Part:BBa_K3598048

AOX1 Promoter_α factor secretion signal_CEN1HC-Br_AOX 1 Terminator

The circuit we transformed into Pichia Pastoris to produce AMP CEN1HC-Br.

Usage and Biology

Figure 1. Part demonstration

The AOX1-CEN1HC-Br is a composite part consisting of an AOX1 promoter, a CEN1HC-Br sequence, and an AOX1 terminator. It is designed for the expression of our AMP CEN1HC-Br. 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.
We tested the antimicrobial potency of CEN1HC-Br 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 CEN1HC-Br on P. acnes

Figure 3. Plate verification of CEN1HC-Br on E. coli

Then, we verified CEN1HC-Br'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 4. OD600 verification of 2.5% fermentation broth CEN1HC-Br on E. coli

Figure 5. OD600 verification of 25% fermentation broth CEN1HC-Br on E. coli

Figure 6. OD600 verification of 25% fermentation broth CEN1HC-Br on P.acnes

Sequence and Features

Figure 1. Part demonstration

The AOX1-Snake cathelicidin BF is a composite part consisting of an AOX1 promoter, a Snake cathelicidin BF sequence, and an AOX1 terminator. It is designed for the expression of our AMP Snake cathelicidin BF. 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

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 verified Snake cathelicidin BF'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 4. OD600 verification of 2.5% fermentation broth Snake cathelicidin BF on E. coli

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

Figure 6. OD600 verification of 25% fermentation broth Snake cathelicidin BF on P.acnes

Assembly Compatibility:

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

  Illegal NotI site found at 1311
  
• 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 1377
  
• 1000
  COMPATIBLE WITH RFC[1000]