Abstract

The use of the periplasmic alkaline phosphatase (PhoA) reporter protein from E. coli has been critical for definition of the topology of transmembrane proteins of multiple bacterial species. This report demonstrates development of a PhoA reporter system in B. burgdorferi. Codon usage of the E. coli phoA in B. burgdorferi was analyzed and an optimized version of the gene was obtained. In order to assess the differential activity of the reporter system, two optimized PhoA-fusion construct using B. burgdorferi proteins were engineered: one using the periplasmic protein OppAIV and one using the cytoplasmic protein PncA. The activity of PhoA requires periplasmic localization. The periplasmic OppAIV-PhoA fusion as well as the cytoplasmic PncA-PhoA fusion produced detectable PhoA protein in E. coli and in B. burgdorferi. The periplasmic fusion construct, but not the cytoplasmic fusion construct, resulted in functional alkaline phosphatase (AP) activity in E. coli, as observed by blue colonies on agar plates containing a chromogenic substrate for AP. In contrast, both of the fusion constructs produced limited detectable levels of functional alkaline phosphatase activity in B. burgdorferi, as observed by yellow color change in liquid protein lysate containing a chromogenic substrate for AP. Development of a PhoA fusion reporter system for use in B. burgdorferi will provide a new molecular genetics tool for analyzing the topology of B. burgdorferi transmembrane proteins. These types of studies are critical for understanding the function of B. burgdorferi transport systems and may identify novel molecular approaches for the treatment of Lyme disease.

Notes

If this is your Honors thesis, and want to learn how to access it or for more information about readership statistics, contact us at STARS@ucf.edu

Thesis Completion

2013

Semester

Spring

Advisor

Jewett, Mollie

Degree

Bachelor of Science (B.S.)

College

Burnett School of Biomedical Sciences

Degree Program

Molecular Biology and Microbiology

Subjects

Dissertations, Academic -- Medicine;Medicine -- Dissertations, Academic

Format

PDF

Identifier

CFH0004343

Language

English

Access Status

Open Access

Length of Campus-only Access

5 years

Document Type

Honors in the Major Thesis

Download

Share

COinS