A few days after transformation the moss protoplasts was observed under a fluorescent microscope and YFP-expression was visible in moss protoplasts for the antifreeze and the STS construct (fig. 1A and 1B). This confirms that the transformation was a success and highly suggests that the genes of interest - antifreeze and STS - are expressed. It demonstrated that the gene 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.
The transformed moss protoplasts were then moved to PhyB-plates containing Kanamycin (50 mg/ml) and were left to grow for a few weeks. One month after transformation, there was ten growing clumps of antifreeze transformed moss. Seven of those clumps of moss were expressing YFP (fig. 1). This suggests stable integration of the antifreeze gene construct.
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.
The Antifreeze Experiment
While the presence of YFP in the 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. 3 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. 3 B1/C1) and red autofluorescence can be seen with 480/40 nm excitation and 510 nm emission (fig. 3 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 3: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. 4 A2/B2). This suggests that part of the antifreeze moss was still alive.
Figure 4: Results of freeze experiment at -20*c in a freezer. A) Moss transformed with the UNIK antifreeze gene 2 days after being frozen. B) WT moss 2 days after being frozen. 1) UV filter 350/50 excitation and 420 emission. 2) GFP2 filter with 480/40 excitation and 510 emission.
While these results are not conclusive, they suggest a slight increase in the survivability of transformed moss after being frozen at -20*c and -60*c. This increase in survivability could be due to the UNIK antifreeze gene.
Validation of nptII-resistance cassette in P. patens
The transformed 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 5: A moss protoplast a few days after transformation and one month after
To further validate this, an additional experiment were outlined where WT moss was grown 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. 6). The WT moss planted on Kanamycin containing plates grew very little or not at all and is seen under the microscope as withering (fig. 6 B2). This is a stark contrast to the transformed moss that was able to grow from protoplasts to full clumps and the more vibrant WT moss grown on nonselective media (fig. 6 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 6: 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 moss protoplasts transformed with the STS-construct were left to grow for 6 weeks. After 6 weeks the protoplasts had grown into small moss clumps. Liquid chromatography-mass spectrometry (LC-MS) was then used for the purpose of detecting resveratrol. 100% methanol was used for extraction and both WT moss as a negative control and transformed moss was extracted. Pure resveratrol dissolved in 100% methanol was used as a postive standard. A peak corresponding to resveratrol without a hydrogen atom 227,1 g/mol, is seen on the standard (fig. 7). However, the LC-MS machine was not able to detect resveratrol in the 3 STS transformed moss samples. This suggests that resveratrol is not produced in the transformed moss. It may also be that the amounts of resveratrol was simply to small to be detected or that there was not enough sample mass.
Figure 7: On top a graph showing peak intensity versus time. A big peak corresponding to 227,1 g/mol is present for the pure resveratrol sample (green line). No such peak is present for WT or transformed STS moss samples (red, purple lines).
Testing a new promoter
The first moss transformation confirmed the function of the Zea Mays ubiquitin promoter (ZmUbi) since YFP was present in the transformed moss protoplasts. Unfortunately, the ZmUbi promoter sequence had four illegal restriction sites towards the 3' end, which prevented it from being added to the registry as a biobrick. Since a promoter is not converted to amino acids, silent mutation was not an option. We would then try to verify the promoters function without the 3' end sequence with the illegal restriction sites. A DNA part similar to piece A, but with a partial promoter sequence without illegal restriction sites was amplified with PCR. This would have an overlapping part to a DNA part similar to piece D, which contains YFP and a terminator. Ideally, the new promoter would express YFP. After transformation of moss protoplasts no transformants expressing YFP was observed. It is therefore unlikely that the promoter works without the 3' part, even though single transformants may have been missed under the microscope.
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 :)
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.
Parts we have added to the registry:
Parts we have improved on: