Generation Of An Amplifiable Biosensor Based On Conjugation For Detection Of Salmonella In Food Samples
Master of Science
Physiology and Pharmacology
Rieder, Michael J.
Foodborne illness caused by ingestion of pathogens represents a significant, increasingly complex global health challenge. The gold-standard of food pathogen detection is becoming outdated, representing a reasonable target for reducing foodborne illness. The work in this thesis aims to develop and characterize an amplifiable biosensor system based on conjugation from a non-pathogenic E. coli to Salmonella for detection in food samples. The assay functions via conjugation of a plasmid pDETECT to Salmonella, inducing production of luxI and homoserine lactones, which induce fluorescent readout via pLux-driven transcription of thermal green protein (TGP) in an E. coli sensor. With optimized conditions for conjugation and fluorescent readout, the estimated current limit of detection of the assay is 104-105 CFU/mL in spiked food samples, with a total assay time ranging between 11-13 hours.
Summary for Lay Audience
Foodborne illness (FI), commonly known as ‘food poisoning’, is caused by consuming contaminated food. Contamination can be caused by several factors, but most cases are caused by bacteria. Your food’s journey from the farm to your kitchen table is a complex one, involving imports and exports from all over the globe, handling by many different people and equipment, and distribution networks with many moving parts. All these factors provide ample opportunity for bacterial contamination of food products and/or equipment used in food production. Food producers perform tests to evaluate whether contamination has occurred, but they are limited in their capacity to provide fast, reliable results. The work in this thesis aims to develop a test that can address these limitations. The potential design uses non-harmful (NH) E.coli to detect harmful bacteria in food samples. This thesis investigates the potential utility of this test using Salmonella, a widespread bacterium and one of the most common causes of FI. The proposed design relies on a DNA communication system between NH E.coli and Salmonella. E.coli delivers a piece of DNA to Salmonella in the sample, which leads to the production of messengers in Salmonella. These messengers relay a signal back to E.coli, triggering a cascade of signals that ultimately results in production of thermal green protein (TGP) that can be measured to estimate levels of contamination. This research begins by assessing TGP production by the DNA communication system, and determining optimal conditions to produce a fast, strong signal. Next, the transfer of the DNA signal between E.coli and Salmonella is optimized. Finally, the system was put to the test by artificially spiking leafy greens with Salmonella – and established initial proof of principle that the assay design could detect concentrations >10,000 Cells/mL with a total assay time 11-13 hours. Future work will focus on making this system specific for Salmonella and trying to improve assay sensitivity.
Keer, Kyra, "Generation Of An Amplifiable Biosensor Based On Conjugation For Detection Of Salmonella In Food Samples" (2023). Electronic Thesis and Dissertation Repository. 9143.
Available for download on Monday, January 01, 2024