Team:iTesla-SoundBio/ProjectStory

Summary (heavy text warning)

The biomedical industry relies heavily on the benefits provided by the horseshoe crab. As infection and sanitation are extremely important and pertinent issues addressed by biomedical companies and professionals alike, the horseshoe crab’s unique blood, which can be used to

detect bacterial endotoxins for medical purposes, have become a staple in the biomedical world. Their blood contains amebocytes which can fight against pathogens, and these amebocytes are then used to make Limulus amebocyte lysate, or LAL. With such beneficial and life-saving potential, horseshoe crab blood can go for close to $15,000 per quart. However, this practice is unsustainable and detrimental to the horseshoe crab species. In order to extract the amebocytes and make LAL, the horseshoe crabs’ pericardiums are stabbed and bled. The aim of our project is to provide an alternative to this process by designing e.coli with factor C, a protein that activates the coagulation response in horseshoe crab blood. [Talk about what we want to use to signal when endotoxin is detected/factor C is cleaved] ← still figuring this out

1: WHAT ARE HORSESHOE CRABS?

things to discuss: their habitat, population, anatomy, role in ecosystem, etc

Citation Numbers 1-60

Notes:

Range: atlantic coast of north america from nova scotia to the pucatan peninsula

Size: 10 to 20 in (25-50 cm)

Diet: worms, small mollusks, algae

Description: they have five legs arranged around the mouth

 

We used the Japanese Horseshoe Crab

  • They have a long evolutionary history [1]
    • Horseshoe crabs have been around since 450 million years ago, but now finally their population is in danger [4]
  • Many other animals rely on the horseshoe crab for food and in some regions, are keystone species [2]
    • Keystone species in coastal regions as their eggs are the primary source of food for millions of shorebirds (only 10 out of the 90,000 eggs laid by females survive) [3]
  • In some areas along the East Coast, the horseshoe population was nearly wiped out due to being ground-up for fertilizer and chopped up for bait


Their importance

  • Animals like the red knot, ruddy turnstone, sanderling depend on horseshoe crabs for nutrition
  • Other algae, worms, and barnacles live on their back
  • Ecosystem blah-blah they’re important to sustain see here:https://www.nature.com/scitable/knowledge/library/biodiversity-and-ecosystem-stability-17059965

These sites may have been used:

https://www.americanpharmaceuticalreview.com/Featured-Articles/167236-The-Incredible-Horseshoe-Crab-Modern-Medicine-s-Unlikely-Dependence-on-a-Living-Fossil/

http://www.inlandbays.org/

http://www.horseshoecrab.org/anat/circ.html


2: WHAT ARE THE CURRENT PROCESSES/PRACTICES?

things to discuss: what happens to horseshoe crabs before and after bleeding, why are horseshoe crabs bled, what happens with the blood (that it is used for LAL)

Citation Numbers 61-100

Notes:

  • 500,000 a year are caught
  • Horseshoe crab blood goes for $15,000 quart
  • Biomedical industry is extremely reliant
  • short-term changes in their behavior and physiology that could exacerbate the crabs' population decline in parts of the east coast [60]
    • Primarily environmental devastation results in the steady decline of the population
  • A recent trend shows an increase in horseshoe crab blood harvest
  • Mortality rates for crabs who have been drained of their blood has risen to 30% [60]
  • For female horseshoe crabs, their reproductive abilities decline, and both male and female crabs have a larger chance of dying after release [60]
    • Crabs are most often caught during their breeding cycle, when its easiest to capture them on beaches
    • Bleeding of crabs -> disorientation -> might affect their ability to breed
  • blue color of their blood is due to the presence of copper in hemocyanin
  • A large animal can yield 200 - 400 mL of blood
  • The horseshoe crab has an open circulatory system, which means all internal surfaces and organs are bathed in blood. Because of this, any pathogen introduced to the body can easily access all vital organs. [61]
    • Picture [64]
    • this requires it to have an immune system that can clot toxins as fast as possible and seal the rest of the body from the pathogens [62]
    • Its blood contains amoebocytes that can detect bacterial toxins and presence, trap them by clotting with coagulin (a process called coagulation), and isolate them [61]
    • LAL uses those amoebocytes to detect toxins (maybe use this as a segway into the next section)
  • Stab the pericardium ---> centrifuge blood, separate from serum ---> pellet is washed then lysed ---> freeze-dried into powder
  • Horseshoe crab harvested during spawning season.
  • Tests using LAL cost $195/$250 [70]
  • We we use horseshoe crabs as opposed to to methods [63]
    • Before they used rabbits which wasn’t feasible [63]
    • When they harvest 30% of the hemolymph (combined blood and interstitial fluid) is taken
    • Fewer females show up to mate and they are preferentially bled because they are larger

