Part:BBa_K3598049

AOX1 Promoter_α factor secretion signal_Snake Cathelicidin-BF_AOX 1 Terminator

The circuit we transformed into Pichia Pastoris to produce AMP Snake Cathelicidin-BF.

Demostration

Figure 1. Part demonstration

This part is a composite part consisting of an AOX1 promoter, a Snake cathelicidin BF coding sequence, and an AOX1 terminator. It is designed to produce our AMP Snake cathelicidin BF. 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 Snake cathelicidin BF fermentation.

Experiments and Results

The fermentation was carried out in BMMY medium, 5% methanol was added every 24 h to induce the expression of Snake cathelicidin BF. We tooke samples during the fermentation every 24 h, and the supernatant was used to test the antibacterial effect for AMP was secreted extracellularly.

We tested the antimicrobial potency of Snake cathelicidin BF as fermentation product of our Pichia pastoris by adding its solution to plates inoculated with P. acnes and E. coli MG1655. It can be inferred from the results that the fermentation product's effect of killing E. coli MG1655 and P. acnes are not significant. Only a certain amount of bacteria within contact of the products were eliminated and the inhibition zones are quite unclear due to low volume of AMPs dissolved in solutions.

Figure 2. Plate verification of Snake cathelicidin BF on P. acnes

Figure 3. Plate verification of Snake cathelicidin BF on E. coli

Then, we added the fermentation broth to liquid culture to test its antibacterial potency, both E.coli MG1655 and P.acnes were inoculated to the culture. 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 for its low growth rate. The results were shown in Figure 5&6, from which we can infer that the fermentation product was effective against E. coli MG1655 and P. acnes.

Figure 5. OD600 verification of 25% fermentation broth Snake cathelicidin BF on E. coli

Figure 6. OD600 verification of 20% fermentation broth Snake cathelicidin BF on P.acnes

Sequence and Features

Assembly Compatibility:

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

  Illegal NotI site found at 1317
  
• 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 1383
  
• 1000
  COMPATIBLE WITH RFC[1000]