Development of DNA-encoded cell-free biosensors for the detection of bacterial infections

Abstract

This thesis describes the development of cell-free biosensors for the detection of quorum- sensing molecules in human clinical samples. The biosensor devices were comprised of a transcription factor and a regulated GFP reporter output that was expressed in the presence of the target molecule. The genetic circuit was encoded on a DNA plasmid and expressed in a cell-free system, consisting of the basic transcription and translation machinery of Escherichia coli in an open reaction. A biosensor device specific for the 3-oxo-C12-HSL quorum-sensing molecule from Pseudomonas aeruginosa was implemented in cell-free systems and used to measure the presence of acyl homoserine lactones in sputum samples collected from cystic fibrosis patients. Mass spectrometry analysis of sputum samples was also conducted to validate the biosensor results. It was found that not all samples from Pseudomonas-infected patients had detectable levels of AHL, and that the detection limit of the biosensor was greater than the concentration of AHL measured in many of the analysed clinical samples. Optimisation of the sample preparation and cell-free assay improved the ability of the biosensor to detect 3-oxo-C12-HSL in cystic fibrosis sputum. The limitations of the assay were identified so that the biosensor can be further developed into a functional tool for the detection of AHLs in clinical samples. In addition to the clinical testing, two new biosensors were constructed for the detection of the AHL C8-HSL and the intracellular signalling molecule c-di-GMP, and both devices were shown to be functional in cell-free systems.