Part:BBa_K1825000

UNIK antifreeze protein

This is the coding region of a small antifreeze protein. The antifreeze protein used in this project is normally produced by an insect, the Spruce Budworm, Choristoneura fumiferana. It works by binding to the ice crystals as they form inside the cell, and by doing so prevents the ice crystals from enlarging and penetrating the cell membrane. Through Uniprot we found the amino acid sequence of the protein, where it had the entry name Q9GTP0⁠. We then converted the sequence of amino acids into DNA codons. This DNA sequence were then codon optimized for expression in Physcomitrella patens. Lastly, we sent the DNA sequence to IDT where the gene was synthesized.

We transformed this gene into Physcomitrella patens as a part of a larger DNA construct. 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), a nptII-resistance cassette, 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.

Gene construct with antifreeze gene

A few days after transforming this gene construct into P. patens protoplasts we observed YFP expression in several moss protoplasts. This confirms that our transformation was successful and highly suggests that our antifreeze protein is expressed. One month later, we had small clumps of moss still expressing YFP. This suggest a stable integration of our construct and expression of the antifreeze protein in a stable line of transformed P. patens.

Transformed P. patens. A) Transformed moss protoplast one a few days after transformation. Scale bar: 0.05 mm B) Transformed clump of moss one month after transformation. Scale bar: 0.6 mm C) wild type moss Scale bar: 0.5 mm. 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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 242
  Illegal BglII site found at 296
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]