Coding

Part:BBa_K1825005

Designed by: Jonathan Asmund Arnesen   Group: iGEM15_UNIK_Copenhagen   (2015-09-03)

nptII

This sequence codes for neomycin Phosphotransferase and it is almost a twin of BBa_K1021001. But since it is not identical, we had to add this sequence to the registry since we use it in the composite biobrick BBa_K1825006. Nevertheless, here is the description of our experiments with the nptII-gene which also can be found under BBa_K1021001.

The gene was a part of a larger construct used for transforming moss Physcomitrella patens. We transformed this gene into Physcomitrella patens as a part of a larger DNA construct (fig. 1). This DNA construct contained in the following order. A homologous region to the 108 locus on the moss genome (to ensure stable integration into P. patens genome), the nptII-resistance gene driven by the 35s cauliflower mosaic virus, the Zea mays ubiquitin promoter driving the antifreeze protein (AFP) linked to yellow fluorescent protein (YFP) with the LP4 linker sequence, terminator and lastly another 108 region.

Figure 1) Gene construct with a gene encoding for an antifreeze protein. The nptII-gene is expressed with the 35s Cauliflower Mosaic virus promoter












Our moss was able to grow from single transformed 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 (fig. 2).

Figure 2) Moss protoplast a few days after transformation and one month later when grown on kanamycin














To further validate this, we outlined an additional experiment where we grew wild type (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. 3). The WT moss planted on kanamycin containing plates grew very little or not at all and is seen under the microscope as withering (fig. 3 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 without kanamycin (fig. 3 A2). This experiment validates the function of the the neomycin phosphotransferase II gene in P. patens.

Figure 3) 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.





































Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 629
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//chassis/eukaryote/ppatens
Parameters
None