Part:BBa_K1763007

Honeybee silk protein driven by T7 promoter

This part is the Apis Mellifera (Honeybee) silk protein coding region, with a composite t7 promoter element and ribosome binding site upstream, part BBa_K525998. In order to express honeybee silk protein, a bacterial strain containing the gene for T7 polymerase must be used. For example, the Bl21 (DE3) strain.

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).

Characterization

1. Protein Expression

• Here is the SDS gel of our expression of the honeybee silk protein. It was purified using [http://wolfson.huji.ac.il/purification/PDF/Protein_Expression_Extraction/NOVAGEN_BugBuster_protein_extraction.pdf this inclusion body protocol]
• [http://2015.igem.org/Team:UCLA/Notebook/Honeybee_Silk/28_July_2015 Here] is the link to the protocol that we used for expressing the protein starting from a colony. (We were using Kanamycin as our antibiotic, but chloramphenicol should be used if using this biobrick.)

Fig. 1 Expected size of product is 40.0 kDA

• There are contaminating bands present on the SDS PAGE gel, indicating that the purification is not completely effective.
• We also ran BCA protein concentration assays on our purified honeybee proteins. Based on this assay we were able to get around 12 mg of protein from a 300 ml cell culture.
  • However, this 12 mg might not be composed entirely of honeybee protein because based on the SDS PAGE results, the purification was not 100% complete.

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]