge synthetic biology project we can help maintain a rational dialogue dispelling the misinterpretation of ideas, concepts and goals that have impacted upon other new areas of science in the past. As tax-payers providing funding for UK scientific research, we are all an important stake-holder that needs to be directly engaged with goals and outputs of the work they are helping fund in order to maintain public support for science.