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| - | <p>TALEs are modular proteins for which the amino acid to nucleotide binding code has been solved | + | <p><b>TALEs</b> are modular DNA binding proteins for which the amino acid code that binds to each nucleotide has been solved. Additionally, these DNA binding domains are found in repeat sequences called the repeat variable diresidues (RVDs). Since TALEs are well characterized proteins whose nucleotide binding code has been solved they are easy to engineer. To increase the modularity of our system we also added <b>coils</b> at the 3' end of the TALEs. These synthetic E and K coils are small and therefore do not misfold proteins that they are fused to such as TALEs. In addition they bind to each other with high affinity making it easy for the reporter with the opposite coil to interact with the TALE . For our plasmid submitted to the registry with the TALEs (BBa_XXXXX) has a KasI res |
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Revision as of 03:01, 19 October 2013
Our Sensor
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 treat our sample collected from the supershedders with a TALE-Ferritin complex (a FerriTALE!). Then this DNA-TALE-Ferritin complex would be flown over our strip which contains the second 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 TALEs we will see a colour change on the strip.
Figure 1. Our system is composed of both an immobile element on the strip that will capture our target DNA and a mobile element that will report the presence of our target DNA.
FerriTALEs: A platform technology
TALEs are modular DNA binding proteins for which the amino acid code that binds to each nucleotide has been solved. Additionally, these DNA binding domains are found in repeat sequences called the repeat variable diresidues (RVDs). Since TALEs are well characterized proteins whose nucleotide binding code has been solved they are easy to engineer. To increase the modularity of our system we also added coils at the 3' end of the TALEs. These synthetic E and K coils are small and therefore do not misfold proteins that they are fused to such as TALEs. In addition they bind to each other with high affinity making it easy for the reporter with the opposite coil to interact with the TALE . For our plasmid submitted to the registry with the TALEs (BBa_XXXXX) has a KasI res
