Coding

Part:BBa_M36996:Design

Designed by: Nathan Barnett, Meghan Bowler, Kian Torabian   Group: Stanford BIOE44 - S11   (2011-04-28)

Non-Glycolated Antifreeze Protein (AFP) - 10-Repeated Segment


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 32
    Illegal PstI site found at 98
    Illegal PstI site found at 170
    Illegal PstI site found at 182
    Illegal PstI site found at 281
    Illegal PstI site found at 329
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 32
    Illegal PstI site found at 98
    Illegal PstI site found at 170
    Illegal PstI site found at 182
    Illegal PstI site found at 281
    Illegal PstI site found at 329
    Illegal NotI site found at 133
    Illegal NotI site found at 232
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 360
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 32
    Illegal PstI site found at 98
    Illegal PstI site found at 170
    Illegal PstI site found at 182
    Illegal PstI site found at 281
    Illegal PstI site found at 329
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 32
    Illegal PstI site found at 98
    Illegal PstI site found at 170
    Illegal PstI site found at 182
    Illegal PstI site found at 281
    Illegal PstI site found at 329
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The antifreeze protein (AFP) sequence was mostly copied from Solomon and Appels' experiments. They derived this Type I AFP from the white flounder, Pseudopleuronecta americanus, and created a recombinant AFP (rAFP). This rAFP is comprised of five 11-amino acid repeat units. According to their paper, more units increases the antifreeze capabilities of the protein. The rAFP final structure is an a-helix. The first 11-amino acid unit is slightly different from the rest (TASDAAAAAL as opposed to TAANAAAAAAL). The long alanine chain of the first unit was also reduced from six to five alanines. We will investigate if this changes the helical structure and function of the rAFP; we expect it will not. According to Solomon and Appels, the aspartic acid residue in the first unit may play a role in helical dipole stabilization. Similarly, the TAR amino acid sequence at the C-terminus contains an arginine residue that is thought to contribute to helical dipole stabilization. The DNA sequence has been optimized for expression in E.coli while maintaining some degeneracy to avoid incorrect looping and splicing during transcription. Solomon and Appels had done this in the context of degeneracy to increase accuracy during oligo ligation in PCR annealing.


This specific sequence has 5 extra repeating units of "TAANAAAAAAL" before the TAR amino acid sequence at the C-terminus.

Source

Based on the 5-repeated Segment AFP from the white flounder, Pseudopleuronecta americanus.

References

1. Antifreeze Protien Sequence Information: Solomon, R. G., and Appels, R. (1999) "Stable, High-Level Expression of a Type I Antifreeze Protein in Escherichia Coli." Protein Expression and Purification 16: 53-62.

2. Secondary Structure Prediction Tool: Jones, David T., Daniel Buchan, Sean Ward, Anna Lobley, Tim Nugent, and Kevin Bryson.PSIPRED: Protein Structure Prediction Server. PSIPRED. BioInformatics Group - University College London, Oct. 2010. Web. Apr. 2011. <http://bioinf.cs.ucl.ac.uk/psipred/>.

3. Codon Optimization Tool: Richardson, S. M., S. J. Wheelan, and J. D. Boeke. "GeneDesign: Codon Juggling." The Bader Lab. Dec. 2009. Web. Apr. 2011. <http://genedesign.thruhere.net/cgi-bin/gd/gdCodJug.cgi>.