Beth's Proposal

TITLE
The Role of Toxin-Antitoxin Gene Pairs in Cell Death/Cell Survival in Escherichia coli


ABSTRACT
Toxin-antitoxin pairs are pairs of genes that encode the information necessary for the generation of a toxin and an antitoxin. Within a healthy cell, toxin and antitoxin proteins are simultaneously produced, and bind together to form a complex that is harmless to the cell; however, in an abnormal cell, the toxin protein accumulates, disrupting normal cellular functions and ultimately causing cell death. For this reason, toxin-antitoxin pairs have been implicated as key components of programmed cell death mechanisms and the plasmid addiction system (a mechanism in which a cell dies if it does not receive a copy of a small, circular piece of DNA called a plasmid). Understanding the basic mechanisms underlying programmed cell death and plasmid addiction is an important step in gaining insight into disorders/diseases caused by uncontrolled cell proliferation like cancer, and may yield important molecular tools for initiating death in specific, undesired cells in laboratory experiments.

The aim of this project is to generate a miniature database documentary about toxin-antitoxin pairs, and more specifically toxin-antitoxin pairs in the bacterium Escherichia coli. This will be a referential and educational tool that includes a review of toxin-antitoxin pair literature, as well progress on toxin-antitoxin pair research being conducted in the Finkel laboratory at the University of Southern California.


PROJECT DESCRIPTION
DEFINITION OF PROJECT
Genre and Delivery Format
Data presentation is one of the most important aspects of the scientific process, regardless of whether the project is a literature review or laboratory-based research. The publication of papers and the presentation of works at conferences and symposiums allow researchers to present their findings to the scientific community, as well as the general public. This generates a forum for the discussion and exchange of ideas, which is inherently important to scientific progress.
There are three primary modes of presentation in the sciences: paper publications, oral presentations, and poster presentations. Despite the inherently scholastic nature of science, which demands careful note-taking, attention to detail, and the analysis and interpretation of data, there are many aspects of science that are better explained through images, especially for lay audiences or those unfamiliar with the research topic. These components include physical, chemical, or physiological mechanisms, experimental procedures, and data. For instance, it is much easier to understand how a neurotoxin can cause paralysis via a schematic drawing or an animation, than it is to read a dense passage detailing each step in the process. Likewise, it is much easier to understand data presented as a graph than as a list of numbers.

For this reason, this project will be a multimedia-based project in the form of a database documentary. It is critically important to maintain a textual, scholarly component in the presentation of this project because of the scholastic nature of the research: at the core of this project are a literature review of toxin-antitoxin pairs in the bacterium Escherichia coli and data collected from ongoing research. However, there are also multiple components of this project that are far better served by visual aids, including pictures, animations, interviews, and data. A database documentary will allow for the incorporation of scholastic and multimedia components, as well as allow the audience to explore and learn about toxin-antitoxin pairs in E. coli at their leisure and personal level of understanding.

Research Methodology
The specific aim of this research project is to conduct an extensive literature review of toxin-antitoxin pairs in the bacterium Escherichia coli, and compile the information in a cohesive and understandable multimedia format that is accessible to both the layperson and the professional researcher. This project will be comprised of five major components: (1) general information about toxin-antitoxin pairs, (2) toxin-antitoxin pairs in E. coli, (3) proposed theories for the role of toxin-antitoxin pairs in E. coli death and/or survival, (4) potential applications of toxin-antitoxin pair research, (5) progress with research that I am conducting in the laboratory.

The first component of this project will present a broad overview of toxin-antitoxin pairs – the background information. It will begin with two basic concepts that are crucially important to toxin-antitoxin pair action, programmed cell death and plasmid addiction (a mechanism in which a cell dies if it does not receive a copy of a circular piece of extra-genomic DNA called a plasmid). The discussion will continue with the identification of toxin-antitoxin pairs on bacterial genomes, what they are, how they are thought to act, and their potential significance for cell death or survival.

The second component of this project will concentrate on the main focus of this project, toxin-antitoxin pairs on the E. coli genome. This section will include information about each of the toxin-antitoxin pairs identified in E. coli, their locations on the E. coli genome, their putative modes of action, and other pertinent information.

The third section will be focused on the major theories regarding the purposes of these toxin-antitoxin pairs in E. coli. The theories proposed by the two major camps of toxin-antitoxin pair research, the Gerdes laboratory and the Engelberg-Kulka laboratory, will be discussed here, as well as the theory proposed by the Finkel laboratory.

The fourth, and final section of the literature review, will be concerned with the potential applications of toxin-antitoxin pair research. Understanding the mechanisms of toxin-antitoxin pairs is a major step forward in understanding programmed cell death mechanisms, and possibly the mechanisms underlying the termination of programmed cell death abnormal proliferation of cells (e.g. cancer). Additionally, toxin-antitoxin pairs have the potentially of being a very useful molecular technique that can be used to terminate certain, unwanted cells in controlled experiments.

The final component of this project will be concerned with my own research. This section will consist primarily of the protocols and processes that are involved in my research, with a little bit of data. Realistically, there will be little data to present, as current research endeavors are focused on the generation of mutant E. coli strains required for the study of toxin-antitoxin pairs.


