Difference between revisions of "Team:UNIK Copenhagen/Results"

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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. 1A and 1B). This confirms 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 1: 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) UV filter 350/50 excitation and 420 emission. 2) GFP2 filter with 480/40 excitation and 510 emission. 3) YFP filter with 500/20 excitation and 535/30 emission.

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 antifreeze transformed moss. Seven of those clumps of moss were expressing YFP (fig. 1). This suggests stable integration of our gene constructs.

Figure 2: A clump of antifreeze transformed P.patens showing YFP-expression grown on kanamycin containing plates (50 mg/ml). A) UV filter 350/50 excitation and 420 emission. B) GFP2 filter with 480/40 excitation and 510 emission. C) YFP filter with 500/20 excitation and 535/30 emission.

Validation of nptII-resistance cassette in P. patens

Our moss was able to grow from protoplasts to full clumps on media containing kanamycin (50 mg/ml), which suggests that the nptII-resistance cassette provides P. Patens with resistance to kanamycin.

Figure 3: A moss protoplast a few days after transformation and one month after

To further validate this, we outlined an additional experiment where we grew WT moss on either non-selective PhyB-media (3 plates) or PhyB-media containing kanamycin 50 mg/ml (3 plates). After 7 days, there was a visible difference in growth between WT on non-selective media and WT on kanamycin plates (fig. 4). The WT moss planted on kanamycin containing plates grew very little or not at all and is seen under the microscope as withering (fig. 4 B2). This is a stark contrast to our transformed moss that was able to grow from protoplasts to full clumps and the more vibrant WT moss grown on nonselective media (fig. 4 A2). This experiment validates the function of the nptII-cassette consisting of the neomycin phosphotransferase II gene driven by the 35s Cauliflower Mosaic virus promoter.

Figure 4: Wild type P.patens grown on PhyB-edia with or without kanamycin (50 mg/ml). A) Wild type moss on media without kanamycin. B) Wild type moss grown on kanamycin containing plates (50 mg/ml). C) Wild type moss without kanamycin on top and with kanamycin on bottem. Photo taken 8 days after the moss was planted.

The antifreeze experiment

While the presence of YFP in our transformed antifreeze moss suggest expression of the antifreeze protein, the question remains if the antifreeze protein improves the moss ability to survive cold temperatures. To answer this question, we filled our self designed PhyscoFreezer (see Red lab for more information) with dry ice and put a plate containing 3 transformed clumps of P.patens and a plate of WT moss straight down into the dry ice. From arduino we got a consisting reading of nearly -60*c and we left the box overnight. The next day the PhyscoFreezer still contained dry ice and we replated our transformed on moss on new PhyB-media. We also took 3 equal sized clumps of WT moss and put them on new PhyB-media. After 7 days both WT and antifreeze moss looked brown and withered. However, under a fluorescent microscopes, a number of differences were observed. First, the wild type moss clumps looked amorphous and with the rhizoids in a collapsed mass (fig. 5 A1), while the antifreeze moss clumps seemed to have conserved their structure. Secondly, what seems to be cells still containing living chloroplasts were observed on one of the antifreeze clumps. The small concentrations of green colour can be seen with UV-filter (fig. 5 B1/C1) and red autofluorescence can be seen with 480/40 nm excitation and 510 nm emission (fig. 5 B2/C2). This suggest a few, still live cells. The same bright autofluorescence and green colour was not observed for the frozen WT moss.

Figure 5:Results of freeze experiment at nearly -60*c in dry ice. A) WT moss 7 days after being frozen. B and C) Moss transformed with the UNIK antifreeze gene 7 days after being frozen. White arrowheads indicate green colour or autofluorescence D) WT moss that has not been frozen. For reference with same microscope settings. 1) UV filter 350/50 excitation and 420 emission. 2) GFP2 filter with 480/40 excitation and 510 emission.

In addition, a similar experiment were conducted but only at -20*c in a normal freezer for 8 hours. 2 days after, strong autofluorescence was observed in one clump of antifreeze moss but not in WT (fig. 6 A2/B2). This suggests that part of the antifreeze moss was still alive.

Figure 6: Results of freeze experiment at -20*c in a freezer. A) WT moss 2 days after being frozen. B) Moss transformed with the UNIK antifreeze gene 2 days after being frozen. 1) UV filter 350/50 excitation and 420 emission. 2) GFP2 filter with 480/40 excitation and 510 emission.