Difference between revisions of "Part:BBa K1825000"

 
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This is the coding region of a small antifreeze protein. 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. We wanted to test if there would be a significant difference in the growth rate of moss, Physcomitrella patens, with the antifreeze gene, compared to the one without. This would be done at a low temperature to test the in-vivo function of the antifreeze protein.
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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.
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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, 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.
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[[File:AFP construct.jpg]]
  
 
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[[File:Example.jpg]]

Revision as of 13:54, 12 September 2015

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, 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. AFP construct.jpg

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]


Example.jpg