Team:US AFRL CarrollHS/Design
In order to combat the fungi predominant in biofilms, a vast array of mechanisms were needed to effectively eliminate the microbial contaminants. The LabPats started with the premise that they would use cinnamaldehyde in order to puncture the cell membrane of the bacteria and fungi present in the biofilm. However, fungi have an additional cell wall that must be penetrated. To effectively infiltrate this cell wall, the team aimed to weaken it structurally. The LabPats chose to use chitinase to make a hydrolysis reaction with the chitin in the cell wall. With chitin being the main structural component of the fungal cell wall, degradation of the cell wall allowed access to the cell membrane. After extensive testing and research, they found that in order to get the concentration of cinnamaldehyde and chitinase in the fuel high enough to destroy the biofilms, they would need an unreasonably high amount of both substances. In order to mitigate this problem, the team knew that we’d need to find a way to transport the cinnimaldehyde and chitinase towards the biofilms.
With all of this in mind, we started designing our plasmids in order to express all of these desired characteristics. We designed 4 plasmids in total: our delivery plasmid, our cinnamaldehyde plasmid, and two different chitinase plasmids. In the delivery plasmid, the team decided on utilizing both quorum sensing and chemotaxis to reach the biofilms. Expressing these would allow for the engineered cell to swim towards the biofilm and then activate the mechanism necessary to break down the cell walls of the fungi and bacterial contaminants. C4-HSL is a quorum sensing molecule commonly expressed by Pseudomonas aeruginosa, a very common component of biofilms. When our microbe sensed a large concentration of C4-HSL, the gene CheZ will be expressed, allowing the flagella motors of the cell to activate and rotate in a clockwise direction. Even if the Microbe left the concentration gradient of C4-HSL, the bacteria’s natural ability to tumble will be expressed and eventually the microbe should end up back into the concentration gradient to swim towards the biofilm. The next plasmid was the cinnamaldehyde plasmid. The team perused the iGem registry for previously made parts. The parts utilized Phenylalanine commonly found in E. coli to create cinnamaldehyde. Finally, the last two plasmids were the chitinase plasmids. The team decided on two different chitinases to express so that there could be a wider range of chitin that could be destroyed. In one plasmid, the C-1 chitinase was expressed, and in the other, B4A was expressed. These chitinases were chosen because they both came from bacteria and should thus express well in E. coli, and because both were shown in the literature to have antifungal properties.
By deploying our modified E. coli cell with all of the engineered plasmids expressed, the biofilm growth would theoretically be reduced. The quorum sensing and chemotaxis mechanisms expressed would allow the team's microbe to swim towards the biofilm. Then, the teams two chitinases would allow for the cinnamaldehyde to puncture the cells of the biofilm effectively killing it. Additionally, the cinnamaldehyde would also kill the engineered cell itself, leaving behind no living organisms within the fuel.