Team:Fatih Turkey/Sporocide

Left: fenton reagent + aqueous dissolved oxygen
Right: fenton reagent + distilled water
The reason that we chose B.subtilis in our project is the LPS binding character of LALF. Gram positive bacteria such as B.subtilis have no LPS layer on their cell wall. This obligation of choosing alternative bacterium leads to another problem: B.subtilis has the ability to form endospores.
B.subtilis, normally, has no pathogenic character unless it forms endospores under some circumstances like high temperature, limit pH levels or deoxygenized media. The inhalation of endospores is very dangerous and must be avoided in the case of study on B.subtilis.
Decontamination of biological endospores is very important considering the necessary of the safety in synthetic biology laboratories. Especially in iGEM, teams may need alternative creatures to plan and perform their projects properly like in our project. Because of this, we began to look through the web and hope to find a solution for this safety problem. Fortunately, some specific dilutions are characterized to solve possible safety problems in order to assemble aseptic 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)1
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.
The effectiveness of modified Fenton Reagent and the advantages/disadvantages of different Fenton Reagent solutions are characterized during our experiments. For more information about Fenton Reagent experiments, please visit Fenton Reagent Experiments page.
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 decided to extract the oxygen ingradient 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.
Considering the importance of safe and secure labs in synthetic biology, especially in iGEM, prospective projects including B.subtilis may confront such problems. The need of sporicide will increase in the next iGEM competitions. To have safer labs, we recommend our chemical decontaminator to all iGEM teams.

DISCUSSION
As a conclusion, we observed distilled water included Fenton Reagent (DFR) is more effective than oxygenated water included Fenton Reagent (OFR) against E.coli. When 25 uL OFR and DFR have been added in E.coli cultures, 82% of DFR treated culture was dead and 47% of OFR treated culture was dead. We did not see any colony which included 50 uL and more volumes of DFR. In contrast, after 75 uL and more addition of OFR, no colony formation has been observed.

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

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 was dead but 25 uL OFR and DFR treated E.coli culture was still alive. 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