PROJECT GOALS
The primary goal of this multimedia project is to generate a miniature database of information regarding toxin-antitoxin pairs in Escherichia coli. This will be an interactive project that can be used as both a source of reference for people conducting research on toxin-antitoxin pairs in E. coli and an educational experience for people of all educational backgrounds who have an interest in learning about toxin-antitoxin pairs.

To accomplish this goal, the information contained within this project will be made accessible to both the layperson and the professional researcher. a researcher can browse through the project, looking at current information and research about toxin-antitoxin pairs and bypassing explanations and images that describe basic knowledge (e.g. transcription and translation), while a lay person can browse through the project, bringing up annotations (textual and/or visual) that explain concepts they do not know or understand.


SIGNIFICANCE
Toxin-antitoxin pairs have been implicated as key components of two important cell death mechanisms, programmed cell death and the plasmid addiction system. Programmed cell death (PCD; apoptosis) is a normal regulatory process that allows for the destruction and removal of unneeded or damaged cells. PCD results from highly regulated biochemical processes that are activated by a variety of mechanisms, including damage to DNA and external chemical signals, and trigger death. In the plasmid addiction system, a type of programmed cell death, a toxin-antitoxin pair resides on a circular piece of DNA (called a plasmid) in the cytoplasm of a bacterial cell. During replication, those bacterial cells that do not receive the plasmid undergo cell death, while those that do receive the plasmid survive and replicate. Understanding these mechanisms are extremely important in diseases where cell proliferation goes unchecked, such as cancer, because in many of these diseases, there is abnormal suppression of PCD.

It has been recently discovered that these toxin-antitoxin pairs not only reside on plasmids, but are also integrated into the genomes of many bacterial species. The Escherichia coli genome has been found to contain at least ten of these toxin-antitoxin pairs at seven different locations. The incorporation of these toxin-antitoxin pairs into the E. coli genome suggests they play an important function in regulating cell death and / or cell survival. Utilizing E. coli provides us with a highly accessible way of gaining insight into the mechanisms underlying cell death / cell survival mechanisms.


TIMELINE
27 July 2007 Written / visual draft of all necessary project components
28 September 2007 Draft of all textual aspects of this project submitted to Biology Advisor
26 October 2007 Final draft of all textual aspects of this project
28 December 2007 Completed compilation of audio / visual components
29 February 2008 Rough draft of final multimedia project
28 March 2008 Second draft of final multimedia project
18 April 2008 Third draft of final multimedia project
May 2008 Final draft of multimedia project


BUDGET
To be determined.


CONFIRMED ADVISORS
Steven Finkel, Ph.D.
Assistant Professor
Molecular and Computational Biology
University of Southern California
1050 Childs Way
Ray Irani Hall 319B, Mail Code 2910
Los Angeles, CA 90089-2910
Phone: (213) 821-1498
Email: sfinkel@usc.edu


PRESENTATION PLANS
This project will be presented at the end of the Spring 2008 semester, as a part of the Institute for Multimedia Literacy Honors Program. This multimedia project will be accompanied by a written thesis that will be submitted to the University of Southern California Biology Department for a letter grade.


PRODUCTION RESOURCES
SOFTWARE / HARDWARE
Literature review and laboratory research will be conducted in Ray Irani Hall in the USC Department of Molecular and Computation Biology. The multimedia aspect of this project will be primarily Macromedia Flash-based. Animations and images will be downloaded from the Internet or they will be generated in Macromedia Flash or Microsoft PowerPoint, and interviews and videos of the experimentation process will be shot and captured and edited in Final Cut Pro.


COLLABORATORS
Finkel Laboratory, University of Southern California
Journal of Visualized Experiments (Tentative)


PRESENTATION NEEDS
This multimedia project will be designed to be viewed on a computer and the final project will be delivered in Flash on DVD.


BACKGROUND INFORMATION
Toxin-antitoxin (TA) pairs are gene pairs that coexpress a stable toxin and a labile antitoxin (Brown and Shaw, 2003). Within the cell, toxin and antitoxin proteins bind to form a toxin-antitoxin complex that is harmless to the cell; however, when free toxin is allowed to accumulate in the cell (via disruption of TA transcription and/or translation, deletion of the TA pair, etc.), it dies. For this reason, TA pairs have been implicated as key components of programmed cell death mechanisms and the plasmid addiction system, though the modes of action of most TA pairs remain undetermined.

Ten toxin-antitoxin pairs have been identified at various loci on the E. coli genome, and seem to be associated with cell death mechanisms triggered by large chromosomal deletions in long-term cultures. Preliminary studies suggest that these toxin-antitoxin pairs play a key role in cell survival, as E. coli strains mutant for seven TA pairs put into direct competition with the wild type strains show reduced competitive ability and large population reductions (Finkel laboratory, unpublished).

REFERENCES
Brown, J. M., and K. J. Shaw. 2003. A Novel Family of Escherichia coli Toxin-Antitoxin Gene Pairs. J. Bacteriol. 185(22): 6600-6608.

Datsenko, K. A., and B. L. Wanner. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97:6640-6645.