Date of Award

Spring 2011

Document Type

Restricted Thesis

Terms of Use

© 2011 Nicole C. Machac. All rights reserved. Access to this work is restricted to users within the Swarthmore College network and may only be used for non-commercial, educational, and research purposes. Sharing with users outside of the Swarthmore College network is expressly prohibited. For all other uses, including reproduction and distribution, please contact the copyright holder.

Degree Name

Bachelor of Arts

Department

Biology Department

First Advisor

Amy Cheng Vollmer

Abstract

Bdellovibrio bacteriovorus is a predatory Gram-negative bacterium whose life cycle consists of a highly mobile attack phase and an intraperiplasmic growth phase. While in attack phase, B. bacteriovorus seeks other Gram-negative bacteria which serve as their prey and subsequently invades the prey's periplasm, thus entering the intraperiplasmic growth phase. B. bacteriovorus quickly kills the prey, consumes the available nutrients, grows into a filament which septates into progeny and causes lysis of the prey cell in order to enter attack phase again. B. bacteriovorus has been shown to translocate a porin which resembles outer membrane proteins (OMPs) found in other species, to the prey's cytoplasmic membrane following invasion as a method for obtaining nutrients from the prey cell. The gene Bd0427 has been determined to encode a protein of similar weight and isoelectric point as the translocated protein. In order to study the connection between the Bd0427 gene expression and the translocated protein, we have devised a reporter system using bioluminescence. The Bd0427 promoter has been PCR-amplified and ligated in front of a promoterless luxCDABE operon carried on the pUCD615 plasmid. This construct has been transformed into Escherichia coli and subsequently into B. bacteriovorus. Bioluminescence assays have been conducted with both species to determine if the cloned sequence functions as a promoter in both E. coli and B. bacteriovorus and can direct transcription of luxCDABE. It was found that exogenous addition of a substrate for the luxCDABE protein product was necessary in order for B. bacteriovorus HI to produce bioluminescence. B. bacteriovorus cells were synchronized such that assays at specific points in the life cycle could be performed. These assays suggest Bd0427 is always expressed but may increase throughout the time period measured. Due to many difficulties, this study has shown that the luxCDABE system may not be the ideal method of studying gene expression in B. bacteriovorus. Other methods must be developed in order to study gene expression in an effective manner.

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