Team:Fatih Turkey/LALF

As we know, seawater is a virtual "bacterial soup". Typical near-shore areas that form the prime habitat of the horseshoe crab can easily contain over one billion Gram-negative bacteria per milliliter of seawater. Thus, the horseshoe crab is constantly threatened with infection. Unlike mammals, including humans, the horseshoe crab lacks an immune system; it cannot develop antibodies to fight infection. However, the horseshoe crab does contain a number of compounds that will bind to and inactivate bacteria, fungi, and viruses. Anti-LPS factors that are synthesized in the blood cells of crab are part of this primitive "immune" system.
Lots of horseshoe crabs are collected by some manufacture companies and their blood is extracted in some laboratories. During the extraction process, up to 30% of the animal's blood is removed. Research has shown that once returned to the water, the horseshoe crab's blood volume rebounds in about a week. But, it is also noted that a number of crabs which cannot be undervalued are also dying during the process.
                                        

In our project, we aim to produce those factors by using the synthetic biology. In this way, we can obtain them without extracting blood and hurting any crab. Additionally, the possible medication will be gathered cheaper.
In our project, mainly we study on a protein that is gathered from horseshoe crab (limulus polyphemus), limulus anti lipopolysaccharide factor (LALF). By the help of this protein, we planned to stop bacteria growth in vitro situations. 

Lipopolysaccharide (LPS), or endotoxin, is the major mediator of septic shock, a serious complication of Gram-negative bacterial infections in humans. Molecules that bind LPS and neutralize its biological effects or enhance its clearance could have important clinical applications. Limulus anti-LPS factor (LALF) binds LPS tightly, and, in animal models, reduces mortality when administered before or after LPS challenge or bacterial infection. The wedge-shaped molecule has a striking charge distribution and amphipathicity that suggest how it can insert into membranes. The binding site for LPS probably involves an extended amphipathic loop, and it has been proposed that two mammalian LPS-binding proteins will have a similar loop. The amphipathic loop structure may be used in the design of molecules with therapeutic properties against septic shock.
To make it possible, we use Bacillus Subtilis as a host; because it is supposed that this bacterium cannot be affected by LALF. Preventing such possibilities during our experiments will help us to get the best and the clearest results. 
On the other hand, we want to apply our anti-LPS factor on a surface as a coat in order to obtain an anti-gram negative bacterial surface. Normally, B.subtilis has the ability to produce biofilm. This complex media may also include some components and protein that are synthesized by bacteria. We think that after the production of LALF, the protein can pass to biofilm with the help of signal peptides we added. As long as LALF remains in the biofilm, the surface that is covered with that biofilm material will not be infected by gram negatives.

The LPS coat of gram negative bacteria is an important reason of endotoxin and septic shock. LALF binds to that LPS coat and inhibits the growth of bacteria. This ability is very crucial and may be very useful considering the diseases and pandemics that are caused by gram negatives. Our project suggests a possible prevention for such circumstances.
Also, by killing spores of B.subtilis on the biofilm surface with aqueous dissolved oxygen, ascorbic acid, and copper ions, we will try to perform a sterilized coating material which will possess a protective feature against infectious gram negative bacteria.

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