Cell-free systeMs for synthetic biology: development of characterisation tools and biosensors
Creators: name: Chappell, James Edward
Publisher: name: Imperial College London
Year: 2016

description: Synthetic biology is an application field attempting to apply a rational engineering approach to the re-design of biological systems, to produce valuable and novel biological functions. To date the field has largely focused on the engineering of living systems, most notably prokaryotic organisms such as Escherichia coli but eukaryotic orgnanisms for example Saccharomyces cerevisiae and even mammalian cells. An alternative approach to engineering living organisms is cell-free synthetic biology, whereby complex biological systems are studied in a reduced in vitro experimental setup, omitting the limitations and complexity of living organisms. To date cell-free synthetic biology has been used to study genetic circuits and metabolic pathways within a reduced context. This project aims to explore the use of cell-free transcription and translation reactions for two distinct applications. The first application aims to validate an entirely in vitro approach for the generation and characterisation of DNA regulatory elements. A major limitation of our ability to predictably design biological devices is the limited availability of well-characterised DNA parts from which to build. Current methods to characterise DNA part libraries rely upon living systems and as such are inherently time consuming and low throughput. As an alternative a completely in vitro approach to both the generation and characterisation of DNA regulatory elements in cell-free transcription and translation reactions has been validated which is significantly quicker than current characterisation approaches. First the study of DNA regulatory elements in cell-free systems is validated by the demonstration of a clear correlation between characterisation measurements in vivo and in vitro. Secondly, the importance of DNA template structure for characterisation in cell-free systems is investigated. Finally a completely in vitro method to create circular DNA templates for the characterisation in cell-free systems is developed. The second application explored was the development of in vitro biosensors for the detection of Pseudomonas aeruginosa (P. aeruginosa). P. aeruginosa is a Gram-negative bacterium that is an opportunist pathogen able to infect humans with compromised natural defences. Of particular concern is the infection of cystic fibrosis patients, where once an infection establishes it is rarely eradicated. There is strong evidence that the persistence and resistance of chronic infections is due to the ability of P. aeruginosa to exist as biofilms within the lungs of infected cystic fibrosis patients. In this study biosensors have been designed to detect the presence of quorum sensing signals, a cell-to-cell signalling mechanism that has been shown to be important for the formation and maturation of biofilms in P. aeruginosa. The biosensors were constructed from the endogenous quorum sensing responsive transcription factors and promoters, which are used to produce detectable outputs in response to the presence of quorum sensing signals. These biosensors were tested against purified signaling molecules and demonstrated to be highly sensitive, to have a rapid response and to be specific for the P. aeruginosa variants of quorum sensing signaling molecules. Importantly, it was demonstrated that these biosensors are capable of detecting endogenous levels of signaling molecules from laboratory grown P. aeruginosa. As such this in vitro biosensor could provide a better alternative to current detection methods for the presence and the state P. aeruginosa infections.