Masters Theses

Date of Award

8-2003

Degree Type

Thesis

Degree Name

Master of Science

Major

Microbiology

Major Professor

Gary Sayler

Committee Members

John Sanseverino, Pamela Small, Stephen Oliver

Abstract

In light of the recent anthrax attacks of 2001in the United States, the need for a rapid, sensitive, and real-time surveillance system for detecting pathogenic bacteria has become readily apparent. A proof-of-principle study involving development of a bacteriophage-based bioreporter system for the detection of Bacillus anthracis spores has been initiated in the surrogate strain Bacillus thuringiensis 4Q7. The final goal of the project is to develop an engineered bacteriophage with the luxI gene from Vibrio fischeri, which upon infection of the target organism, would produce the signal molecule, N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), capable of inducing bioluminescence in a neighboring OHHL bioluminescent bioreporter cell. Studies done in this project involve expression of a plasmid borne luxI gene in B. thuringiensis, quantification of OHHL produced, and examination of the effect of the OHHL degrading enzyme lactonase found in B. thuringiensis.

The luxI gene was expressed under a xylose-inducible promoter in B. thuringiensis 4Q7. This strain was capable of producing a detectable amount of OHHL within 3 hours post-induction as determined by bioluminescent assays with the OHHL bioreporter, RoLux. In addition, OHHL concentrations of approximately 500 nM were reached during late-log phase growth of the luxI engineered B. thuringiensis strain. Other aspects of the study included optimization of the detection of OHHL with the RoLux bioreporter. Finally, the aiiA gene, or lactonase, from B. thuringiensis 4Q7 was detected, cloned, and sequenced, and determined to be functional. Assays involving incubation of OHHL with growing cultures of B. thuringiensis provided evidence that the degradation of OHHL by lactonase would not have an effect on detecting the molecule from the engineered strain at early log-phase of growth.

Recommendations for advancement of this project are made, along with future work in this area is provided.

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