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

This part is a composite part consisting of an AOX1 promoter, a tridecapeptide sequence, and an AOX1 terminator. It is designed for the expression of our AMP tridecapeptide. 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 tridecapeptide production.

Experiments and Results

Tridecapeptide was produced by fermentation in BMMY medium, 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 AMP's potency by adding the fermentation broth to liquid culture inoculated with the two E.coli MG1655 and P.acnes, 25% was added in verification to E. coli and 20% added to P. acnes. The OD600 were measured at 12 h for E.coli, while P.acnes was measured at 48h. The results were shown in Figure 5&6, from which we can infer that the fermentation product was effective in inhibting the growth of 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]