Team:US AFRL CarrollHS/Demonstrate
Chitinase and Cinnamaldehyde Testing
To determine the efficacy of our destroy mechanism, we decided to test the ability of cinnamaldehyde and chitinase to inhibit the growth of Yarrowia lipolytica. We used Yarrowia lipolytica for our testing because it was one of the fungal isolates from fuel tanks that Dr. Wendy Goodson provided us. We obtained the cinnamaldehyde and chitinase we used from Sigma. We used a chitinase from Streptomyces griseus for the testing. One of the chitinases we designed our microbe to produce also originated from Streptomyces griseus, which should act very similarly to the chitinase we tested with, even though they are not exactly the same chitinase. For our experiment, we plated dilute fungi culture on TSA plates, let them dry, and then pipetted spots of cinnamaldehyde, chitinase, and a combination of both onto the plates (the full protocol for our testing can be found on our Experiments page). After letting them incubate at 27°C for 1-2 days, we examined the plates for zones of clearing. Plates from one of our tests are shown below.
Figure 1. Chitinase test plate. The concentration of chitinase for the top row is 10 mg/ml, 1 mg/ml for the middle row, and 0.1 mg/ml for the bottom.
Figure 2. Cinnamaldehyde test plate. The concentration of cinnamaldehyde for the top row is 0.66 mg/ml, 0.33 mg/ml for the middle row, and 0.16 mg/ml for the bottom.
Figure 3. Combined test plate. The concentrations of chitinase and cinnamaldehyde respectively for the top row are 10 mg/ml and 0.66 mg/ml, 10 mg/ml and 0.33 mg/ml for the second row, 1 mg/ml and 0.66 mg/ml mg/ml for the third row, and 1 mg/ml and 0.33 mg/ml for the bottom row.
The fungi did not grow very evenly on the chitinase test plate, so no real conclusions can be made from it. The cinnamaldehyde plate did not have total coverage by the fungi, but the cinnamaldehyde by itself did not seem to inhibit the growth of the fungi. Finally, the plate with both cinnamaldehyde and chitinase did not have perfect coverage, but it showed some indication of a zone around some of the spots with a 10 mg/ml concentration of chitinase (the spot in the middle column and top row exhibits the clearest zone). None of the plates turned out well enough to make conclusive statements, but the plate with both cinnamaldehyde and chitinase indicates that there is potential for the combination of both to inhibit fungal growth.
Cinnamaldehyde Biofilm Assay Testing
In order to test cinnamaldehyde’s preventative abilities against biofilm formation, multiple experiments were conducted against Nissle E. coli, a bacteria known for growing biofilms. The goal of the experiment was to discover the half maximal effective concentration (EC 50 ) of cinnamaldehyde that would be effective in preventing biofilm formation. The results of the experiments showed that on average, the lowest concentration of cinnamaldehyde required was 0.26 mg/mL to remove all growth. Further testing needs to be completed in order to find the range between 0 and 0.26 mg/mL that will give the EC 50 .
Figure 3. Graph showing the effect of different concentrations of cinnamaldehyde on biofilm formation.
N-butanoyl-L-Homoserine lactone (C4-HSL) Testing with Green Fluorescent Protein
The RhlR gene will be used as the receptor gene for the quorum sensing molecule N-butanoyl-L-homoserine lactone (C4-HSL). Once C4-HSL is bound to the RhlR gene, the plasmid will begin the production of a green fluorescent protein (GFP). Once constructed, the team wanted to test in order to verify the plasmid was working the way we intended. Four tubes were filled with LB and DH5-Alpha E.coli cells containing the RhlR and GFP plasmid. C4-HSL was then added to two of the tubes and incubated overnight. The next day, the tubes were placed under UV light in order to see if any of the GFP was being produced. As seen in the picture below, the two tubes containing the C4-HSL are glowing green, indicating that the protein was being produced and the plasmid was working correctly.
Figure 4. Test tubes from the C4-HSL testing. The two control tubes are not glowing, while the tubes with C4-HSL are glowing!