Team:iTesla-SoundBio

Template:ITesla-SoundBio

Project Description

Every year, hundreds of thousands of horseshoe crabs are caught each year and drained of up to to 30% of their blood. Why? To collect LAL, a chemical that is crucial in the detection of endotoxins in everything from drugs to medical equipment.

LAL, or Limulus Amebocyte Lysate, found in Horseshoe Crab amebocytes, is extremely sensitive to LPS, a membrane component of gram-negative bacteria. The chemical detects these harmful endotoxins by coagulating in their presence, thus creating a visible signal of bacterial contamination. The reaction proceeds as follows: First, a present endotoxin cleaves and activates a zymogen in LAL, named Factor C. Cleaved Factor C then activates Factor B, which in turn converts a proclotting enzyme in LAL into a clotting enzyme. This clotting enzyme cleaves coagulen, another protein present, and turns it into coagulin, turning the whole mixture into a coagulated signal communicating that endotoxin is present.

While this process has been relied upon for the approval of any FDA drug, the confirmation of safe pacemakers, and the sanitation of medical equipment, it is the procurement of LAL itself that has proven inefficient and dangerous of the Horseshoe Crab population.

iTesla-SoundBio is working towards creating a viable synthetic equivalent of the LAL Assay. We have chosen to focus on Factor C, a crucial part of the LAL reaction cascade, but a valuable component by itself as well. Since Factor C cleaves in the presence of endotoxin, we are working towards synthetically producing Factor C and incorporating it into a fusion protein-system that will give off a detectable signal when exposed to endotoxin.

Our project has divided itself into two phases. The first is the actual production of synthetic Factor C by bacillus subtilis. This phase has provided its own considerations, for example taking into account the large coding region, as well as inducing expression in a gram-positive system to avoid automatic cleavage of the protein itself. The second phase comprises the conception and assembly of a signal amplification system that will produce a detectable signal when Factor C is cleaved.

While there are already companies who have developed an alternative to the LAL Assay using synthetic Factor C, the high price point has encouraged us to try to produce a simpler, cheaper alternative.

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