Part:BBa_K1763008

Promoter + Silk + SpyCatcher

This part is a fusion protein of Apis Mellifera (Honeybee) silk and SpyCatcher affinity domain, which binds specifically to the short Spytag peptide sequence. When this is protein is expressed, it can be processed into various silk materials including films, gels, or fibers, and can be customizably conjugated to anything modified with the SpyTag. It is under control of a Lac Promoter and is ready for expression upon induction with IPTG.

Usage and Biology

Silk from Apis Mellifera represents an intriguing alternative to silks from spiders or silkworms. Although it is not quite as strong as these other types of silks, working with honey bee silk has certain advantages over spider and silkworm silk (Weisman). The size of the honey bee silk protein gene is considerably smaller than the silk genes of spiders or silkworms. More importantly, the gene sequence is non repetitive, which allows us to synthesize and make modifications to the gene without the complications that are inherent to repetitive DNA sequences. Honey bee silk also has a very different secondary and tertiary structure than spider and silkworm silks. It forms primary alpha helices, and four silk proteins come together to form a coiled coil structure these coiled coils are formed from four similar, yet unique proteins, Amelf 1-4 (Sutherland 2007). However, a study has shown that using one of these proteins, (Amelf3) is sufficient to reproduce the physical properties of the wild type fibers (Sutherland).

A major goal of this project is to be able to functionalize our fibers. Adding SpyCatcher to our silk theoretically would allow us to conjugate peptides to our silk that have been modified to cotain the short SpyTag peptide. This strategy of functionalizing honeybee silk is advantageous because it does not depend on creating a different fusion protein for each functionalized fiber you want to create. The SpyCatcher SpyTag system instead, only requires the addition of a small peptide to your protein of interest, SpyTag, which allows it to bind to the silk.

Characterization

• This construct has been sequence verified.
• We have not yet expressed this part.

References

• Weisman, S., Haritos, V., Church, J., Huson, M., Mudie, S., Rodgers, A., Dumsday, G., Sutherland, T. Honeybee silk: Recombinant protin production, assembly, and fiber spinning. Elsevier Ltd. 2009.
• Sutherland, T., Church, J., Hu, X., Huson, M., Kaplan, D., Weisman, S. Single Honeybee Silk Protein Mimics Properties fo Multi-Protein Silk. PLoS ONE 2011. e16489
• Sutherland, T. Conservation of Essential Design Features in Coiled Coil Silks. Mol Biol Evol 2007;24:2424-2432

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 221
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]