A Synthetic Biology approach for the development of in vitro based biosensors and tools

View all posters

James Chappell, Kirsten Jensen

Imperial College London, United Kingdom

My PhD project aims to investigate the use of cell-free systems as an alternative chassis for synthetic biology. Two applications have been explored, firstly for the characterisation of DNA regulatory elements and secondly for the development of in vitro biosensors. Both aspects of my project are explained below. Current approaches to characterise DNA regulatory elements are mostly tied to living systems and are inherently time-consuming and low-throughput. I have developed a completely in vitro assay to assemble and characterise libraries of regulatory DNA regulatory regions that is significantly quicker than current in vivo approaches. First a PCR coupled to a USER-ligase reaction is used to generate libraries of DNA regulatory regions within 2-3 hours, omitting the need for transformation into e.coli. These DNA templates are then expressed in a cell-free system for 2-3 hours and characterisation performed. I have demonstrated a correlation for the data collect in cell-free systems to that of living systems for the most frequently used DNA regulatory regions.  The second part of the project is to developed an in vitro biosensor to detect the presence of pathogenic bacterial biofilms. It is based upon a DNA template being expressed within the context of a cell-free systems. This DNA biosensor once expressed is able to detect biomarkers that have been shown to be both essential and present in pathogenic biofilms, and give a detectable output. Currently, a proof of principle has been shown for detection of the formation and presence of Pseudomonas aeruginosa biofilms.