Difference between revisions of "Part:BBa K3598050"

Revision as of 13:05, 25 October 2020

AOX1 Promoter_α factor secretion signal_tridecapeptide_AOX1 Terminator

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

Usage and Biology

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 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 tredicapeptide 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 tredicapeptide on P. acnes

Figure 3. Plate verification of tredicapeptide 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 4. OD600 verification of 2.5% fermentation broth tredicapeptide on E. coli

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

Figure 6. OD600 verification of 25% fermentation broth tredicapeptide on P.acnes

Sequence and Features