Part:BBa_K1763015

T7 Promoter+Silk+sfGFP

This part is a fusion of the Apis mellifera (Western Honey Bee) silk protein coding region and the superfolder GFP coding region. A T7 promoter regulatory element has been added upstream, along with a ribosomal binding site. It is essentially a fusion between sfGFP (BBa_I746916) and BBa_K1763007 , which is honeybee silk driven by T7 promoter. This part produces Apis mellifera silk protein that can fluoresce green under blue and UV light.

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 goal of our project is to add new functionality to these fibers. As a proof of principle, we fused sfGFP to the honeybee silk protein to see if physical properties could be maintained after fusion to a foreign protein. sfGFP is a version of green fluorescent protein that provides a visual way for us to assess whether or not we were able to successfully functionalize the honeybee silk.

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
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]