Team:UNIK Copenhagen/Results
Project Results
A few days after transformation we observed the moss protoplasts under a fluorescent microscope and saw YFP expression in moss protoplats for the antifreeze and the STS construct (fig. 4A and 4B). This confirms the that our transformation was a success and highly suggests that our genes of interest, antifreeze and STS, are expressed. It demonstrated that our construct design works and that moss can combine different DNA pieces with matching overhangs using homologous recombination.
Figure 4: Fluorescence microscopy pictures of P. patens transformed with our genetic constructs. A) A moss protoplast transformed with the antifreeze construct. B) A moss protoplast transformed with our STS construct. C) A moss protoplast transformed with a vector expressing YFP. A positive control. D) WT moss. A negative control. 1) Bright field picture. 2) Filter showing autoflouorescence (red) and YFP-expression (green). 3) Filter showing only YFP-expression (green).
Our transformed moss protoplasts were then moved to to PhyB-plates containing kanamycin (50 mg/ml) and were left to grow for a few weeks. One month after transformation we had ten growing clumps of transformed moss. Seven of those clumps of moss were expressing YFP (fig. 4). This suggests stable integration of our gene constructs. Our moss was able to grow from protoplasts to full clumps on media containing kanamycin, which suggests that the nptII-resistance cassette provides Physcomitrella Patens with resistance to kanamycin. To further validate this, we outlined an additional experiment, where we grew WT moss on either nonselective PhyB-media (3 plates) or PhyB-media containing kanamycin (3 plates). After 9 days, there is a visible difference in growth between WT on nonselective media and WT on kanamycin plates. XXXX. This is a stark contrast to our transformed moss, that was able to grow from protoplasts to full clumps. This experiment validates the function of the nptII-cassette consisting of the neomycin phosphotransferase II gene driven by the 35s Cauliflower Mosaic virus promoter. (new figure)
Figure 5: A clump of transformed P. Patens a few weeks after transformation showing YFP-expression, grown on kanamycin containing plates (50 mg/ml). A) Bright field picture. B) Filter showing autoflouorescence (red) and YFP-expression (green).
C) Filter showing only YFP-expression (green).