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 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 autofluorescence (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 antifreeze transformed moss. Seven of those clumps of moss were expressing YFP (fig. 4). This suggests stable integration of our gene constructs.
Figure 5: A clump of antifreeze transformed P.patens showing YFP-expression grown on kanamycin containing plates (50 mg/ml). A) Bright field picture. B) Filter showing autofluorescence (red) and YFP-expression (green).
C) Filter showing only YFP-expression (green).
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 6: 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 nonselective 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 nonselective media and WT on kanamycin plates (fig. 7). The WT moss planted on kanamycin containing plates grew very little or not at all and is seen under the microscope as withering (fig. 7 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. 7 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 7: 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.
Considerations for replicating the experiments
Doing PCRs can be surprisingly hard. Usually not when amplifiyng small DNA fragments like Piece B or C but for longer pieces it gets a lot more technical difficult. Piece A was around 5000 bp and piece D was around 3000 bp and that made the process difficult. From around the time we started in the laboratory, we spent a lot of time just optimizing the PCR reactions for Piece A and C. Not only was it hard to just get a band on the gel, it was also difficult to amplify a high enough amount of each piece, since you need 30 ug of DNA total for our moss transformation protocol. Looking in the hind mirror, we could have potentially have amplified Piece A in two different overlapping pieces and the same for Piece D. But we suspect that having more separate DNA pieces (4-6) for one transformation would have affected our transformation efficiency negatively. Moss is fun, but as we already mentioned, it grows very slowly. So buckle up and plan ahead :)
Future plans
Our future plans is to grow the transformed lines of P.patens so that they can be used as a starting point for next year’s teams. We imagine that future teams can do experiments with our antifreeze moss and/or do additional transformations with our lines, to make them even more adapted to the martian environment.
Confirming the function of stilbene synthase
The antifreeze experiment
Testing a new promoter
Parts we have added to the registry:
Parts we have improved on: