Characterisation of cellular assemblies in microfluidic systems (synthetic biology to obtain novel antibiotics and optimized production systems)

The proposed research project forms an integral part of the larger international proposal SYNMOD (Full name: Synthetic biology to obtain novel antibiotics and optimised production systems), which is a collaborative project with five partners in Europe and one in the UK. SYNMOD seeks to integrate the basic science of Synthetic Biology with the necessary bioprocess engineering in order to achieve new and improved antibiotics to overcome the growing problem of antimicrobial resistance. In order to realise this potential, it is necessary to undertake research to develop protocols and technologies that allow to quickly and easily characterise biological parts and cells, and to understand their behaviour in different growth conditions. Developing such technologies and protocols is the goal of this proposed research project. Currently, some tools, such as shaker flasks, are easy to set up for an experiment, but provide relatively little information per experiment. Other tools, such as bench-scale bioreactors with multiple probes, are costly and labour-intensive to set up for an experiment. It is therefore difficult to perform the large number of experiments required to fully understand the behaviour of biological parts and cells. The proposed research addresses this by using micro-fabrication methods to create chambers with gas permeable membranes to provide the oxygen cells need. Through such 'microfluidic' reactor systems for the cultivation of cells have been fabricated, they have mainly been designed to operate in 'batch' mode. This is a reactor operation mode where the culture conditions vary considerably during an experiment. Accordingly, the properties of the cells, such as size, composition, and functional characteristics vary considerably, too. In contrast, in a 'chemostat' operation mode, the cells are kept in the same growth condition, i.e. the cell biomass, the nutrient and the product concentrations in the culture remain constant. This is achieved by a controlled and continuous supply of nutrient combined with a controlled and continuous removal of product and of 'excess' cells. Therefore, in a 'chemostat' operation mode, it becomes possible to correlate a cellular behaviour with a particular growth condition. In this research, we propose to build 'microfluidic' reactor systems that operate in 'chemostat' mode. Once this is solved, it will then become possible to use the general advantages of 'microfluidic' systems, such as parallelisation and automation, to create a cost-effective tool with which cell behaviour can be rapidly investigated and characterised.

The European Collaborative Research Project (CRP) SYNMOD, Synthetic biology to obtain novel antibiotics and optimised production systems, proposes to apply a comprehensive synthetic biology approach to the design and production of novel antibiotic molecules. An important part of the SYNMOD project is to provide thoroughly characterized expression tools for an at least semi-rational assembly of pathways in the future production chassis S. carnosus TM300. These tools will be selected from appropriate libraries by screening and then be subjected to physiologically and statistically rigorous analysis. Such rigorous characterisation must today either be produced in simple flasks or tube batch cultures which provide little information per experiment or in costly and labour-intensive fully controlled bench-scale bioreactors. This leads not only to a limitation on the number of culture conditions that can be investigated in reasonably short time frames but also makes interpretation of the data obtained difficult due to the dynamically changing growth conditions in batch cultures. We propose in this individual project (IP5, Characterisation of cellular assemblies using microfluidic systems) to develop a microfluidic methodology to culture bacteria and cells with dramatically fewer resources and in parallelised and automated configurations. In particular, we will develop microfluidic chemostats to culture Gram-positive S. carnosus clones in steady-state conditions and detect the influence of specific cultivation parameters on expression status via read out of fluorescence due to produced fluorescent proteins. This will lead to complete platforms for rapid statistical analysis of engineered cells under precisely controlled growth conditions. The information on the impact of growth conditions for the different promoter expression strengths will then be transferred back to other PIs of SYNMOD, for example to optimise the pathway designs.

Synthetic Biology (SB) is an emerging multidisciplinary field with very broad economic, social, and ethical impacts on the UK and worldwide. The research proposed in the Individual Project 5 (IP5) will develop underpinning technologies that enable researchers in the SB community to accomplish their specific goals in a more timely and efficient manner. The combination of expertise and technologies in the Collaborative Research Project (CRP) SYNMOD will help SB realise its full economic potential by reducing the effort associated with developing new products. The project is highly integrated in terms of required competencies and work flow. It can be clearly stated that none of the participating countries could launch a SYNMOD-like project with only national groups, because no single country has available the required combined expertise in lantibiotic genetics, staphylococci genetics, microfluidics for cultivation purposes, large-scale lantibiotic production, gene synthesis and ELSI- competence. The European perspective is the only perspective to bring all competencies together. Given the potential practical outcome of the overall project, a set of novel antibiotic molecules effective against prominent pathogens, it is clear that the European perspective has here a special importance. Research into novel antibiotic medicines has been reduced over the last 2 decades and correspondingly current problems with hospital-acquired infections of resistant bacteria are particularly worrying. SYNMOD follows a promising route towards novel antibiotic molecules and demonstrates that interaction at the European level can be an excellent way to address pressing societal problems with direct impact for society. The resulting advances will ultimately benefit the economy, health and the quality of life of Europe and the UK general public and beyond. Furthermore, using this project as a concrete example, IP6 of the CRP will analyze the potential impact of synthetic biology on the safety of biotechnological processes and its ethical implications for our society. These considerations will be shared with the public to institute a constructive dialogue about a potentially transformative novel technology.