Team:Cornell/project/wetlab/fungal toolkit/antifungals

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Ecovative has encountered problems with mold and other fungal contamination during the growth phase of their biomaterial. These once mixed with the culture, these contaminants can 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 <i>Aspergillus</i> species (especially <i>Aspergillus fumigatus, Aspergillus niger</i>, and <i>Aspergillus brasiliensis</i>) 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 <i>Aspergillus</i> species.  
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Ecovative has encountered problems with mold and other fungal contamination during the growth phase of their biomaterial. These once mixed with the culture, these contaminants can 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 <i>Aspergillus</i> species (especially <i>Aspergillus fumigatus, Aspergillus niger</i>, and <i>Aspergillus brasiliensis</i>) 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 <i>Aspergillus</i> species.
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To test the activity of afp1 on <i>Aspergillus niger</i>, we ran a growth assay.  <i>E.coli</i> BL21-AI cultures with sequence confirmed PT7 and afp1 plasmids were used as an antibiotic spread on CYM agar plates.  Equal masses of <i>Aspergillus niger</i> were plated on these as well as empty and <i>E.coli</i> 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 <i>Aspergillus niger</i>, 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 <i>E.coli</i> BL21 with arabinose to produce higher concentrations of afp1 and to experiment with a more susceptible species like <i>Aspergillus fumigatus</i>.
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To test the activity of afp1 on <i>Aspergillus niger</i>, we ran a growth assay.  <i>E.coli</i> BL21-AI cultures with sequence confirmed PT7 and afp1 plasmids were used as an antibiotic spread on CYM agar plates.  Equal masses of <i>Aspergillus niger</i> were plated on these as well as empty and <i>E.coli</i> 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 <i>Aspergillus niger</i>, 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.   
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To further test this construct, we plan induced the <i>araBAD</i> promoter within <i>E.coli</i> BL21 with arabinose to produce higher concentrations of afp1. Because the glucose within CYM represses the <i>araBAD</i> 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 <i>A.niger</i>.  We hypothesize that because LB media (that allows for growth of <i>E.coli</i>) was used in this assay, the BL21 was able to outcompete <i>A.niger</i> regardless of production of afp1. 
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                                Luria Broth
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                                <i>E.coli</i>  BL21
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                                afp1
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Another attempt was made to quantify the activity of antifungal protein 1 using inoculation disks.  As with the last test, <i>E.coli</i> 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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                                Left: afp1  Middle: LB  Right: BL21
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<h3> References </h3>
<h3> References </h3>
1. Bormann, C., Baier, D., Horr, I., Raps, C., Berger, J., Jung, G., & Schwarz, H. (1999). Journal of bacteriology.Characterization of a Novel, Antifungal, Chitin-Binding Protein from <i>Streptomyces tendae</i> Tü901 That Interferes with Growth Polarity,181(24), 7421-7429.
1. Bormann, C., Baier, D., Horr, I., Raps, C., Berger, J., Jung, G., & Schwarz, H. (1999). Journal of bacteriology.Characterization of a Novel, Antifungal, Chitin-Binding Protein from <i>Streptomyces tendae</i> Tü901 That Interferes with Growth Polarity,181(24), 7421-7429.

Latest revision as of 02:39, 29 October 2013

Cornell University Genetically Engineered Machines

Antifungals


Ecovative has encountered problems with mold and other fungal contamination during the growth phase of their biomaterial. These once mixed with the culture, these contaminants can 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 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.
Luria Broth
E.coli BL21
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.

Left: afp1 Middle: LB Right: BL21

References

1. Bormann, C., Baier, D., Horr, I., Raps, C., Berger, J., Jung, G., & Schwarz, H. (1999). Journal of bacteriology.Characterization of a Novel, Antifungal, Chitin-Binding Protein from Streptomyces tendae Tü901 That Interferes with Growth Polarity,181(24), 7421-7429.

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