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Team:Cornell/project/wetlab/fungal toolkit/antifungals
From 2013.igem.org
Antifungals
Ecovative encountered problems of mold and other fungi contamination during the growth phase of the biomaterial. These contaminants can contaminate and outcompete the growing mycelium. These fungi compete in standoff where opposing fungal species will secrete enzymes designed to halt the growth of the competitor. During our collaborations with Ecovative we identified various Aspergillus species (especially Aspergillus fumigatus, Aspergillus niger, and Aspergillus brasiliensis) as likely and potentially harmful contaminants. To help reduce the harmful effects of these contaminants on the growing mycelium, we decided to pursue expressing antifungals in the growing mycelium to combat the Aspergillus species.
Aspergillus niger
Expressing an antifungal within a fungus seems rather risky, as it could very well harm the desired mycelium as much as or even more than the contaminants. In our research, however, we found an antifungal protein from the bacteria Streptomyces tendae that has specific activity toward Aspergillus species and is benign to many other fungi tested [1]. This antifungal protein will contribute to the standoff phenomenon and should give our mycelium a competitive advantage over its contaminants.
To test the activity of afp1 on Aspergillus niger, we ran a growth assay. E.coli BL21-AI cultures with sequence confirmed PT7 and afp1 plasmids were used as an antibiotic spread on CYM agar plates. Equal masses of Aspergillus niger were plated on these as well as empty and E.coli BL21 control plates. The mass of the plate after growth was measured and compared to the original mass of the CYM agar plate. The governing principle behind this test is that as the fungi grow, the rate of respiration will increase, converting solid carbon compounds from the agar plate into CO2 gas and decreasing overall mass. If our construct was effective in inhibiting the growth of Aspergillus niger, then the afp1 plates should not decrease in mass as much as the controls. Our test revealed that mass loss was similar for all tests. We hypothesize that afp1 was not present at a high enough concentration to substantially inhibit growth under basal expression conditions. To further test this construct, we plan on inducing E.coli BL21 with arabinose to produce higher concentrations of afp1 and to experiment with a more susceptible species like Aspergillus fumigatus.