The horseshoe crab is the backbone of the denver coastal shore ecosystem

Why is the test done

  • Tests the quality of food

People helping out horseshoe crabs

  • "They were coming to the beaches and hauling off tractor-trailer loads of 10,000 at a time," Hall says.
  • When the census showed the population was declining, a management plan was created regulating the number of animals that could be harvested. Hall says that last year's survey finally gave some indication that the population is recovering. "That's what we figured it would take," he says, "a minimum of 10 years of management for the population to recover, since it takes 10 years for them to mature."


3: WHAT IS LAL AND FACTOR C?

things to discuss: what are the medical/actual uses of LAL, where does factor c fit in with LAL, what happens to factor c

Citation Numbers: 101-180

Notes:

  • Amebocytes (mobile cells) that horseshoe crabs use to detect pathogens
  • Proteins in the amebocytes are released when gram-negative bacteria is detected, these proteins bind to endotoxins and inactivate them [181]
  • A clotting cascade is invoked in the presence of endotoxin
  • Each person who has received a medical injection has been impacted by LAL and by extent the horseshoe crab  
  • No other practice is as accurate as the LAL test for testing purity of medicines [4]
  • The limulus amebocyte lysate (LAL) has been widely used for ~30 years for the detection of endotoxin in the quality assurance of injectable drugs and medical devices. The LAL constitutes a cascade of serine proteases which  which are triggered by trace levels of endotoxin, culminating in a gel clot at the end of the reaction. The Factor C, which normally exists as a zymogen, is the primer of this coagulation cascade. [102]
  • It is used to test for contamination during the manufacture of anything that might go inside the human body: every shot, every IV drip, and every implanted medical device.
    • Use of LAL ensures the sterilization of medical equipment and supplies
  • The unassuming horseshoe crab is indispensable to human health

[101]

  1. The endotoxin binds with the LPS receptor on the cell, granules in the cell (containing factor) travel to the surface of the cell to release the granules contents kinda like vesicles and neurotransmitters
    1. Factor C is triggered by LPS, the zymogen1 factor C has a H chain2 (80 kD)3 and L chain is (43 kD), in autocatalyitcally becomes activated AKA DESIGNATED FACTOR C
    2. factor C becomes cleaved at its light chain, the Phenylalanine-isoleucine bond gets cleaved resulting in a B chain and A chain
  2. Zymogen factor B is activated by factor c which activates the proclotting enzyme, and an activation peptide is released
    1. Coagulation cascade is separate so its illustrated here, the serine protease zymogen Factor G is activated activations to proclotting enzyme
  3. The clotting enzyme cleaves 2 peptide bonds in coagulogen (a fibrinogen molecule specific to arthropods) to form a insoluble coagulin gel
  4. Coagulogen a 175 chain chain polypeptide is converted to insoluble coagulin with limited proteolyses at 2 place. Excision of intermediate peptide C results in coagulin monomer (a smol molecule) called AB which self aggregates to make a gel like substance.

4: WHAT IS THE GOAL OF OUR PROJECT?

things to discuss: what is the final stage of this project, what are we going to do with factor c, is it better than current processes, what are the benefits

Citations 181-210

The aim of our project is to provide an alternative to the harvest of horseshoe crab blood by designing e.coli with factor C, a protein that activates the coagulation response in horseshoe crab blood. This will also help protect marine ecosystems by reducing the number of horseshoe crabs killed by the blood harvesting process. [181]

Notes:

  • Rather than bleeding horseshoe crabs, our team wants find a more sustainable, less harmful way to detect the presence of life threatening pathogens and bacteria.
  • Focus on the cleavage of Factor C instead of the entire coagulation cascade
  • Produce Factor C in Bacillus subtilis and create a mechanism for detecting cleaved fc


5: WHAT IS THE DESIGN OF OUR PROJECT?

