Our Sensor

The goal of our project is to design a biosensor to rapidly identify cattle known as super shedders. Super shedders are cattle that excrete extremely large numbers of E.coli O157:H7 a subgroup of E. coli referred to as Enterohemorrhagic E. coli (EHEC). EHEC organisms produce a toxin called Shiga toxin or verotoxin. This toxin binds to blood cells and lyses them resulting in hemolytic-uremic syndrome (HUS) in humans and can be deadly. Supershedders excrete in the range of 10 ⁷ to 10 ¹⁰ E. coli O157:H7 exceeding normal cattle by 3-6 logs and can contaminate other cattle in the same holding pen as well as the meat downstream. Super-shedders are thought to be the reason for 95% of the E. coli O157:H7 contamination in the industry.

We are building a DNA-based biosensor that specifically detects the gene called stx2. Our device will be useful for the detection of not only E.coli O157:H7 but also other EHEC strains. Click on the components below to learn more about their design and function. View our animation below to see how the system would actually work!

The FerriTALE System:

As seen in the video above our goal is to develop a strip based assay that can alert us upon detection of pathogenic DNA sequence, namely Stx 2. We will immobilize a TALE-Ferritin scaffold (a FerriTALE!) to a nitrocellulose strip. The TALE protein that has been immoblized will bind to a unique oligonucleotide sequence that is approximately 18-20 base pairs in size on the Stx 2 gene. This immobile component of our system (Figure 1) will effectively capture our target DNA on the strip. A second TALE attached to our reporter (Prussian blue ferritin or β-lactamase) that targets a different unique sequence on the Stx 2 gene will then be run along the nitrocellulose as a mobile component. The TALE-reporter complex will only bind if the second target DNA sequence is present. Following this we will add a substrate solution and if both of the TALE proteins are bound to the Stx 2 gene we will see a colour output on the strip.

A Platform Technology for iGEM

TALEs are modular and can be used platform technology implying TALEs can be engineered to detect any DNA of interest. We are using TALE proteins as our sensory element. TALEs are very powerful tools since they can be engineered to bind to any 12 to 31bp sequence of DNA (source: Golden Gate TALEN and TAL Effector Kit 2.0). Therefore, by just swapping the TALEs in our PLASMID XYZ, one can detect any DNA of interest. To further ease this process, we have incorporated a KasI restriction cut site at the end of all TALEs in all the constructs. Using the KasI restriction enzyme and one of the biobrick prefix enzymes one can incorporate their own engineered TALE into the part, and use it for detecting their DNA of interest.