Team:TU-Munich/Project/Transformation

From 2013.igem.org


Transformation in Physcomitrella patens

To transform Physcomitrella patens, the moss material has to be taken from the liquid culture and its cell walls have to be digested with driselase dissolved in mannitol to obtain protoplasts. The protoplasts are isolated by passing the digested material through sieves and the enzyme is washed off with mannitol and then resuspended in mannitol.

The number of protoplasts is determined with a hemocytometer and the material is suspended in the right amount of 3M medium to adjust the concentration. The linearized and purified DNA is mixed with PEG4000 and the protoplast solution and incubated while regularly mixing. After incubation, the mixture is diluted with 3M medium, centrifuged and resuspended in regeneration medium.

The protoplasts are put into 6-well plates, left in the dark over night and then left for 10 days for the regeneration of the cell walls at standard conditions. After moving the protoplasts onto solid medium covered with a layer of cellophane for three days, they are transferred to solid selection medium plates for two weeks. To ensure stabile integration, repeat the two weeks of selection after a two week release phase.

Trips to Freiburg

We had the great chance to perform our Physcomitrella patens transformations at Prof. Dr.Reski´s lab in Freiburg, with Dr. Wiedemann as our expert instructor. Our first trip started in a great hurry, because we worked on our DNA preparations until the very last minute. Ingmar even pulled an all-nighter to get the DNA ready. We would have missed our intercity bus if it wasn’t for Rosario who drove us to the bus station, all squished together in the “pizza mobile” with the trunk full with our medium bottles and lab equipment. After five hours on the bus, we fell into bed to get some rest, because we had a very long day ahead of us.

We arrived at the Reski lab early in the morning to meet with Dr. Gertrud Wiedemann, who instructed us throughout the day and gave us many tips how to proceed with the moss. Because of the incubation times and because it was our first try, it took us ten hours without a break until we had two boxes stacked with 6 well plates. We quickly went to get some beers and chips and met with the Freiburg iGEM team for a really nice barbecue. When we finally left, public transport wasn’t running anymore, so we didn’t miss the chance to take a midnight sightseeing tour through Freiburg, where Volker showed us around.

Ten days later, Johanna and Andi visited the Freiburg lab to transfer the then regenerated protoplasts onto agar plates and soon after, we came back for our second and final round of transformation. At our first try, we didn’t get enough moss protoplasts, so we worked through two batches and therefore had to prepare another batch of driselase. Our handling had improved, yet it still took twelve hours and again, there was no time left for a break. After we said good bye, we celebrated with a couple of beers and some yummy flammkuchen at the UC uni café of Freiburg. We had learned so much and got much closer to our goal. A big successful step for our team!

References:

  1. [[1]] Hohe, A., T. Egener, J. Lucht, H. Holtorf, C. Reinhard, G. Schween, R. Reski (2004): An improved and highly standardised transformation procedure allows efficient production of single and multiple targeted gene knockouts in a moss, Physcomitrella patens. Current Genet. 44, 339-347.



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