Difference between revisions of "Team:NYU Abu Dhabi/Description"

Why is STEC a major concern?

Globally, 1 in every 10 people is affected by foodborne diseases each year. According to 2010 data, 600 million were affected by such diseases and 420,000 died as a result. Countries of low- and middle-income are the most affected due to unsafe practices of food production and storage. Enteropathogenic E. coli is one of the main causes of death due to foodborne disease. Shiga toxin-producing Escherichia coli (STEC) causes over 70,000 infections per year in the United States alone. A portion of these individuals will experience kidney failure after 6 days, 50% of which will require renal replacement therapy.¹

What is the mode of action?

Shiga toxin is an exotoxin comprised of a toxic A subunit and a cell-binding B subunit. The B subunit binds to a globotriaosylceramide Gb3 receptor. This receptor is expressed on the surface of cells. Binding of the B subunit to the Gb3 receptor causes the shiga toxin to be endocytosed into the cell. Once inside, shiga toxin inhibits protein synthesis and induces apoptosis. [4]

What are the symptoms of STEC infections?

Shiga toxin-producing E. coli (STEC) is one of the most prevalent enteropathogenic E. coli strains. In the US alone, this strain causes 70,000 infections a year. The most common symptom of such infections is diarrhea. Even more worrying is the fact that 6-9% of those cases derived in life-threatening hemolytic uremic syndrome (HUS). 5-7% of the individuals affected by HUS did not survive. [5]

What are we doing?

We have produced a rapid, affordable and portable device that allows for the detection of Shiga toxin-producing Escherichia coli using loop-mediated isothermal amplification (LAMP).

How are we doing it?

LAMP is a highly specific, efficient and rapid DNA amplification technique that uses 4-6 primers that bind to 6-8 distinct regions of target DNA. This technique was shown to be more specific than colony PCR without the need for heat lysis or centrifugation steps. The selectivity of our system was tested using the rfbE gene, a non-toxic coding sequence required for O157-antigen synthesis. The presence of the O-antigen confers resistance against phagocyte killing.

The samples are contained in a PDMS chip that contains wells for positive control, negative control, and three samples. Heating is supplied by a Peltier Modular Cooling. The reaction temperature of 65˚C was achieved using the Peltier system supplied with a 6V, 1.5A external power supply. The reaction was visualized under UV and blue light. It is envisioned that a smartphone will suffice for capturing the output of the reaction. [6]

Why E.coLAMP?

iGEM NYU Abu Dhabi 2016 Project

Award: Silver medal

In many developing countries, people depend on reasonably priced and conveniently available street food. However, lack of action taken by governments to regulate street food vendors has led to the prevalence of severe street food-related illnesses. One of the primary microbial contaminants in street food is E. coli O157:H7, which acts by secreting the Shiga-like toxin (SLT). Currently, there is no detection method for SLT outside of a lab setting, thus putting the consumers of foods at risk. Our project aims to develop a device that would be used by street vendors and restaurant owners to verify the safety of their products. Through our device, we exploit the binding of Gb3 to subunit B of the Shiga toxin, and compare the migration pattern of the bound Gb3-subunit B complex to a non bound subunit B. A shift in the migration pattern on a PAGE gel will occur when Gb3 is bound, indicating the presence of the toxin in the food sample. If no shift occurs in the SLT migration pattern, this implies the absence of the toxin within the sample, and reflects the safety status of the food.

Our improvements

Shiga toxin is an exotoxin that consists of two subunits. Subunit B binds to Gb3 receptor expressed in the surface of target cells and permits the entry of subunit A, which inhibits protein synthesis.² 2016 Team NYU Abu Dhabi exploited the binding of Gb3 to subunit B to detect for the presence of STEC. Their prototype compared the migration pattern of a bound Gb3-subunit B complex to that of free subunit B using a PAGE gel. Their device was estimated to take 45 minutes and their prototyping process ran into several issues that negatively impacted the specificity, affordability, and accessibility of the product. However, based on feedback from food vendors, we discovered that very few individuals would wait so long for to obtain results. Since they also had issues with expressing their protein of interest, we decided to target a DNA sequence for our device.