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June 3
Fusion PCR was initiated.
June 6
The samples from the PCR are run on an agarose gel.
Aspergillus nidulans American strain was grown overnight in two 1-liter Erlenmeyers, each filled with 250ml transformation medium. Settings: 250 rpm, 30˚C & 250 rpm, 37 ˚C. Both were inoculated with 1*106 spores/mL. Update next day: Did not grow, because no supplements were added. Without them, the strain will not grow at all.
June 7
American strain was grown on transformation medium overnight. This time we tried to eliminate pellet formation by adding talc powder. One Erlenmeyer was filled with 250 mL transformation medium and incubated at 30˚C, a similar one was incubated at 37˚C. Both contained no talc powder. Two remaining Erlenmeyers were treated the same way, but this time talc powder was added. Each of these 4 Erlenmeyers was inoculated with 1*106 spores/mL.
June 8
Protoplasts were recovered from the grown mycelium using the standard protocol(32). The mycelium grown from the 30˚C without talc powder was used, cause this one showed to have the best mycelium. (decent growth and least amount of pellets). This resulted in 5 mL protoplast solution (1*108 protoplasts/mL), which is good for 25 transformations. (200 µL solution was aliquoted in each Eppendorf)
June 9
A transformation was carried out on the protoplasts, to see whether they are transformable. A co-transformation accompanied with a negative and a positive control was carried out using standard transformation protocol. Youri had some plasmid stocks available that were used for this purpose. For the positive control a Pyr plasmid was used. Update after four days: No transformants grew, not even the positive control worked. Though, some Aspergillus niger colonies grew on the plates, which indicates contamination.
June 14
Re-did co-transformation of protoplasts. Next to this, a second test was carried out to check the amount of viable protoplasts in the protoplast solutions and to check for mycelia contamination. Update after three days: No transformants grew. The second test did however show good results.
June 20
Inoculated 350 ml of transformation medium in two 2-liter Erlenmeyers with 0.5*106 spores/mL and grew them overnight at 30˚C.
June 20
Tubular shaped mycelial clumps were visible in both cultures. protoplasts were recovered. This resulted in 100 aliquots.
June 23
In the morning, 3 MM plates and 3 MMS plates were treated with all supplements needed for the strain to grow (Parabenzoic acid, Riboflavine-5p & Uridine) and inoculated with either 10, 100 or 1000 protoplasts. This test was carried out to check the amount of viable protoplasts in the protoplast aliquots and to check for mycelia contamination. In the afternoon, a co-transformation was carried out accompanied with a negative and a positive control using standard protocol. Update after four days: No protoplasts grew in both tests, only slight mycelial growth observed.
June 28
Inoculated 250 ml of transformation medium in a 1-liter Erlenmeyer with 1*106 spores/mL and grew overnight at 30˚C.
June 29
This time, no pellets were observed in the grown medium. Protoplasts were recovered. This however resulted in a very low amount of 2 aliquots.
