Project Description

Many gram-negative bacteria naturally create compounds called endotoxins, which induce pathological symptoms including septic shock in humans. Limulus Amebocyte Lysate (LAL) testing, the gold-standard endotoxin detection test, is used in virtually every area of biomedical product development. The test is derived from horseshoe crab blood, including coagulation Factor C, the primary effector protein. Many horseshoe crabs die each year due to the bleeding process, straining populations and ecosystems along the US Atlantic Coast and in Asia, where it is less sustainable. Moreover, LAL testing is expensive, creating a barrier to biomedical innovation in low-resource settings. For these reasons, our team sought to synthesize a codon-optimized sequence of Factor C and integrate it into Bacillus subtilis (a gram-positive bacterium) using a pAX01 backbone with a xylose-inducible promoter. In the future, we hope to design a detection mechanism to signal for the cleavage of Factor C and the presence of endotoxin.

What Are Horseshoe Crabs

Horseshoe crabs are large, fourteen legged marine arthropods that grow up to 10 to 20 inches and diet on worms, small mollusks, and algae. They have been around since 450 million years ago and are often called the “living fossils.” Due to the fact that they show few signs of genotypic evolution, they are incredibly useful and interesting creatures.

Atlantic horseshoe crabs (Limulus polyphemus) can be commonly found along the Atlantic coast of North America from Nova Scotia to the Yucatan Peninsula near the Gulf of Mexico. Other species like the red knot, ruddy turnstone, and sanderling rely on horseshoe crab eggs for nutrients. Additionally, the horseshoe crab serves as a mini ecosystem, housing algae, worms, and barnacles on their back shell.

Yet as of today, we find that horseshoe crab populations all over the world are being threatened. In terms of man-made stressors, the common practice of using horseshoe crabs as fertilizer and bait has nearly wiped out horseshoe crab populations along the East Coast and overharvesting is an extreme issue in Asia.

When our team talked with Dr. Thomas Novitsky, the retired President/CEO of Cape Cod Biosystems, about horseshoe crab harvesting in the United States, he explained the effects of the United States’ regulations:

The horseshoe crab harvest in the US is currently sustainable. Mortality from bleeding is estimated to be about 15% mainly due to handling, i.e. transport, storage conditions, time out of water, etc. Dr. Stanley Watson [President/Owner of Associates of Cape Cod Inc], myself, and Dr. Jack Levin [who discovered LAL] with whom I still collaborate, believe that bleeding in itself does not kill the crab. As I said, the horseshoe crab harvest in the US is sustainable and that includes crabs used for eel and whelk bait, which sadly is 100% lethal. In addition, the majority of crabs harvested are used for bait. Thus, if bait use were eliminated and bleeding practices were improved (and regulated), horseshoe crab populations would most likely increase.

Why Are Horseshoe Crabs Bled

What Is LAL and Factor C