Part:BBa_K1763001:Design

Apis mellifera (honeybee) silk fibroin 3 with regulatory elements

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]

Design Notes

• [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

1. Primers 3 and 8.
   • 5' AGCTAGAATTCGCGGCCGCTTCTAGAGCAATACGCAAACCGCCTC 3' (fwd) and 5' CGATCTGCAGCGGCCGCTACTAGTAGAACTTCTTGTCCTCGATGTTCTC 3 (rev)
     • These primers amplify the lac promoter and silk coding region and add on the biobrick prefix and suffix respectively. The template is the aforementioned IDT sequence that we ordered.

Source

Apis mellifera genome. Reference Genbank FJ235090.1