things to discuss: why b. Subtilis, about our vector, what are our biobricks, how are we getting to the final stage

Citations 211-280

As stated above, we are trying to replicate the LAL assay using synthetically Factor C, a component of the reaction cascade that LAL uses to coagulate in the presence of endotoxin. To do this, our workflow should proceed as follows (I’m pretty sure this is correct?):

  • we found the sequence for Factor C produced by the Japanese Horseshoe Crab, or Tachypleus tridentatus. Unfortunately, the free synthesis deal iGEM has with IDT forced us to split this geneblock into two halves, which we named Factor C Fragment 1 and Factor C Fragment 2.
    • Thus, our project encounters an additional step, we first need to create a continuous factor c before we can push for expression in our expression vector.
    • When we split up our Factor C gene into two fragments, we split them so as to create a restriction digest site, BglII, right where they join. We will use this restriction digest site in the future to join the two fragments together
    • We inserted biobricking prefixes and suffixes that would straddle each fragment.
    • We also made sure that the restriction sites BamHI SacII straddled the theoretical completed Factor C gene as well. These restriction sites lie inside of the biobricking prefixes and suffixes for future digestion and ligation into our expression vector.
  • we digest fragment 1 with PstI and EcoRI, and digest our biobricking vector, psb1c3, with PstI and EcoRI, as well. We ligate those two components together. We transform that fragment 1 + psb1c3 complex into e.coli, and grow up copies of that plasmid with fragment one inside. We miniprep successful colonies and sequence them to ensure our success.
  • We then should digest the successfully miniprepped and sequenced plasmid/fragment 1 complex with BglII and PstI. This will make an opening that will allow Fragment 2 to ligate to the appropriate side of Fragment 1. After digest Fragment 2 with the same enzymes, and ligating/ transforming those components into e.coli, a successful colony will have fragment 1 and 2 ligated together to form a complete factor c gene in our biobricking backbone, psb1c3.
    • This will be our main biobrick for our project. Secondary biobricks we may send in are the individual fragments.
  • After creating our complete factor C gene in psb1c3, we need to get it into our expression vector, pax01.
    • This expression vector is optimized for bacillus subtilis, a gram-positive bacteria. We are using a gram-positive bacteria because of the role factor C plays in the reaction cascade of LAL. Factor C cleaves in half in the presence of endotoxin, which itself is a component of gram-negative bacteria. If we used e.coli, a gram-negative bacteria, as our expression mechanism, any successful expression would yield already cleaved and useless Factor C protein. By using B.subtilis, we would express Factor C without inducing it to automatically cleave.
    • We obtained the pax01 expression vector through the advice of Dan Ziegler of the Bacillus Genetic Stock Center (http://www.bgsc.org). Pax01 is an integration vector, which means that it won’t exist in B.subtilis as a free-floating plasmid, but rather it will integrate itself directly into the chromosomes of B.subtilis.
  • To get our complete factor C gene into our expression vector, pax01, we need to digest both the miniprepped psb1C3 and pax01 with BamHI and SacII. By digesting the miniprepped psb1c3 with these two enzymes, we free the completed factor C gene from the biobricking prefixes and suffixes.
  • Finally to finish off our expression mechanism for factor C, we need to follow some integration protocol that I’m not sure where it is yet, to integrate this completed expression plasmid into B.Subtilis’ genome.
  • After we have integrated pax01 into B.subtilis, we can start expressing Factor C, and then we can start creating out detection mechanism.
    • Fusion protein? Nanobodies? Who knows.
  • Abbreviated complete phase 1 workflow: https://docs.google.com/document/d/1zlNOwnXDt9q61xQ4Qzu2YrE8aYZ_0aFuMorKVKnwy3U/edit

Other biobricks we need to consider in order to fulfill silver requirements:

  • Optimized promoter for b.subtilis
  • Pax01 vector


6: WHAT’S NEXT?

things to discuss: what happens after our project, how can our project be taken further/adapted, are there any long-term goals

Citations: 281-370

Notes:

  •   Phase 2
    • Carrying on the LAL cascade in a shorter version
    • Shortening the process that involves factor b, the clotting factor, and other reactions
    • This could involve: the sushi domains in factor c, Non cleaved factor C binding site 281 , Diisopropyl_fluorophosphate282
      • It probably binds to the sugars of the endotoxin (outer core part)