Difference between revisions of "Part:BBa K3598050"
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The circuit we transformed into Pichia Pastoris to produce AMP tridecapeptide. | The circuit we transformed into Pichia Pastoris to produce AMP tridecapeptide. | ||
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− | === | + | ===Demonstration=== |
− | [[File:T--BEIJING 4ELEVEN--Atri 1.png| | + | |
− | + | [[File:T--BEIJING 4ELEVEN--Atri 1.png|400px|thumb|center|Figure 1. Part demonstration]] | |
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− | We tested the antimicrobial potency of | + | This part is a composite part consisting of an AOX1 promoter, a tridecapeptide sequence, and an AOX1 terminator. It is designed for the expression of our AMP tridecapeptide. 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 tridecapeptide production. |
− | [[File:T--BEIJING 4ELEVEN--Atri 2.png| | + | |
− | [[File:T--BEIJING 4ELEVEN--Atri 3.png| | + | ===Experiments and Results=== |
− | Then, we verified | + | |
− | + | Tridecapeptide was produced by fermentation in BMMY medium, 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. | |
− | [[File:T--BEIJING 4ELEVEN--Atri 5.png| | + | |
− | [[File:T--BEIJING 4ELEVEN-- | + | We tested the antimicrobial potency of tridecapeptide 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. |
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+ | [[File:T--BEIJING 4ELEVEN--Atri 2.png|400px|thumb|center|Figure 2. Plate verification of tridecapeptide on P. acnes]] | ||
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+ | [[File:T--BEIJING 4ELEVEN--Atri 3.png|400px|thumb|center|Figure 3. Plate verification of tridecapeptide on E. coli]] | ||
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+ | Then, we verified AMP'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. | ||
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+ | [[File:T--BEIJING 4ELEVEN--Atri 5.png|400px|thumb|center|Figure 5. OD600 verification of 25% fermentation broth tridecapeptide on E. coli ]] | ||
+ | |||
+ | [[File:T--BEIJING 4ELEVEN--tri 6.png|400px|thumb|center|Figure 6. OD600 verification of 20% fermentation broth tridecapeptide on P.acnes]] | ||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> |
Latest revision as of 14:25, 27 October 2020
AOX1 Promoter_α factor secretion signal_tridecapeptide_AOX1 Terminator
The circuit we transformed into Pichia Pastoris to produce AMP tridecapeptide.
Demonstration
This part is a composite part consisting of an AOX1 promoter, a tridecapeptide sequence, and an AOX1 terminator. It is designed for the expression of our AMP tridecapeptide. 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 tridecapeptide production.
Experiments and Results
Tridecapeptide was produced by fermentation in BMMY medium, 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 tridecapeptide 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 AMP'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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 1266
- 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 1332
- 1000COMPATIBLE WITH RFC[1000]