Development of sensitive reporter phage systems for detection of bacteria
Date
2022-08
Authors
Wijeratne, Shalini
Major Professor
Advisor
Talbert, Joey
Acevedo, Nuria
Shaw, Angela
Gomes, Carmen
Dickson, James
Committee Member
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Abstract
Reporter phage assays are a promising technique for bacterial detection in many fields, especially in food and water quality assessments. However, additional development and engineering of these assays are required when considering the high sensitivity expected by these test standards. Therefore, the goal of this research was to improve the sensitivity of phage-based bacteria detection assays by enhancing the understanding and application of reporter systems. To achieve this goal, two studies were carried out. In the first study, alkaline phosphatase (ALP*)/DynaLight™ and NanoLuc luciferase (NLuc)/Nano-Glo® luminescence-based reporter systems were compared through an engineered T7 reporter phage-E. coli detection assay to determine which is the more sensitive system and why. Based on the results, the NLuc reporter system was determined to be more sensitive than the ALP* reporter system. The enhanced sensitivity of NLuc reporter system is attributed to the greater reporter expression following host infection of the reporter phage and the low detection limit of the NLuc enzyme to Nano-Glo®. In the second study, a heterobifunctional reporter was designed and engineered as an alternative to enzyme-based reporter systems. The designed reporter consisting of SNAP-tag fused with monomeric streptavidin was evaluated on its ability to get immobilized and labeled as a phage-expressed reporter following host infection. Results indicated that this reporter could be incorporated into T7 phage genome, expressed, captured on to magnetic particles, and labeled using biotinylated horseradish peroxidase. The engineered system enabled the detection of E. coli and proved to be a promising technology due to its versatility and potential to be integrated with various established protein detection methods to develop sensitive reporter phage systems. Collectively, this research presented in this dissertation provides new knowledge and solutions to advance the sensitivity of phage-based detection assays.
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dissertation