Part:BBa_K3598047
AOX1 Promoter_α factor secretion signal_GDP20_AOX1 Terminator
The circuit we transformed into Pichia Pastoris to produce AMP GDP20.
Part demonstration
The AOX1-GDP20 is a composite part consisting of an AOX1 promoter, a GDP20 sequence, and an AOX1 terminator. It is designed to produce our AMP GDP20. 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 GDP20 production.
Experiments and Results
GDP 20 was produced by fermentation in BMMY, 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 GDP20 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.
Then, we verified GDP 20'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.
Sequence And Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 1260
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 937
Illegal XhoI site found at 1191 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1326
- 1000COMPATIBLE WITH RFC[1000]
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