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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 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.

On the left two plates with 60µg/mL geneticin and on the rigth two plates with 220µg/mL geneticin are shown. The control plates are also included on the right side of the two pictures.

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), 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.

The iGEM team of NRP UEA Norwich is working on a biosensor to be able to detect antimycins, an anti-fungal compound produced by Streptomyces. For the quality of their project they asked different iGEM teams to send them soil samples from different places in their hometown. Of course we grabbed our bikes, played in the mud and collected samples to help out the iGEM team of Norwich.

Soil samples in Greiner tubes

Gel extraction and DNA purification kits are expensive to come by for most iGEM teams. Few of our team members found a cheaper and effective alternative for the same. These are homemade silica spin columns which have made it into the Elseiver journal, Analytical biochemistry. We sent this protocol to the iGEM team of TU Delft as they had problems with gel extraction and buying expensive kits.

The iGEM team of Purdue asked iGEM teams to fill in a questionnaire about the standardization of the data sheet of the biobricks. They asked more than 50 teams around the world about their opinion and iGEM Wageningen was one of them. Of course we were happy to fill in this questionnaire and help them. Especially because it will help us and future iGEM teams in submitting their parts and search for other interesting parts.

Why are you joining the iGEM and what experiences do you want to get and actually get? This is something the iGEM team Paris was wondering about and that is why they sent around a survey we were happy to fill in. Every team member was asked to do it separately as it was a survey about your motivation.