Team:Fatih Turkey/Sporocide

In our Project, we designed a cover sheet made of bacterial biofilm that possess Limulus anti lipopolysaccharide factor (LALF); thus, the surface that is covered with it, has an aseptic character specially against the gram negative bacteria. However, there was still a problem in the isolated layer; LALF protein does not affect gram positive bacteria. Therefore, we decided to use a specific sporicide that kills gram positive bacteria; even in their endospore forms. Sporicide is called “Fenton reagents”.

Decontamination of biological endospores is very important considering the necessary of the safety in synthetic biology laboratories. Thus, some specific dilutions are characterized to gain septic surfaces. Fenton reagents, which kill septic microorganisms with mechanism of hydroxyl radicals, were traditionally being used. However; its major ingredient, hydrogen peroxide, was examined and was compared with aqueous dissolved oxygen about their capability of killing the spores. (J. B. Cross, 2003)

It is said that in addition to aqueous dissolved oxygen, cupric chloride, ascorbic acid, sodium chloride and a little sulfactant dilution which are used as modified Fenton reagent are more effective on endospores of gram positive bacteria. In contrast; classic Fenton reagent, which possess hydrogen peroxide and copper ion, shows that gram negative bacteria are more vulnerable to this formulation. The mechanism of these dilutions is not well-characterized; however, comparing with other dilutions that include specific oxidizing agents such as ozone, some differences in the images that are gained from effected bacteria are indicated. It is deduced that modified Fenton reagent affects the inside mechanism of the bacteria; although the dilutions with ozone damages the coat of the bacteria. After formation of hydroxyl radicals within the spore, these highly reactive molecules attack either the DNA or enzymes necessary for the spore conversion to a bacterial cell. Further investigations of ion transport into spores and oxidation-reductions with in spores will be needed to determine precisely the mechanism of spore kill under these conditions.

Another problem we faced was that the sporicide can also kill gram negative bacteria in their non-endospore forms. Our LALF protein is there for this reason; but we should be sure that gram negative bacteria are terminated just because of LALF protein. To achieve this, we determine to extract the oxygen in gradient which is the main component of killing mechanism in the Fenton reagent. Thus, hydroxyl radical mechanism of their agents would not work and only mechanism on the surface as terminator would be our anti-LPS factor against the gram negative bacteria, especially E.coli in our project.

DISCUSSION

As a conclusion we observed Distilled water including Fenton Reagent (DFR) is more effective than Oxygenated water including Fenton Reagent (OFR) in E.coli. When 25 uL OFR and DFR have been added in E.coli cultures, 82% of DFR treated culture is dead and 47% of OFR treated culture ‘s dead. We didn’t see any colony which included 50 uL and more DFR. However, after 75 uL and more addition of OFR, any colony formation has not been observed

https://static.igem.org/mediawiki/igem.org/1/15/Resimecoli.JPG

On the other hand we observed OFR is more effective than DFR in Bacillus subtilis.When 10 uL OFR and DFR have been added in Bacillus subtilis cultures,38% of DFR treated culture is dead and 98.3% of OFR treated culture’s dead. Not only the 25 uL OFR treated but also 25 uL DFR treated culture is dead.

https://static.igem.org/mediawiki/igem.org/7/7e/Resimbacillus.JPG

If we want to compare E.coli and B. subtilis, we can say that Fenton Reagent application is more fatal for Bacillus subtilis. Both 25 uL OFR and DFR treated Bacillus subtilis culture is dead but 25 uL OFR and DFR treated E.coli culture is still alive.

REFERENCES

1. Killing of Bacillus Spores by Aqueous Dissolved Oxygen, Ascorbic Acid, and Copper Ions; J. B. Cross, R. P. Currier, D. J. Torraco, L. A. Vanderberg, G. L. Wagner, and P. D. Gladen Chemistry Division1 and Biosciences Division,2 Los Alamos National Laboratory, Los Alamos, New Mexico 87545