Team:Bio Without Borders/Description

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Project motivation

Our team is from New York City and the Atlantic Ocean is essentially in our backyard. Horseshoe crabs are seen a lot throughout the shores especially in Jamaica Bay. Unfortunately, throughout the years the horseshoe crab population has been decreasing. We wondered why this was happening and the reason why this was happening is that pharmaceutical companies are harvesting one third of the HorseshoeCrab Blood since their blood is good at detecting endotoxins by Limulus Amebocyte Lysate (LAL) in injectable medicines, which is required by the FDA.

Defining the project challenges

Even though the patent on Factor C assay has expired and new recombinant methods have been created — for example, Lonza bioscience has created an rFC assay — the assay still remains monopolized and inaccessible (in order to be able to view the Lonza protocol, you have to request permission from the company). It is also inaccessible in the sense that the protocol is very demanding in terms of the dollar amount, and requires expensive equipment and reagents. Therefore, the challenge of our project was to create a more accessible and easy-to-use diagnostic tool with the rFC in a way where we could visualize the self-cleavage of the protein on a simpler system, such as a paper strip.

How did we combat the challenges

We decided that our best course of action would be to create a two-part system with Limulus Factor C and a second protein that would function as a reporter. We envisioned a fusion protein between green fluorescent protein (GFP) and a cellulose binding domain (CBD), linked together by a short sequence of amino acids that includes the cleavage site from the natural substrate of Factor C (Factor B) consisting of an Arg-Ser. When applied to paper the CBD should anchor this protein. The hope was that when Factor C encountered endotoxin, it would undergo self-cleavage into its proteolytic active form and act on the linker, freeing the GFP to diffuse away from the paper (or potentially wicked up a strip giving a visual signal under a blacklight flashlight). We also decided to use the Pichia pastoris yeast strain in order to express our product, as the yeast directly releases the protein without having to lyse or sift through damaged cell parts.