Part:BBa_K3598051

AOX1 Promoter_α factor secretion signal_Human CathelicidinLL-37_AOX 1 Terminator

The circuit we transformed into Pichia Pastoris to produce AMP Humancathelicidin-37.

Demonstration

Figure 1. Part demonstration

This part is a composite part consisting of an AOX1 promoter, a Human cathelicidin LL-37 sequence, and an AOX1 terminator. It is designed for the expression of our AMP Humancathelicidin-37. We inserted the sequence of the system onto vector pPIC9K and transferred the resulting plasmid into Pichia pastoris inoculated on BMMY culture, then added 5% methanol every day to induce its expression.

Experiments and Results

In our experiments, Humancathelicidin-37 was produced by fermentation in BMMY medium, 5% methanol was added every day to induce its expression. During the fermentation, we took sample every 24 h, and the supernatant was used to test the antibacterial effect for AMP was secreted extracellularly.

In the plate test, we add Humancathelicidin-37 fermentation supernatant directly to the plate with P. acnes and E. coli MG1655. From the results (Figure 2&3), we found the inhibition zone were not clear, which means their efficiency against bacteria are not obvious. That may be caused by the low concentration of antimicrobial peptides in the fermentation broth.

Figure 2. Plate verification of Human cathelicidin LL-37 on P. acnes

Figure 3. Plate verification of Human cathelicidin LL-37 on E. coli

Then, we verified Humancathelicidin-37'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 Human cathelicidin LL-37 on E. coli

Figure 6. OD600 verification of 20% fermentation broth Human cathelicidin LL-37 on P.acnes

Sequence and Features

Assembly Compatibility:

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

  Illegal NotI site found at 1335
  
• 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 1401
  
• 1000
  COMPATIBLE WITH RFC[1000]