Part:BBa_K1763000:Design

Apis mellifera (honeybee) silk fibroin 3

Assembly Compatibility:

[http://www.sciencedirect.com/science/article/pii/S0142961209013805 This paper] referred us to the some of the honeybee silk sequences. There are 4 different honeybee silk sequences in native silk that come together to form a silk fiber. However, it was shown that by just expressing one of the three sequences, you could obtain silk with comparable physical properties to the native honeybee silk (Weisman). So we chose Apis mellifera silk fibroin 3 and we synthesized the sequence, making sure to omit the signal peptide at the 5' end of the protein.
Here is the sequence that we had synthesized by IDT. It includes some regulatory elements and is also fused to Spy Catcher protein for future experiments. However, we designed primers to isolate just the silk portion for cloning. You can see some of the primers we designed and where they attach to the template sequence.
Here are the sequences and purposes of the pertinent primers we designed

Primers 5 and 6.
- 5' ATGGGCGTCGAGGAGTTC 3' (fwd) and 5' GAACTTCTTGTCCTCGATGTTCTC 3' (rev)
  - These primers amplify just the silk coding region (no stop codon).
Primers 7 and 8.
- 5' GAACTTCTTGTCCTCGATGTTCTC 3' (fwd) and 5' CGATCTGCAGCGGCCGCTACTAGTAGAACTTCTTGTCCTCGATGTTCTC 3' (rev)
  - These primers amplify the silk coding region and add on the biobrick prefix and suffix respectively.

Weisman, Sarah, et al. "Honeybee silk: Recombinant protein production, assembly and fiber spinning." Biomaterials 31.9 (2010): 2695-2700.
Sutherland, Tara D., et al. "Single honeybee silk protein mimics properties of multi-protein silk." PLoS One 6.2 (2011): 16489.
Hepburn, H., Duangphakdee, R., & Pirk, O. (2013). Physical properties of honeybee silk: A review. Apidologie, 44(5), 600-610.