Introduction

Filamentous fungi are only rarely used in iGEM. This is partly because genetic modifications take a lot more time, but also because there are few (if any) parts available in the iGEM registry. Fortunately, we found that the 2013 Cornell team (provide link) has selected a fungus as organism of choice. They are working on Basidiomycetes and are making a toolkit for this chassis that has not been used in iGEM before. We decided that, since Cornell is making selection markers for filamentous fungi, we would use their constructs and validate them in our organism of choice: Aspergillus niger. These constructs encoded antibiotic resistance cassettes against hygromycin and geneticin. In addition a pki promoter was send to the iGEM team of Cornell.

Aim

The goal was to test the geneticin resistance gene in A. niger. Cornell was kind enough to provide us with pure geneticin to perform the experiment. As hygromycin could not be sent via standard shipping as it is considered a hazardous material, we only worked with the geneticin resistance construct.

Approach

The construct we received from the iGEM team from Cornell University was present in E. coli and the organization of the construct was as followed: promoter, the geneticin resistence gene and a terminator. The first step was to isolate the plasmid from E. coli, and after obtaining sufficient quantities of the vector it was used to transform A. niger. The transformants were grown on two different concentrations of geneticin and one controle plate for a period of 3-4 days at 30°C. One plate contained a control and no geneticin was present in the agar. The other two plates contained different concentration of geneticin: 60µg/mL and 220µg/mL.

Results

Transformation of A. niger requires the generation of protoplasts, which is a delicate process, and somewhat more cumbersome than the making of competent E. coli cells. After transformation the protoplasts take 3-4 days to grow. Interestingly, we observed growth on all the plates, including the negative controls, in which untransformed protoplasts were left to grow on plates containing high doses of geneticin.

Discussion

In literature study it was mentioned that geneticin has an inhibitory concentration between 60 µg/mL and 200 µg/mL for Trichoderma atroviride. For this reason we selected the concentrations mentioned above. We conclude that our A. niger strain, which is closely related to the standard (and fully sequenced CBS strain, http://www.ncbi.nlm.nih.gov/pubmed/17259976), is not sensitive to geneticin. Though it is possible that the geneticin resistance gene works properly in other Eukaryotes, we conclude that it is unsuited as a selection marker for Aspergillus niger.