Part:BBa_K1021004:Experience

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Applications of BBa_K1021004

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.

To further test this construct, we plan induced the araBAD promoter within E.coli BL21 with arabinose to produce higher concentrations of afp1. Because the glucose within CYM represses the araBAD promoter, LB media was used for the growth assay with induction. The results from this assay were very interesting, as both BL21 culture and afp1 producing culture completely inhibited the growth of A.niger. We hypothesize that because LB media (that allows for growth of E.coli) was used in this assay, the BL21 was able to outcompete A.niger regardless of production of afp1.

Another attempt was made to quantify the activity of antifungal protein 1 using inoculation disks. As with the last test, E.coli BL21 without antifungal protein had a similar affect to cultures expressing antifungal protein. There zone of inhibitions was similar for BL21 and antifungal protein 1 trials while plain LB had no associated zone of inhibition. The image to the right shows a typical plate for this experiment. The disks with afp1 expressing culture and plain BL21 culture have very clear zones of inhibition while the LB plate has none.

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