Introduction

In our project we want to apply our proteins (Reflectin 1A and anti-LPS factor) on a surface as a coat because our aim is to obtain an anti-gram negative bacterial surface with the help of our anti-LPS protein. We use Bacillus Subtilis as a host because this bacteria is expected to produce biofilm containing the anti-LPS protein that can be applied on a surface and this might be the best coating material for our project. 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 coating material which will possess a protective feature against infectious gram negative bacteria.

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. The components of LAL are part of this primitive "immune" system.

The LPS coat is an important reason of endotoxin and septic shock. And gram negative bacteria contain this coat. The Limulus anti-LPS factor (LALF) binds with gram negative’s LPS coat and with this function, this protein can use on the medical and pharmaceutical areas.

Lots of horseshoe crabs are collecting by some manufacture companies and they are bleeding in some laboratories. During the bleeding process, up to 30% of the animal's blood is removed. Researchs has shown that once returned to the water, the horseshoe crab's blood volume rebounds in about a week.

It takes longer for the crab's blood cell count to return to normal, about two to three months. Theoretically, crabs can be bled several times a year, but LAL manufacturers bleed them only once per year.

In our project we aim that produce the LALF by using the synthetic biology. With this way we can obtain LALF without bleeding and hurting any crab. Also, with this way the damage will decreased.

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.

Photographs provided by Associates of Cape Cod

Horseshoe crabs (Limulus polyphemus and Tachypleus tridentatus) are ancient arachnids that possess a primitive circulatory system, the hemolymph, containing only one kind of cell, the hemocyte. Exposure of hemocytes to bacterial endotoxins [lipopolysaccharide (LPS)] results in the activation of an intracellular coagulation cascade (Iwanaga et al., 1986), a defense against microbial invasion. The system consists of several proteins, including one that may inhibit the cascade, called anti-LPS factor(Morita et al., 1985). Limulus anti-LPS factor (LALF) is a small (101 amino acids), basic protein (Aketagawa et al., 1986; Muta et al., 1987), which binds and neutralizes LPS (Wainwright et al., 1990) and has a strong anti-bacterial effect on the growth of Gram-negative R-type bacteria (Morita et al., 1985)(22). Our interest in determining the crystal structure of LALF arose from its potential in designing molecules that would have therapeutic properties in humans. It has been proposed that LALF has sequence similarity with ct-lactalbumin, a protein that binds LPS in vitro (Aketagawa et al., 1986).

LALF recognizes the lipid A portion of individual soluble LPS molecules (Warren et al., 1992), which are obtained below the critical micellar concentration. The simplest molecules that bind lipid A with high affinity are the polymyxin family of antibiotics; these are positively charged amphipathic cyclic oligopeptides linked to a single fatty acid (Morrison and Jacobs, 1976).

Fig3, Fig4 (A. Hoess, S. Watson, G.R.Siber and R.Liddington.Crystal structure of an endotoxin-neutralizing protein from the horseshoe crab, Limulus anti-LPS factor, at 1.5 A resolution.) Freeze fracture electron micrographs of LPS. Alone (A), in the presence of LALF supernatant (B) and precipitation (C).( Mechanism of interaction of optimized Limulus-derived cyclic peptides with endotoxins: thermodynamic, biophysical and microbiological analysis Jorg ANDR¨A, Jorg HOWE, Patrick GARIDEL, Manfred R OSSLE, Walter RICHTER§, Jos´e LEIVA-LE ´ON_, Ignacio

Endotoxin-neutralizing protein protects against Endotoxin-Induced Endothelial Barrier Dysfunction. Endotoxin-neutralizing protein, a recombinant peptide that is derived from Limulus antilipopolysaccharide factor and targets lipid A, could block the effects of lipopolysaccharide on protein tyrosine phosphorylation, actin organization, and movement of 14C-bovine serum albumin across bovine pulmonary artery endothelial cell monolayers. endotoxin-neutralizing protein crossprotected against lipopolysaccharide derived from diverse gram-negative bacteria. LALF is a 11.8-kDa protein isolated from the amebocyte, the single blood cell type found in the horseshoe crab (13). The amebocyte-derived LALF as well as its recombinant form, endotoxin-neutralizing protein (ENP), each binds to and neutralizes LPS (13, 18). The LPS-binding site is 32 to 50 amino acids in length and forms an amphipathic loop which binds to the lipid A portion of LPS (11, 15, 18). We therefore studied whether a molecule such as ENP, which binds to lipid A and has been shown to confer protection against the deleterious effect of LPS in vivo, could block one or more of the sequential LPS-induced events leading to increased EC monolayer permeability. In this work, we have studied whether ENP protects against LPS-induced protein tyrosine phosphorylation, actin reorganization, and loss of endothelial barrier function. the importance of the amphipathic loop structure can be investigated by introducingappropriate disulfide bonds to create a cyclic conformation. However, previous investigations with cysteine- and non-cysteine- containing peptides derived from TALF, a protein highly homologous with LALF, showed little difference in LPS binding activity (23).

Considering that Limulus anti-lipopolysaccharide factor (LALF) reduces the growth of gram negative bacteria and terminates them; we determine that it can be used as a surface protector. In our Project, we plan to design LALF protein producing system in specific bacteria that will not be harmed by this bactericide itself. To do this, we needed a bacteria type that has no lip-A structure which is bound by LALF. Thus, we decided to use Bacillus subtilis colonies which are the most prefered gram positive bacteria in iGEM. In conclusion, our “protector” bacteria affects on E. Coli mortally, therefore our remaining region is cleared from E.coli. As a future aspect, a special covering material, a biofilm for example, may be used as a cover sheet on surfaces that leads to protection of bacterias.

Crystal structure of recombinant anti lipopolysaccharide factor protein, which is homologous type of our Limulus anti polysaccharide factor (LALF) protein.NMR structure of rALF-Pm3, an anti-lipopolysaccaharide factor from shrimp: model of the possible lipid A- binding site.Yang Y, Boze H, Chemardin P, Padilla A, Moulin G, Tassanakajon A, Pugniere M, Roquet F, Destoumieux-Garzon D, Gueguen Y, Bachere E, Aumelas A) Binding of 1 mg/ml biotinylated LALF-14 to lipid A and different kinds of lipopolysaccharide adsorbed to a microtiter-plate (1.0 mg/ml).(From MorphoSys GmbH, 80807 Munich, Germany, the Institute of Medical Microbiology and Hygiene, Technical University of Munich, 81675 Munich, Germany, and the Institute of Medical Immunology, Humboldt University of Berlin, 10098 Berlin, Germany. High Affinity Endotoxin-binding and Neutralizing Peptides Based on the Crystal Structure of Recombinant Limulus Anti-lipopolysaccharide Factor).The important indication of this figure for our project is E.coli’s OD on 450 nm.

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24. http://www.horseshoecrab.org