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 renal cells and lyses them, resulting in hemolytic-uremic syndrome (HUS) in humans and can be deadly. Supershedders excrete in the range of 10⁷ to 10¹⁰ 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 two regions of the shiga toxin (stx2) gene, present in not only E.coli O157:H7, but other EHEC strains as well. This means our detector will not only be specific to one specific strain of EHEC, but instead target a broad group of EHEC organisms. 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 stx2. We will treat our sample collected from the supershedders with a TALE-Ferritin complex (a FerriTALE!). This DNA-TALE-Ferritin complex would then be flown over our strip that contains a second, immobilized TALE that recognizes yet another 18-20 bp region on the stx 2 gene. This second TALE will immobilize our DNA-TALE-ferritin complex on the strip. Following immobilization we will add substrate to our strip. Only in the presence of both the EHEC sequences we get a colour change on the strip. If there is only one of the sequences present, the colour change does not occur either due to lack of immobilization or due to lack of presence of a reporter.

FerriTALEs: A platform technology

TALEs are modular DNA binding proteins for which the nucleotide binding amino acid code has been solved. This means through alterations of the protein primary structure in regions called repeat variable di-residues (RVD), the nucleotide-binding region, we can easily engineer the TALEs for practically any sequence of interest.

We also added E and K coils to the registry. These coils are synthetic binding domains that only bind to each other with an extremely high-affinity. Fusing these coils with larger proteins allows the proteins to fold properly because these coils are small and therefore do not interfere with the tertiary structure of the proteins. Fusing these coils with our detector and reporter system allows for easy swapping of the components such that the final system can be customizable.

Because the customizability of this protein relies on the nucleotide sequence, we added KasI restriction sites to the 3’ end of the TALEs, upstream of the coils in a fusion backbone. This backbone could then be used by other teams to swap both the promoter and TALEs, making the nucleotide-binding sequence and expression level tunable by teams using it. In addition, the TALE protein is typically expressed in eukaryotes, indicating the presence of a Kozak sequence. This Kozak sequence interfered with the protein’s expression in bacteria, therefore we removed it to make expression in bacteria viable.