Part:BBa_K1763007:Design

Honeybee silk protein driven by T7 promoter

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]

Design Notes

Keep in mind that in order for the promoter to work as expected, the plasmid must be transformed into a cell line that contains a gene coding for T7 polymerase.

• [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 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.

• In order to create this part, we simply added biobrick prefix and suffix via pcr to the silk gene using the aforementioned primers and gene template. We then proceeded to use standard iGEM assembly to add this construct downstream of biobrick BBa_K525998.
  • Specifically, we double digested K525998 with Spe1 and Pst1, and digested our construct with Xba and Pst1. We then ligated them together and transformed into our BL21 (DE3) cells.

Source

Genbank FJ235090.1 Genomic sequence from Apis Mellifera