Date of Award

12-31-2022

Document Type

Campus Access Thesis

Degree Name

Master of Science (MS)

Department

Biology

First Advisor

Michael Shiaris

Second Advisor

Kim Hamad-Schifferli

Third Advisor

Katherine Gibson

Abstract

Vibrio parahaemolyticus (Vp) is a Gram-negative bacterium found worldwide in estuaries and coastal environments. Vp growth rate is highly impacted by temperature; thus, Vp populations increase from undetectable levels in the water column in the winter to millions per ml in the warm summer months. As the global production of farmed shellfish, specifically oysters, continues to rise so does the incidence of Vp infections. When contaminated shellfish are ingested raw, symptoms take effect within 12 to 24 hours causing gastrointestinal distress and, in some severe cases, convulsions and death. The pathogenicity of Vp is largely related to their coordinated communication system of quorum sensing (QS). When the bacterial concentration reaches a critical threshold, or “quorum”, it releases hormone-like compounds called autoinducers (AIs) to alter gene expression. Due to the high incidence of infections, the major objective of this thesis was to create an E. coli plasmid to act as a bacterial reporter to detect quorum sensing of Vp.Constructed using the Gibson Assembly method, the reporter plasmid contains components of the AI-1 quorum sensing system modeled after Vibrio fischeri with the goal of having β-galactosidase activity responsive to the presence of acylated homoserine lactones (AHLs). Preliminary data showed non-specific expression of β-galactosidase without exposure to Vp. This behavior was minimized with the addition of glucose to the growth medium as glucose works as an “off switch” for the lac operon. The presence of glucose in all growth conditions allowed for either Vp or purified AHL to act as an activator for the bacterial reporter. lacZ encodes the β-galactosidase enzyme that cleaves the synthetic compound X-gal, producing a blue pigment that is observed on agar plates and in liquid media. Therefore, when exposed to Vp, the reporter system specifically produces a blue pigment in the presence of Vp. The next steps are to adapt the assay to an inexpensive paper strip modality.

Comments

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