From 2012.igem.org

Project Overview

Abstract

Diarrhetic pathogens including E.coli O157:H7 serotype, Campylobacter, Shigella, and Salmonella often contaminate drinking water supplies in developing nations and are responsible for approximately 1.5 million worldwide annual deaths. Current technologies for detection of bacteria include DNA hybridization FRET signaling, electrical detection via immobilized antimicrobial peptides, and PCR amplification followed by gel visualization. Our method of bacterial detection fills a niche in biosensor technology. Our design implies lower costs, higher portability, and a more rapid signal output than most bacterial biosensors. Additionally, our interchangeable DNA probe confers modularity, allowing for a range of bacterial detection. Using a novel split beta-galactosidase complementation assay, we have designed three unique chimeric proteins that recognize and bind to specific pathogenic markers and create a functioning beta-galactosidase enzyme. This functioning enzyme unit then cleaves X-gal and produces a colorimetric output signal. Our research demonstrates success in initial stages of chimeric protein assembly.

Synopsis of Project

Diarrheic pathogens account for approximately 1.5 million annual deaths worldwide with most of these incidents occurring in third-world countries. Unfortunately, there are currently no biosensors available that can detect for these pathogens in a fast, cheap, and efficient manner. As such, Arizona State University’s 2012 iGEM team aims to develop a water-borne pathogen biosensor that is cheap, portable, robust, easily customizable, and produces a quick response. Our vision is to build a user-friendly device that does not require any technical expertise to operate.

To achieve both specificity and portability in our biosensor, our team is constructing two biosensors, each capable of detecting a specific pathogenic marker in water-borne bacteria. The sensor design is made of three parts: the enzyme, the linker, and the sensor. Both of the systems are based on a split-enzyme engineered protein that induces a colorimetric response in the presence of pathogens. In the first system, the split-enzyme engineered fusion protein selectively binds to the membranes of pathogenic bacteria in water samples and induces a colorimetric response. The second system will detect specific DNA sequences in pathogenic bacteria and activate a similar colorimetric change.

Figure 1: In the absence of pathogens, the split-enzyme system will not come together, and there would be no colorimetric response.

Figure 2: In the presence of pathogens, the split-enzyme system will come together, and there will be a blue colorimetric output.

In the end, we wish to create a biosensor production pipeline, where the sensors are expressed in E. Coli, purified, and added to a heat-treated water sample.

Figure 3: Biosensor Production Pipeline