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Revision as of 21:30, 1 November 2017
Design
Background
What is Lipopolysaccharide?
"Endotoxin" is often used as a general term to describe any bacterial toxin, but is more properly used to describe components of the cell surface of Gram-negative bacteria, particularly the lipopolysaccharide complex. Lipopolysaccharide (LPS) is composed of a core oligosaccharide, O-antigen a glycan polymer and Lipid A. The lipid component, Lipid A is a phosphorylated glucosamine disaccharide with multiple fatty acids and is the primary cause for endotoxin toxicity. When gram-negative bacteria enter the human body a complement immune response is initiated. Once the cell wall and/or bacteria are destroyed endotoxins are released which can lead to endotoxemia, the symptoms of which are vomiting, nausea, diarrhea, fever, disseminated intravascular coagulation, vascular collapse, organ failure and possibly death. Antibiotics will not inactivate the endotoxins, therefore detection of the endotoxin before they enter the body is prudent.
What do horseshoe crabs have to do with endotoxin?
![Horseshoe Crab in Aquarium](https://static.igem.org/mediawiki/2017/c/cf/T--Georgia_State--horseshoebranfromaquarium.jpg)
In the presence of endotoxin, a clotting cascade is invoked to activate the proclotting enzyme which is used to transform coagulogen into coagulin. The first step in this cascade involves activation of zymogen Factor C, a 123 kDa glycoprotein that becomes enzymatically active in the presence of bacterial endotoxin. Factor C consists of an H chain (80kD) and L chain (43kD); in the presence of LPS the enzyme becomes active and undergoes autocatalysis, the phenylalanine- isoleucine bond on the L chain is cleaved resulting in a B chain (34kD) and an A chain (8.5 kD). Activated Factor C then activates Factor B which activates the proclotting enzyme. The activated proclotting enzyme then converts coagulogen into coagulin, resulting in a clotting-like reaction which isolates the bacterial pathogen.
![Cotting Cascade](https://static.igem.org/mediawiki/2017/9/9a/T--Georgia_State--FactorCcascade.jpg)
![Factor C diagram](https://static.igem.org/mediawiki/2017/1/1d/T--Georgia_State--factorcdiagram.jpg)
What is LAL assay?
![Clotting Cascade](https://static.igem.org/mediawiki/2017/2/24/T--Georgia_State--LALcascadeandBLUEblood.jpg)
![horseshoe crab bleeding](https://static.igem.org/mediawiki/2017/5/52/T--Georgia_State--horseshoecrabbleeding.jpg)
So where does GSU come in to play?
Using horseshoe crab blood is an unsustainable practice due to the impact on the crabs and the expense of the LAL assay. A third of the blood is taken from the horseshoe crab before they are realsed back into the water. The theory is that the crabs eventually heal however up to 30% of bled crabs die. The total population is decreasing rapidly while producers are forced to increase harvests to keep up with global demand. The LAL test is expensive to make; a quart of blood is sold for $15,000. Part of affordable healthcare means that the production of the products used also need to be affordable.
Creating an alternative form of testing is unavoidable. Instead of creating a recombinant version of the entire clotting cascade it is more efficient to create a recombinant version of factor c and include a detection mechanism to detect the autocatalysis in the presence of LPS. Human chorionic gonadotropin (hCG) beta subunit is detected through a human pregnancy test. Using the subunit in tandem with the pregnancy test allows for an efficient and inexpensive autocatalysis detection method. The goal of our project is to create a fusion protein of Factor c with hCG and immobilize it in a solution. When the LPS is exposed to the factor c it will undergo autocatalysis and release hCG into the solution where it is then detected by the pregnancy test.
![factorc1](https://static.igem.org/mediawiki/2017/4/4b/T--Georgia_State--factorcdiagrampart1.jpg)
![factorc1](https://static.igem.org/mediawiki/2017/a/a3/T--Georgia_State--factorc2.jpg)
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References
- - -B. Akbar John, K.C.A. Jalal, Y.B. Kamaruzzaman and K. Zaleha, 2010.Mechanism in the Clot Formation of Horseshoe Crab Blood during Bacterial Endotoxin Invasion (July 10, 2010). Journal of Applied Sciences, 10: 1930-1936. Retrieved June 11, 2017, fromhttp://scialert.net/fulltext/?doi=jas.2010.1930.1936
- - - Elizabeth Cox. (2017,September, 21). Why do we harvest horseshoe crab blood? [Video file]. https://www.youtube.com/watch?v=VgEbcQxFUu8/
- - - Ecological Research & Development Group. (2013).
Horseshoe Crabs and Endotoxin Testing . . Retrieved October 31, 2017, from http://www.horseshoecrab.org/med/sustainable.html
- - - Ecological Research & Development Group. (2013). [Copper Based "Blue blood" and Cascade of Enzymes and Proteins ]. JRetrieved October 31, 2017, from
http://www.horseshoecrab.org/med/testing.html
- - - PBS. (2014, February 26). A still for the PBS Nature documentary Crash [Digital image]. Retrieved October 31, 2017, from
https://www.theatlantic.com/technology/archive/2014/02/the-blood-harvest/284078/
Back to Home
-
References
- - -B. Akbar John, K.C.A. Jalal, Y.B. Kamaruzzaman and K. Zaleha, 2010.Mechanism in the Clot Formation of Horseshoe Crab Blood during Bacterial Endotoxin Invasion (July 10, 2010). Journal of Applied Sciences, 10: 1930-1936. Retrieved June 11, 2017, fromhttp://scialert.net/fulltext/?doi=jas.2010.1930.1936
- - - Elizabeth Cox. (2017,September, 21). Why do we harvest horseshoe crab blood? [Video file]. https://www.youtube.com/watch?v=VgEbcQxFUu8/
- - - Ecological Research & Development Group. (2013).
Horseshoe Crabs and Endotoxin Testing . . Retrieved October 31, 2017, from http://www.horseshoecrab.org/med/sustainable.html
- - - Ecological Research & Development Group. (2013). [Copper Based "Blue blood" and Cascade of Enzymes and Proteins ]. JRetrieved October 31, 2017, from
http://www.horseshoecrab.org/med/testing.html
- - - PBS. (2014, February 26). A still for the PBS Nature documentary Crash [Digital image]. Retrieved October 31, 2017, from
https://www.theatlantic.com/technology/archive/2014/02/the-blood-harvest/284078/