Development Of Novel Genetic Circuits For The Detection Of Disease Biomarkers
The US Centers for Disease Control and Prevention (CDC) estimates chronic diseases account for 86% of the annual health care costs in the US. Monitoring and intervention of chronic diseases such as cardiovascular disease, diabetes, and arthritis, are required to decelerate their progression. Some markers of these diseases are secreted into the intestine, providing an alternative monitoring approach. The development of bacteria based environmental sensors, or bioreporters using synthetic biology techniques provides a novel approach to monitor disease biomarkers. In the studies presented here, we developed two novel bioreporter systems using genetic circuits to monitor disease biomarkers. We identified the peptide fragments of the outer membrane protein, OprF of Pseudomonas aeruginosa which bind interferon-[gamma] (IFN-[gamma]) and interact with tumor necrosis factor-[alpha] (TNF-[alpha]). We engineered sensors for IFN-[gamma] and TNF-[alpha] as fusion proteins of OmpA/OprF, expressed in Escherichia coli. The phage shock protein A, pspA promoter was used for the first time to transduce the interaction of the markers with the OmpA/OprF sensor into a quantifiable signal, measured as [beta]-galactosidase activity. This novel detection system for IFN-[gamma] and TNF-[alpha] was capable of detecting these inflammatory markers. Subsequently, the detection systems were tested in an in vitro model of intestinal barrier dysfunction. Direct application of the bioreporter systems to the intestinal "lumen" portion of the model demonstrated strong detection of the cytokines which diffused through the permeable barrier, showing the promise for the potential advancement to use in vivo models. The threshold detection concertation for the bioreporter systems was in the active range observed in stool samples of patients with Inflammatory Bowel Disease. Secondly, we developed a detection system for elevated concentrations of uric acid, which are implicated in gout, a form of arthritis. We used the transcriptional regulator, PucR of the purine catabolism pathway of Bacillus subtilis to develop a synthetic promoter responsive to uric acid, driving the production of GFP. With this novel system we could differentiate between healthy and hyperuricemic concentrations of uric acid observed in the gut. The sensor systems we developed in these studies provide an essential contribution to the development of future diagnostic systems and active therapeutics.
Synthetic Biology; IFN-gamma; TNF-alpha
Ley,Ruth E.; King,Cynthia A.
Food Science & Technology
Ph.D. of Food Science & Technology
Doctor of Philosophy
dissertation or thesis