Part:BBa_K3598050

AOX1 Promoter_α factor secretion signal_tridecapeptide_AOX1 Terminator

The circuit we transformed into Pichia Pastoris to produce AMP tridecapeptide.

Demonstration

Figure 1. Part demonstration

The AOX1-tredicapeptide is a composite part consisting of an AOX1 promoter, a tredicapeptide sequence, and an AOX1 terminator. It is designed for the expression of our AMP tredicapeptide. We inserted the sequence of the system onto vector pPIC9K and transferred the plasmid into Pichia pastoris GS115 genome. The recombinant strain was cultured for GDP20 production.

Experiments and Results

Tridecapeptide was produced by fermentation in BMMY, 5% methanol was added every 24 h to induce its expression. Sampling the fermentation medium every 24 h, and the supernatant was used to test the antibacterial effect for AMP was secreted extracellularly.

We tested the antimicrobial potency of tridecapeptide fermentation supernatant by adding it to plates inoculated with P. acnes and E. coli MG1655. From the results (Figure 2&3), it can be inferred that the fermentation product's effect of killing E. coli MG1655 and P. acnes are not significant for the inhibition zones are quite unclear. That may be caused by the low concentration of antimicrobial peptides in the fermentation broth.

Figure 2. Plate verification of tridecapeptide on P. acnes

Figure 3. Plate verification of tridecapeptide on E. coli

Then, we verified tredicapeptide's ability as a fermentation product to kill E. coli MG1655 and P. acnes by adding its solution to liquid culture inoculated with the two bacteria. We set the AMP concentration gradient as 2.5% and 25% in verification with E. coli and 25% with P. acnes because 2.5% proved in prior verification to be too low a concentration for the effects to be significant, and measured the OD600 of the bacteria. The results show that the fermentation product is effective in killing E. coli MG1655 and P. acnes.

Figure 5. OD600 verification of 25% fermentation broth tridecapeptide on E. coli

Figure 6. OD600 verification of 20% fermentation broth tridecapeptide on P.acnes

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 1266
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 937
  Illegal XhoI site found at 1191
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1332
  
• 1000
  COMPATIBLE WITH RFC[1000]