Difference between revisions of "Part:BBa K3598049"

Latest revision as of 13:58, 27 October 2020

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 took 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 P. acnes, while its efficiency to E.coli was not obvious.

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]

@@ Line 11: / Line 11: @@
 [[File:T--BEIJING 4ELEVEN--1. Lark20201024-223420.png|400px|thumb|center|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.
+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 took 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.
@@ Line 20: / Line 22: @@
 [[File:T--BEIJING 4ELEVEN--3-49.png|400px|thumb|center|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.
+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 P. acnes, while its efficiency to E.coli was not obvious.
 [[File:T--BEIJING 4ELEVEN--5Snake Cathelicidin-BF Efficiency to MG1655 of 25% Fermentation Broth (2).png|400px|thumb|center|Figure 5. OD600 verification of 25% fermentation broth Snake cathelicidin BF on E. coli ]]