Growing environmental concerns have led to an increase in the use of biofuels. Biofuels contain higher concentrations of organic compounds, making them much more suitable for bacterial and fungal growth than traditional fuels. Consequently, fuel tanks or pipes containing biofuels are much more susceptible to biofilms. Biofilms cause a number of problems, including clogging pipes and filters, degrading the fuel, and corroding fuel tanks and pipes. The goal of this project is to combat biofilms by engineering an E. coli to swim to and destroy the biofilms.
Pseudomonas aeruginosa, a bacterium found in most biofilms, releases the quorum sensing molecule C4-Homoserine Lactone (C4-HSL). By sensing C4-HSL, the RhlR promoter will allow for the transcription of CheZ gene. Expression of the CheZ gene allows the flagella motors of E. coli to rotate counterclockwise in a straight-line path instead of tumbling. It continues to swim throughout the biofilm with the ability to tumble and redirect its path even if it leaves the concentration gradient of C4-HSL. Along with swimming to the biofilm, the engineered E. coli will produce chitinase and cinnamaldehyde to kill the fungi and bacteria. Low concentrations of Cinnamaldehyde destroy bacteria by breaking down the cell membrane. However, fungi have cell walls, which prevents cinnamaldehyde from reaching the cell membrane. Chitinase breaks down chitin, a major component of fungal cell walls, and allows the cinnamaldehyde to reach the cell membrane and kill fungal cells. Two forms of chitinase are used, Chitinase C-1 from Streptomyces griseus and Chitinase B4A from Serratia marcescens. Fusing chitinase and ice nucleation protein tethers the chitinase to the membrane of the engineered microbe, ensuring the chitinase will not diffuse away from the biofilm.