Team:Bio Without Borders
BioTech Without Borders
iGem 2018
Our Project
Our 2018 iGEM project focuses on one of the most ancient of Earth’s inhabitants: the horseshoe crab. The horseshoe crab is a type of chelicerate arthropod and there are several known species, three of the most popular being the Japanese horseshoe crab, the Singapore horseshoe crab, and the Atlantic horseshoe crab (Limulus polyphemus). The horseshoe crab is a particularly relevant species due to their characteristic blue hemolymph, which contains the copper-yielding hemocyanin protein that is responsible for oxygen transportation and delivery in their bodies. For the last 5 decades, horseshoe crab blood has been harvested for pharmaceutical purposes. The crabs' blood is used to perform the Limulus Amebocyte Lysate (LAL) test, which is widely used for the detection of bacterial endotoxins within injectable medicines. The test relies on the horseshoe crab amebocytes’ ability to react with lipopolysaccharides (LPS), a component of negative-gram bacterial membranes, and while in contact, initiate a protein cascade that ends with coagulation and cellular clotting to trap the bacteria. The amebocytes are able to do this because of the endotoxin-sensitive, intracellular, serine protease zymogen Limulus Factor C. Factor C initiates the transduction pathway and in the presence of LPS, Factor C selectively cleaves 103-Arg, 104-Ser, 105-Ile, and 106-Ile to form Factor B. The active form of Factor B cleaves 98-Arg and 99-Ile in the horseshoe crab pro-clotting enzyme, thereby activating it.
All modern injectable medicines are required by the FDA to be tested for endotoxins and other harmful substances using LAL extracts from horseshoe crab blood. To prepare LAL, companies draw about one-third of an individual crab’s blood and release them back into the wild. Recent data shows that harvesting has contributed to an increase in the mortality rate of the species by as much as 30%, which not only poses a threat to their existence but also disrupts the ecological balance of the coast, affecting other species as well. Our solution to this dilemma is attempting to create a recombinant Limulus Factor C in P. pastoris to minimize the pharmaceutical use of horseshoe crabs.
Overview
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Creating Factor C Protein
We used an NEBuilder kit to combine two sequence that IDT sent us.
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Creating a linker protein of the cellulose-binding domain (CBD) and GFP
This functions as the reporter protein which would demonstrate Factor C's ability to cleave.
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Expressing Factor C through Pichia pastoris
After making our parts, we wanted to express the parts in an organism like Pichia.
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Determining the quality of Factor C
We ran a protein gel to ensure that the factor C gene was indeed inside of P. pastoris and that it was working properly.